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Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila

  1. Yan Wang
  2. Menglong Rui
  3. Quan Tang
  4. Shufeng Bu
  5. Fengwei Yu  Is a corresponding author
  1. National University of Singapore, Singapore
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Cite this article as: eLife 2019;8:e39964 doi: 10.7554/eLife.39964

Abstract

Class IV ddaC neurons specifically prune larval dendrites without affecting axons during Drosophila metamorphosis. ddaCs distribute the minus ends of microtubules (MTs) to dendrites but the plus ends to axons. However, a requirement of MT minus-end-binding proteins in dendrite-specific pruning remains completely unknown. Here, we identified Patronin, a minus-end-binding protein, for its crucial and dose-sensitive role in ddaC dendrite pruning. The CKK domain is important for Patronin’s function in dendrite pruning. Moreover, we show that both patronin knockdown and overexpression resulted in a drastic decrease of MT minus ends and a concomitant increase of plus-end-out MTs in ddaC dendrites, suggesting that Patronin stabilizes dendritic minus-end-out MTs. Consistently, attenuation of Klp10A MT depolymerase in patronin mutant neurons significantly restored minus-end-out MTs in dendrites and thereby rescued dendrite-pruning defects. Thus, our study demonstrates that Patronin orients minus-end-out MT arrays in dendrites to promote dendrite-specific pruning mainly through antagonizing Klp10A activity.

Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved (see decision letter).

https://doi.org/10.7554/eLife.39964.001

Introduction

In the developing nervous systems, neurons often undergo remodeling events, such as apoptosis, pruning and regrowth, which are pivotal for the refinement of mature neural circuits in both vertebrates and invertebrates (Luo and O'Leary, 2005; Riccomagno and Kolodkin, 2015). Pruning is a developmental process referred to as selective removal of unwanted neurites, for example axons, dendrites, or synapses, without causing neuronal death. After pruning, neurons often continue to extend their axons or dendrites to form the mature and functional connections. In vertebrates, various cortical, hippocampal and motor neurons prune their excessive neurites and re-wire into mature circuits. Defects in neuronal pruning result in larger dendritic spine density in layer V pyramidal neurons of autism spectrum disorder (ASD) patients (Tang et al., 2014). In invertebrates, such as Drosophila, the nervous systems undergo large-scale remodeling during metamorphosis, a transition from larval to adult stage (Yu and Schuldiner, 2014; Kanamori et al., 2015). In the central nervous system (CNS), mushroom body (MB) γ neurons prune their dorsal and medial axon branches as well as entire dendrites, and subsequently re-extend the medial branches to be part of the adult-specific circuits (Lee et al., 1999). In the peripheral nervous system (PNS), some dendritic arborization (da) neurons, including class I da (ddaD/ddaE) and class IV da (C4da or ddaC) neurons, prune away all their larval dendrites but leave their axons intact (Williams and Truman, 2005; Kuo et al., 2005), whereas class III da neurons (ddaA/ddaF) are eliminated via apoptosis during the first day of metamorphosis (Williams and Truman, 2005). Pruning morphologically resembles axonal or dendritic degeneration following neurological diseases or injury. Thus, understanding the mechanisms of developmental pruning might provide some important insights into neurodegeneration in pathological conditions.

Drosophila C4da or ddaC neurons have been recognized as an appealing model system to unravel the mechanisms underlying dendrite-specific pruning during early metamorphosis. Upon induction of the steroid hormone 20-hydroxyecdysone (ecdysone) at late larval stage, ddaC dendrites are initially severed at their proximal regions as early as 4 hr after puparium formation (APF), followed by rapid fragmentation and debris clearance (Figure 1A) (Williams and Truman, 2005). Ecdysone binds to a heterodimeric receptor complex, which consists of the Ecdysone receptor and its co-receptor Ultraspiracle, to induce several downstream effectors, including a key transcription factor Sox14 (Kirilly et al., 2009; Kirilly et al., 2011), a cytoskeletal regulator Mical (Kirilly et al., 2009), Headcase (Loncle and Williams, 2012), a Cullin1 E3 ligase complex (Wong et al., 2013), and calcium signaling (Kanamori et al., 2013). Cytoskeletal disassembly, especially microtubule (MT) disassembly, is a key step of pruning occurring before the detachment of neurites in both MB γ and ddaC neurons (Watts et al., 2003; Williams and Truman, 2005). In ddaC neurons, MTs in the proximal dendrites break down prior to the dendritic membrane fission. Katanin p60-like 1 (Kat-60L1), a putative MT severing factor, is required for dendrite pruning of ddaC neurons (Lee et al., 2009), whereas other MT severing factors, including Katanin 60 (Kat-60), Spastin and Fidgetin, appear to be dispensable (Lee et al., 2009; Stone et al., 2014; Tao et al., 2016). In contrast, mammalian Spastin mediates branch-specific disassembly of MTs and facilitates synapse elimination in the developing mouse neuromuscular junctions (Brill et al., 2016). A recent study indicated that Par-1, whose mammalian ortholog is a Tau kinase, is required for increased MT dynamics and dendrite pruning in ddaC neurons. Par-1 promotes MT breakdown mainly via Tau inhibition (Herzmann et al., 2017).

Figure 1 with 1 supplement see all
Patronin is required for dendrite pruning in ddaC neurons.

(A) A schematic representation of dendrite pruning in ddaC neurons. (B–F) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. While the wild-type neurons pruned all the dendrites (B), ddaC neurons overexpressing patronin RNAi #1 (C), patroninc9-c5 (D) or patronink07433 (F) MARCM ddaC clones exhibited simple arbors at WP stage and dendrite pruning defects at 16 hr APF. Low-level expression of mCherry-Patronin under the control of the UASp promoter fully restored the elaborate arbors at WP and rescued the pruning defects at 16 hr APF in patroninc9-c5 MARCM ddaC clones (E). Red arrowheads point to the ddaC somas. (G) Quantification of total length of unpruned ddaC dendrites at 16 hr APF. (H) Quantification of severing defects at 16 hr APF. Scale bar in (B) represents 50 μm. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. ***p<0.001 as assessed by two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.002

MTs are polarized cytoskeletal structures that are assembled from heterodimers of α- and β-tubulin, and contain fast-growing plus ends and slow-growing minus ends (Howard and Hyman, 2003; Akhmanova and Steinmetz, 2015). Fast-growing plus ends, which are associated by plus-end-tracking proteins (+TIPs), are highly dynamic, whereas slow-growing minus ends are more stable with the decoration of minus-end-binding proteins (Akhmanova and Steinmetz, 2015; Howard and Hyman, 2003). In differentiated mammalian neurons, axons and dendrites differ in MT orientation: axonal MTs are mainly aligned in a plus-end-out pattern, whereas MTs are mixed at the proximal dendritic region with both minus-end-out and plus-end-out arrays (Akhmanova and Steinmetz, 2015). In Drosophila and C. elegans neurons, axons, like mammalian ones, contain nearly uniform plus-end-out MTs, however, MTs are oriented almost exclusively minus-end-out in major dendrites but mainly plus-end-out in shorter terminal dendrites (Stone et al., 2008; Rolls et al., 2007; Ori-McKenney et al., 2012; Goodwin et al., 2012; Yalgin et al., 2015). Growing studies have begun to unravel how the minus-end-out MTs are organized and maintained in the dendrites. In C. elegans, kinesin-1 was proposed to regulate predominant minus-end-out orientation of dendritic MTs through gliding plus-end-out MTs out of the dendrites (Yan et al., 2013). In Drosophila, minus-end-out orientation of dendritic MTs depends on +TIPs, plus-end-directed motor protein kinesin-1/2 as well as MT regulators γ-Tubulin/Centrosomin (Mattie et al., 2010; Ori-McKenney et al., 2012; Nguyen et al., 2014; Herzmann et al., 2018; Yalgin et al., 2015). However, whether and how minus-end-binding proteins regulate MT orientation in the dendrites remain elusive.

The Patronin family of proteins have been identified as conserved MT minus-end-binding proteins, which contain Patronin in Drosophila, calmodulin-regulated spectrin-associated protein 1/2/3 (CAMSAP1/2/3) in mammals, and PTRN-1 in worms (Baines et al., 2009). Patronin/CAMSAP/PTRN-1 contains a calponin homology (CH) domain at its amino-terminus, three predicted coiled-coil (CC) domains at its central region, and a CAMSAP/KIAA1078/KIA1543 (CKK) domain at its carboxyl-terminus (Figure 3—figure supplement 1A) (Baines et al., 2009). patronin (also known as ssp4) was first identified to regulate the length of mitotic spindle in Drosophila S2 cells (Goshima et al., 2007). The Patronin proteins recognize and stabilize free minus ends by protecting them from kinesin-13-mediated depolymerization (Goodwin and Vale, 2010). Mammalian CAMSAP3/Nezha anchors MT minus ends to adherence junctions and controls the apical-basal MT orientation in cultured epithelial cells (Meng et al., 2008). Both Patronin and CAMSAP3 can polarize MT arrays via actin-crosslinking proteins (Ning et al., 2016; Nashchekin et al., 2016; Khanal et al., 2016). In hippocampal neurons, CAMSAP2 localizes to the MT minus ends and plays an important role in axon specification and dendrite formation (Yau et al., 2014). In C. elegans neurons, PTRN-1 is required for synaptic vesicle localization, neurite morphology and axon regeneration (Chuang et al., 2014; Marcette et al., 2014; Richardson et al., 2014). However, to our knowledge, it is completely unknown about a potential role of Patronin in regulating neuronal development including pruning in Drosophila. Moreover, whether the minus-end-out MT orientation is critical for dendrite pruning remains poorly understood.

Here, we report that MT minus-end-binding protein Patronin plays a crucial role in dendrite pruning in class IV ddaC neurons. Overexpression of Patronin resembles patronin mutants in terms of dendrite pruning phenotypes. The CKK domain is important for Patronin’s function in governing dendrite pruning. Moreover, both patronin knockdown and overexpression resulted in a drastic decrease of the MT minus-end marker Nod-β-gal and a significant increase of anterograde EB1-GFP comets in proximal ddaC dendrites, indicating that Patronin may stabilize uniform minus-end-out MTs in the dendrites. Interestingly, attenuation of Klp10A, a kinesin-13 MT depolymerase, in patronin ddaC neurons significantly restored uniform minus-end-out MTs in dendrites and thereby rescued dendrite pruning defects. Thus, our study demonstrates, for the first time, that the MT minus-end-binding protein Patronin orients uniform minus-end-out MT arrays in dendrites to govern dendrite-specific pruning mainly through antagonizing Klp10A activity in Drosophila sensory neurons.

Results

Patronin is required for Dendrite pruning and arborization in ddaC neurons

Severing, the first step of dendrite-specific pruning, takes place in the proximal dendrites of C4da or ddaC neurons where MT minus ends predominantly localize (Rolls et al., 2007; Zheng et al., 2008; Satoh et al., 2008). To understand a potential role of MT minus-end-binding proteins in dendrite pruning, we conducted a candidate-based RNA interference (RNAi) screen. Patronin was isolated for its requirement in dendrite pruning. Knockdown of Patronin, via three independent RNAi (#1, v108927; #2, BL36659 and #3, NIG18462Ra-1), resulted in consistent dendrite pruning defects in ddaC neurons at 16 hr APF (Figure 1C, G and H, Figure 1—figure supplement 1A). In contrast, all the larval dendrites of control neurons were pruned away at the same time point (n = 16, Figure 1B, G and H). To further verify this requirement, we generated homozygous MARCM clones of patroninc9-c5, a patronin null mutant (Nashchekin et al., 2016). Consistently, mutant ddaC neurons derived from patronin RNAi expression (#1, n = 16, Figure 1C and H) or patroninc9-c5 mutant clones (n = 13, Figure 1D and H) exhibited severe dendrite pruning defects with full penetrance at 16 hr APF. On average, 606 µm and 701 µm of larval dendrites in patronin RNAi and patroninc9-c5 ddaC neurons remained attached to their mutant somas, respectively (Figure 1G). The dendrites of patronin RNAi ddaC neurons were largely removed by 32 hr APF (n = 32; Figure 1—figure supplement 1B), presumably due to extensive apoptosis/migration of the dorsal abdominal epidermis, on which neurons arborize their larval dendrites (Williams and Truman, 2005). Moreover, the dendrite severing defects in patroninc9-c5 mutant neurons were completely rescued by low-level expression of mCherry-tagged Patronin protein under the control of the germline UASp promoter (UASp-mCherry-Patronin) (n = 11, Figure 1E, G and H, Figure 2—figure supplement 1A). As a control, the expression of UASp-mCherry-Patronin did not disturb dendrite pruning in ddaC neurons (n = 16; Figure 3—figure supplement 1B). We also made use of a P-element insertion line patronink07433 and observed similar dendrite severing defects in 64% of mutant neurons (n = 14, Figure 1F and H). An average of 446 µm dendrites remained in the vicinity of patronink07433 ddaC clones (Figure 1G). Thus, Patronin plays a crucial role in dendrite pruning in ddaC neurons. To our knowledge, this is the first observation indicating that the Patronin family of proteins regulate neuronal remodeling during animal development.

The dendritic complexity of patroninc9-c5 ddaC clones was greatly compromised at the wandering 3rd instar larval stage (wL3) (Figure 1—figure supplement 1C). The number of their mutant dendritic termini was significantly decreased (n = 5), compared to that of the wild-type controls (n = 5). However, the number of their primary and secondary dendrites in patroninc9-c5 ddaC clones (53.4 ± 4.7) was slightly fewer but not statistically different, compared to that of the control neurons (61.8 ± 2.0). Likewise, at the white prepupal (WP) stage, simplified dendrite arbors formed in patronin RNAi (#1, n = 8, Figure 1C) or patroninc9-c5 ddaC clones (n = 6, Figure 1D), in contrast to more elaborate dendrite arbors observed in those control neurons (n = 19, Figure 1B). Mammalian CAMSAP2 was reported to regulate dendrite morphology of cultured hippocampal neurons in vitro (Yau et al., 2014). Thus, our in vivo results highlight that Patronin plays a conserved role in regulating dendrite arborization in Drosophila.

Moreover, wild-type ddaD/ddaE neurons, like ddaC neurons, pruned all their larval dendrites at 20 hr APF (n = 14, Figure 1—figure supplement 1D). patronin RNAi-expressing ddaD/ddaE neurons failed to eliminate their dendrites (n = 20, Figure 1—figure supplement 1D), similar to patronin ddaC neurons. However, patronin RNAi-expressing ddaF neurons were still apoptotic (n = 14), similar to wild-type neurons (n = 15, Figure 1—figure supplement 1E), suggesting that Patronin is dispensable for neuronal apoptosis during early metamorphosis.

Taken together, Patronin plays a novel and crucial role in dendrite pruning of Drosophila sensory neurons.

Overexpression of patronin causes dendrite pruning defects in ddaC neurons

Given that Patronin acts as a MT minus-end-binding protein to protect MT minus ends and regulate MT behavior, we hypothesized that elevated levels of Patronin might disturb MT dynamics and thereby impair dendrite pruning. We next attempted to investigate the gain-of-function effects of patronin. We generated an untagged patronin transgene under the control of UASt promoter (UAS-patronin) and also utilized another previously reported UASt transgene (UAS-GFP-patronin) (Derivery et al., 2015), to induce high-level expression in ddaC neurons (Figure 2—figure supplement 1A). Importantly, overexpressing either of patronin transgenes, via Gal44-77 (weak driver) or ppk-Gal4 (stronger driver), resulted in consistent dendrite pruning defects at 16 hr APF (Figure 2B and C), which phenocopied patronin mutants (Figure 1). Overexpression of Patronin, via Gal44-77, led to dendrite severing defects in 70% of ddaC neurons with an average of 511 µm dendrites (n = 34; Figure 2B, F and G). When GFP-Patronin was overexpressed at a higher level by two copies of ppk-Gal4, vast majority of ddaC neurons exhibited stronger dendrite severing defects with the persistence of 884 µm dendrites (87%, n = 15; Figure 2C, F and G), suggesting that overexpressed Patronin behaves as a dominant negative in dendrite pruning.

Figure 2 with 1 supplement see all
Overexpression of Patronin causes dendrite pruning defects in ddaC neurons.

(A–E) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4, Gal44-77 or ppk-CD4-tdGFP at WP and 16 hr APF. While ddaC neurons overexpressing the UAS control construct pruned all the dendrites (A), ddaC neurons overexpressing Patronin (B) driven by Gal44-77 or GFP-Patronin (C) driven by two copies of ppk-Gal4 at a higher level exhibited simple arbors at WP stage and consistent dendrite pruning defects at 16 hr APF. ddaC neurons, in which GFP-Patronin was expressed by GeneSwitch-Gal4-2295 in RU486-induced conditions (E), exhibited normal arbors at WP stage but severe dendrite pruning defects at 16 hr APF, compared to those in non-induced conditions (D). Red arrowheads point to the ddaC somas. (F) Quantification of total length of unpruned ddaC dendrites at 16 hr APF. (G) Quantification of severing defects at 16 hr APF. Scale bar in (A) represents 50 μm. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. ***p<0.001 as assessed by two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.006

We also observed that the dendritic complexity was significantly reduced in GFP-Patronin-overexpressing ddaC neurons at wL3 stage (n = 5, Figure 2—figure supplement 1B), compared to that in the control neurons (n = 5, Figure 2—figure supplement 1B). Likewise, simplified dendrite arbors formed in ddaC neurons overexpressing Patronin (n = 16; Figure 2B) or GFP-Patronin (n = 16, Figure 2C) at WP stage. To rule out the possibility that the dendrite pruning defects are secondary to the initial morphology defects, we induced the expression of GFP-Patronin at the early 3rd instar larval stage (eL3) using the Gene-Switch system. Late-larval induction of GFP-Patronin expression did not affect initial dendrite arborization at WP stage (n = 8; Figure 2E). Notably, dendrite severing defects were observed at 16 hr APF in 75% of GFP-Patronin-overexpressing ddaC neurons derived from RU486-fed animals (n = 16; Figure 2E, F and G), in contrast to no severing defect in non-fed animals (n = 16; Figure 2D, F and G).

Thus, Patronin overexpression phenocopies patronin mutants in dendrite pruning of sensory neurons.

The CKK domain is important for patronin to govern dendrite pruning

Patronin contains a CH domain at its amino-terminus, three CC domains at its central region, and a signature CKK domain at its carboxyl-terminus (Figure 3—figure supplement 1A) (Baines et al., 2009). To dissect functional domains of Patronin that are required for its function in dendrite pruning, we generated a series of patronin transgenes (Figure 3—figure supplement 1A) under the germline UASp promoter, which drives low-level expression in ddaC neurons (Figure 2—figure supplement 1A). As controls, we confirmed that low-level expression of these UASp transgenes did not result in any dendrite pruning defect in ddaC neurons at 16 hr APF (Figure 3—figure supplement 1B). We next examined their abilities to rescue dendrite pruning phenotypes by expressing these Patronin truncates in patroninc9-c5 mutant MARCM ddaC clones. Notably, the expression of CH-deleted Patronin variant (PatroninΔCH) was able to almost fully rescue the pruning defects in patroninc9-c5 ddaC neurons at 16 hr APF (n = 11; Figure 3B, E and F), similar to the full-length protein (Figure 1E, G and H). However, CKK-deleted Patronin variant (PatroninΔCKK) failed to rescue the dendrite pruning defects in patroninc9-c5 ddaC neurons (n = 10; Figure 3C, E and F). These data suggest that the CKK domain, rather than the CH domain, is required for Patronin’s function in dendrite pruning. To further examine whether the CKK domain alone is able to substitute for Patronin’s function in dendrite pruning, we expressed the CKK domain in patroninc9-c5 ddaC MARCM clones. Interestingly, the expression of the CKK domain significantly rescued patroninc9-c5-associated dendrite pruning defects (n = 16; Figure 3D, E and F). Thus, the CKK domain is important for Patronin’s function during dendrite pruning.

Figure 3 with 1 supplement see all
The CKK domain is important for Patronin to govern dendrite pruning.

(A–D, G–J) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 or Gal44-77 at WP and 16 hr APF. Low-level expressions of PatroninΔCH (B) and PatroninCKK (D), but not PatroninΔCKK (C), strongly rescued the dendrite arborization defects at WP stage and the pruning defects at 16 hr APF in patroninc9-c5 (A) MARCM ddaC clones. (G–J) ddaC neurons overexpressing Venus-Patronin (G) or Venus-PatroninΔCH (H) by Gal44-77 exhibited simple arbors at WP stage and the dendrite pruning defects at 16 hr APF. However, overexpression of Venus-PatroninΔCKK (I) or Venus-PatroninCKK (J) had negligible effect on dendrite arborization and pruning. (E, K) Quantification of total length of unpruned ddaC dendrites at 16 hr APF. (F, L) Quantification of severing defects at 16 hr APF. Scale bar in (A) represents 50 μm. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. N.S., not significant; ***p<0.001 as assessed by one-way ANOVA test.

https://doi.org/10.7554/eLife.39964.010

To further investigate gain-of-function effects of the Patronin variants, we generated a second set of patronin transgenes under the UASt promoter for high-level expression in ddaC neurons. Overexpression of PatroninΔCH variant led to prominent dendrite severing defects in ddaC neurons at 16 hr APF (59%, n = 27, Figure 3H, K and L), similar to the full-length Patronin (69%, n = 16; Figure 3G, K and L). In contrast, the expression of PatroninΔCKK variant did not affect normal dendrite pruning at 16 hr APF (n = 16; Figure 3I, K and L), suggesting that the CKK domain of Patronin is important for its gain-of-function effect on dendrite pruning. Interestingly, overexpression of CKK domain (n = 16; Figure 3J, K and L), CH domain (n = 25; Figure 3—figure supplement 1A) or CC1-3 domains (n = 17; Figure 3—figure supplement 1A) had no effect on dendrite pruning of ddaC neurons, suggesting that endogenous Patronin protein may function properly to regulate dendrite pruning even with ectopic expression of these individual domains.

Collectively, both rescue and overexpression results strengthen the conclusion that the CKK domain is indispensable for Patronin to exert its functions in dendrite pruning.

Patronin is required for proper distribution of dendritic and axonal MT markers

We next attempted to understand the mechanisms whereby Patronin regulates dendrite-specific pruning in ddaC sensory neurons. Since Patronin binds and protects MT minus ends in Drosophila S2 cells (Goodwin and Vale, 2010), we hypothesized that Patronin may stabilize MT minus ends and govern MT minus-end distribution in ddaC sensory neurons. The chimeric protein Nod-β-gal, in which the Nod motor domain is fused to the coiled-coil domain region of the conventional kinesin1 (Kin1) and the β-galactosidase (β-gal) tag, localizes to the MT minus ends and is commonly used as a marker of MT minus ends in Drosophila (Clark et al., 1997). In Drosophila neurons including da neurons, this Nod-β-gal chimera localizes specifically in dendrites but not in axons (Rolls et al., 2007; Zheng et al., 2008; Satoh et al., 2008). In wild-type ddaC neurons, Nod-β-gal was predominantly enriched in dendrites but not in axons (n = 6, Figure 4A, D and E). Remarkably, in all patronin RNAi ddaC neurons, Nod-β-gal was no longer enriched in the dendritic arbors and instead highly concentrated in the soma (100%, n = 25, Figure 4B and D). Likewise, all patroninc9-c5 ddaC clones also exhibited robust accumulation of Nod-β-gal in the soma concomitantly with its reduced levels in the distal dendrites (100%, n = 11; Figure 4—figure supplement 1A, Figure 4D and E,). To quantify the alterations of dendritic Nod-β-gal distribution, we measured its intensity in the dendrites which were 40 µm away from the soma. In the dendrites of patronin RNAi or patroninc9-c5 neurons, Nod-β-gal levels were drastically reduced to 12% and 11% of those in the control neurons, respectively (Figure 4E). Reduced levels of dendritic Nod-β-gal signals may reflect a decrease in the number of minus-end-out MTs in the dendrites of patronin mutant neurons. Strikingly, overexpression of GFP-Patronin also resulted in robust accumulation of Nod-β-gal signals in the soma and reduced dendritic distribution in 67% of ddaC neurons (n = 18, Figure 4C, D and E), resembling the patronin mutant phenotypes. No mis-localization of Nod-β-gal signals was observed in the axons of patronin mutant or GFP-Patronin-overexpressing ddaC neurons (Figure 4B and C). These results imply that Patronin might stabilize and distribute MT minus ends into the dendrites of ddaC neurons. Collectively, Patronin regulates dendritic localization of the MT minus-end marker Nod-β-gal in a dose-sensitive manner.

Figure 4 with 1 supplement see all
Patronin is required for proper distribution of dendritic and axonal MT markers.

(A–C, F–H) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal or Kin-β-gal and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and dendrites by curly brackets. Nod-β-gal levels were reduced in the dendrites but enriched in the somas in patronin RNAi #1 (B) or GFP-Patronin-overexpressing (C) ddaC neurons, compared to the control neurons (A). (D) Quantification of the percentage of neurons with defective Nod-β-gal distribution. (E) Quantification of normalized Nod-β-gal intensity in the dendrites. Moreover, Kin-β-gal mis-localized to the dendrites in both patronin RNAi #1 (G) or GFP-Patronin-overexpressing (H) ddaC neurons, compared to the control neurons (F). Arrowheads point to ectopic Kin-β-gal aggregates in the dendrites. (I) Quantification of the percentage of neurons with dendritic Kin-β-gal distribution. Scale bar in (A) represents 20 μm. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. ***p<0.001 as assessed by one-way ANOVA test.

https://doi.org/10.7554/eLife.39964.014

We next examined the distribution of the axon-specific marker Kin-β-gal, which consists of the Kin1 motor and coiled-coil domains fused with the β-gal tag and is often used as a marker of MT plus ends (Clark et al., 1997). Kin-β-gal was localized exclusively to the axons, but not to the dendrites in wild-type ddaC neurons (n = 9; Figure 4F and I) (Zheng et al., 2008). Interestingly, Kin-β-gal often mis-localized to the dendrites as punctate structures in patronin RNAi (89%, n = 19, Figure 4G and I) or GFP-Patronin-overexpressing ddaC neurons (45%, n = 20, Figure 4H and I), implying an increase of plus-end-out MTs in the dendrites. Given that Patronin localizes at MT minus ends in S2 cells (Goodwin and Vale, 2010), we next examined its subcellular localization in ddaC neurons using the anti-Patronin antibody. Patronin localized uniformly to dendrites, axons and soma at larval and WP stages (n = 14 and 14, respectively, Figure 4—figure supplement 1B).

Taken together, Patronin is crucial for proper distribution of dendrite or axon-specific MT markers in ddaC neurons.

Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons

Given that Patronin regulates proper distribution of dendritic or axonal MT markers in ddaC neurons, we next assessed whether dendritic MT minus-end-out orientation depends on Patronin. To this end, we took advantage of the MT plus-end marker EB1-GFP. EB1 is a core plus-end-binding protein that associates with the growing plus ends of MTs during growth and dissociates during shrinkage (Vaughan, 2005). GFP-fused EB1 was reported to track the growing plus ends of MTs in neurons (Stepanova et al., 2003; Rolls et al., 2007). To assess MT orientation in ddaC dendrites, we expressed low-level EB1-GFP via the weak driver Gal44-77 to examine the direction of EB1-GFP comet movement in major dendrites at 96 hr after egg laying (AEL) (Rolls et al., 2007). We first knocked down patronin by using two independent RNAi lines and compared them with the control RNAi line. In the proximal dendrites, 99% of EB1-GFP comets moved predominantly toward the soma (retrograde) in the control RNAi-expressing ddaC neurons (n = 30, Figure 5A and C). These retrograde EB1-GFP events indicate a predominant minus-end-out orientation of dendritic MTs in ddaC neurons (Stone et al., 2008; Ori-McKenney et al., 2012). In contrast, in the control ddaC axons, EB1-GFP comets primarily moved away from the soma (anterograde, 96%, n = 12, Figure 5—figure supplement 1C), suggesting a plus-end-out MT orientation in axons. On average, the percentage of anterograde EB1-GFP comets was increased to 48% and 19% in these patronin RNAi ddaC neurons, respectively (#1, n = 29 neurons, Figure 5B–C; #2, n = 12, Figure 5—figure supplement 1A), compared to that in the control neurons (n = 30; Figure 5C). Notably, the direction of EB1-GFP comets was drastically reversed in 33% of dendrite branches of patronin RNAi #1 ddaC neurons, respectively (n = 43 branches; Figure 5M). In these mutant dendrite branches, over 80% of EB1-GFP comets moved away from the soma, suggesting a predominant plus-end-out MT pattern (Figure 5M). We next quantified the number of EB1-GFP events, which reflects overall MT density and levels of MT nucleation in both cultured cells (Piehl et al., 2004) and Drosophila neurons (Chen et al., 2014). The average numbers of EB1-GFP comets in patronin RNAi #1 expressing ddaC dendrites were not statistically different from that in the control (Figure 5D). patronin RNAi knockdown (#1) also led to normal EB1-GFP track length (Figure 5E) but slightly higher growth speed (Figure 5F).

Figure 5 with 1 supplement see all
Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons.

(A–B, G–H) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In control RNAi (A) or UAS control (G) ddaC neurons, dendritic EB1-GFP comets predominantly moved retrogradely towards the somas. However, in patronin RNAi #1 (B) or Patronin-overexpressing (H) ddaC neurons, dendritic EB1-GFP comets moved bidirectionally (anterogradely and retrogradely). Horizontal arrow indicates the direction towards the soma. Scale bar in (A) represents 10 μm, and each movie was taken for 3 min. EB1-GFP was expressed by Gal44-77. (C, I) Quantification of percentage of dendritic EB1-GFP comets moving away from soma (anterogradely). (D, J) Quantification of average numbers of EB1-GFP comets along 30 µm of dendrites within 3 min. (E, K) Quantification of average track length of EB1-GFP comets along 30 µm of dendrites within 3 min. (F, L) Quantification of average speed of EB1-GFP comets along 30 µm of dendrites within 3 min. (M) Quantification of the percentage of dendrite branches showing different levels of anterograde EB1-GFP comets. The number of dendrite branches (n) examined in each group is shown on the bars. Error bars represent SEM. N.S., not significant; *p<0.05; **p<0.01; ***p<0.001 as assessed by two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.017

The intensity of overall MT levels was significantly reduced in the dendrites of patronin RNAi ddaC neurons (#1, n = 16), compared with the controls (n = 16; Figure 5—figure supplement 1B), as assessed by the antibody 22C10 against Futsch. Moreover, we have also examined EB1-GFP movement in the axons of patronin RNAi ddaC neurons. Compared with 4% of retrograde EB1-GFP comets (predominantly plus-end-out) in the axons of wild-type neurons (n = 12), patronin knockdown led to 41% of retrograde EB1-GFP comets in the axons (n = 17; Figure 5—figure supplement 1C), indicative of a severe defect in the plus-end-out orientation of their axonal MTs upon patronin knockdown. Thus, patronin plays an important role in governing both dendritic and axonal MT polarity in ddaC neurons.

Similar to patronin RNAi neurons, Patronin overexpression also led to a significant increase in anterograde EB1-GFP comets in the ddaC dendrites (10%, n = 15; Figure 5H and I), compared to the control neurons (0%, n = 12; Figure 5G and I). The number of EB1-GFP comets in Patronin-overexpressing ddaC dendrites remained similar to that of the controls (Figure 5I). Patronin overexpression also led to an increase in the number of EB1-GFP comets (Figure 5J) but did not affect normal EB1-GFP track length (Figure 5K) and growth speed (Figure 5L).

Thus, Patronin plays an important role in orienting minus-end-out MTs in ddaC dendrites.

Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotype in MT orientation in ddaC dendrites

A previous study revealed that Patronin stabilizes MT minus ends and protects them against kinesin-13-mediated MT depolymerization in interphase and mitotic S2 cells (Goodwin and Vale, 2010). We next investigated whether mis-orientation of minus-end-out MTs in patronin dendrites is caused by excessive kinesin-13-dependent depolymerization activity. To this end, we double knocked down the Drosophila kinesin-13 Klp10A with Patronin and subsequently examined EB1-GFP movements in the dendrites. In 26% of dendrite branches of ddaC neurons expressing both patronin RNAi (#1) and control RNAi constructs (patronin +control RNAi), EB1-GFP comets predominantly moved away from the soma (n = 23 branches; Figure 6—figure supplement 1A), indicating a nearly uniform plus-end-out MT pattern. On average, 56% of EB1-GFP comets moved anterograde in these dendrites of patronin +control RNAi neurons (n = 12, Figure 6C), similar to patronin RNAi-expressing ddaC (Figure 5C). Double knockdown of patronin and klp10A (patronin +klp10A) completely restored the retrograde movement of EB1-GFP comets (n = 11; Figure 6B and C). Moreover, all dendrite branches exhibited a uniform minus-end-out MT pattern in patronin +klp10A RNAi neurons (n = 27 branches; Figure 6—figure supplement 1A), identical to the control neurons (Figure 5M). Moreover, further knockdown of klp10A resulted in no alteration in the number, track length and movement speed of EB1-GFP comets in patronin RNAi dendrites (Figure 6D–F). As a control, single RNAi knockdown of klp10A did not alter the direction of EB1-GFP movements in ddaC dendrites (n = 12; Figure 6—figure supplement 1B). Thus, these results indicate that MT mis-orientation in patronin mutant dendrites is caused by excessive Klp10A activity.

Figure 6 with 1 supplement see all
Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotypes in MT orientation in ddaC dendrites.

(A, B) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In patronin RNAi #1 and control RNAi co-expressing ddaC neurons (A), dendritic EB1-GFP comets moved bidirectionally (anterogradely and retrogradely). However, knockdown of klp10A via RNAi #1 completely restored the retrograde movement of EB1-GFP comets in the patronin RNAi #1 ddaC dendrites (B). Horizontal arrow indicates the direction towards the somas. Scale bar in (A) represents 10 μm, and each movie was taken for 3 min. EB1-GFP was expressed by Gal44-77. (C) Quantification of percentage of dendritic EB1-GFP comets moving away from soma (anterogradely). (D) Quantification of average numbers of EB1-GFP comets along 30 µm of dendrites within 3 min. (E) Quantification of average track length of EB1-GFP comets along 30 µm of dendrites within 3 min. (F) Quantification of average speed of EB1-GFP comets. (G–I, L–N) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal or Kin-β-gal and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and dendrites by curly brackets. Nod-β-gal levels were reduced in the dendrites but enriched in the somas of ddaC neurons co-overexpressing patronin RNAi #1 and control RNAi (H), compared to the control ddaC neurons (G). However, knockdown of klp10A (RNAi #1) almost completely restored dendritic distribution of Nod-β-gal in patronin RNAi #1 ddaC neurons (I). (J) Quantification of the percentage of neurons with defective Nod-β-gal distribution. (K) Quantification of normalized Nod-β-gal intensity in the dendrites. Moreover, Kin-β-gal mis-localization defects in patronin RNAi #1 ddaC neurons were drastically rescued by knockdown of klp10A (RNAi #1), compared to the patronin, control RNAi neurons (M). (O) Quantification of the percentage of neurons with dendritic Kin-β-gal distribution. Scale bar in (G) represents 20 μm. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. N.S., not significant; ***p<0.001 as assessed by one-way ANOVA test or two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.021

We further explored whether aberrant distribution of MT markers, Nod-β-gal and Kin-β-gal, is attributable to excessive Klp10A activity. While Nod-β-gal was predominantly enriched in the soma and strongly reduced in the dendrites in patronin +control RNAi ddaC neurons (100%, n = 10, Figure 6H, J and K), dendritic distribution of Nod-β-gal was significantly restored in patronin +klp10A RNAi mutant ddaC neurons (n = 12, Figure 6I, J and K) to an extent similar to that in wild-type controls (n = 13, Figure 6G, J and K). As a control, single RNAi knockdown of klp10A, via two independent constructs, did not alter the Nod-β-gal distribution in ddaC dendrites (n = 17 and 17, respectively, Figure 6—figure supplement 1C). Likewise, mis-localization of the axonal marker Kin-β-gal to the dendrites in patronin RNAi neurons was rescued by further knockdown of klp10A (22%, n = 18, Figure 6N and O), compared to the control RNAi construct (85%, n = 20, Figure 6M and O). Thus, attenuation of Klp10A activity restores normal MT orientation in the dendrites of patronin mutant neurons.

These results suggest that Patronin acts against Klp10A-dependent depolymerization to maintain uniform minus-end-out MT orientation in the dendrites of ddaC neurons.

Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites

Given that attenuation of Klp10A is sufficient to restore uniform minus-end-out orientation of dendritic MTs in patronin mutant neurons, we further assessed whether such a restoration of MT orientation also results in a rescue of dendrite pruning defects in mutant neurons. 84% of patronin +control RNAi co-overexpressing ddaC neurons displayed dendrite severing defects and retained an average of 593 µm larval dendrite at 16 hr APF (n = 32, Figure 7A, D and E). We then co-expressed the patronin RNAi #1 line with either of two klp10A RNAi lines, v41534 (#1) and BL33963 (#2). Importantly, the pruning defects associated with patronin RNAi ddaC neurons were almost completely rescued by further knockdown of klp10A (Figure 7B and C). On average, only 73 µm and 71 µm of dendrites were present at 16 hr APF in patronin +klp10A #1 or #2 double RNAi ddaC neurons (n = 20, Figure 7B and D and n = 32, Figure 7C and D, respectively). Moreover, the penetrance of severing defects in patronin +klp10A #1 or patronin +klp10A #2 double RNAi ddaC neurons (Figure 7E) was drastically reduced to 15% and 19%, respectively, in contrast to 84% in the patronin +control RNAi controls (Figure 7E). As controls, knockdown of either klp10A RNAi #1 or #2 did not cause any dendrite pruning defects in 16-h-APF ddaC neurons (Figure 7—figure supplement 1A). Together with the restoration of dendritic MT orientation in patronin mutant neurons by klp10A knockdown, these results suggest that the dendrite pruning defects in patronin mutant ddaC neurons is mainly attributable to mis-orientation of MT minus ends in the dendrites.

Figure 7 with 1 supplement see all
Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites.

(A–C, F–H) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. (A–C) ddaC neurons co-expressing patronin RNAi #1 and control RNAi exhibited strong pruning defects at 16 hr APF (A). The pruning defects in patronin RNAi #1 ddaC neurons were significantly suppressed by knockdown of klp10A via either RNAi #1 (B) or RNAi #2 (C). (F–H) ddaC neurons co-expressing Patronin and the control RNAi construct (F) exhibited strong pruning defects at 16 hr APF. These pruning defects were significantly suppressed by knockdown of klp10A via either RNAi #1 (G) or RNAi #2 (H). (D, I) Quantification of total length of unpruned ddaC dendrites at 16 hr APF. (E, J) Quantification of the severing defects at 16 hr APF. Scale bar in (A) represents 50 μm. (K–L) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In ddaC neurons co-overexpressing Patronin and the control RNAi construct, EB1-GFP comets moved bidirectionally in the dendrites (K). However, klp10A knockdown via RNAi #2 (L) significantly restored the retrograde movement of EB1-GFP comets in Patronin-overexpressing ddaC dendrites. Horizontal arrow indicates the direction towards the soma. Scale bar in (K) represents 10 μm, and each movie was taken for 3 min. (M) Quantification of percentage of dendritic EB1-GFP comets moving away from soma (anterogradely). (N) Quantification of average numbers of EB1-GFP comets along 30 µm of dendrites within 3 min. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. N.S., not significant; **p<0.01; ***p<0.001 as assessed by one-way ANOVA test or two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.025

Previous studies reported a direct interaction between mammalian Patronin (CAMSAPs) and katanin (Jiang et al., 2014; Jiang et al., 2018). We therefore examined the potential genetic interaction between patronin and kat-60 in dendrite pruning. Knockdown of kat-60, via two distinct RNAi lines, did not affect normal dendrite pruning in ddaC neurons (Figure 7—figure supplement 1B). Knockdown of kat-60 did not enhance or suppress the pruning defects in patronin RNAi ddaC neurons (Figure 7—figure supplement 1C). Likewise, although Kat-60L1 and Tau were reported to be involved in dendrite pruning (Lee et al., 2009; Herzmann et al., 2017), double knockdown of kat-60L1/tau and patronin exhibited no significant enhancement or suppression in the dendrite pruning defects (Figure 7—figure supplement 1D–E). These data suggest no genetic interaction between patronin and kat-60/kat-60L1/tau.

To address whether Patronin overexpression, like patronin knockdown, also affects dendrite pruning through excessive Klp10A activity, we knocked down klp10A function in Patronin-overexpressing ddaC neurons. Knockdown of klp10A, via two independent RNAi lines, significantly suppressed the dendrite pruning defects in Patronin-overexpressing ddaC neurons (n = 17 and 24; Figure 7G–J). Moreover, knockdown of klp10A also significantly suppressed the MT orientation defect in the dendrites of Patronin-overexpressing ddaC neurons (n = 14; Figure 7L–N), compared to the control neurons (n = 13, Figure 7K and M–N). Therefore, these data further confirm that Patronin overexpression, like patronin knockdown, affects dendrite pruning and dendritic MT orientation at least partially by upregulation of Klp10A function.

Taken together, Patronin regulates dendritic minus-end-out MT orientation and dendrite pruning at least partially by antagonizing Klp10A function.

Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation

To further confirm whether the dendrite pruning defects in patronin mutant neurons is due to increased Klp10A activity, we overexpressed Klp10A in ddaC neurons via two independent transgenes, UAS-klp10A and UAS-GFP-klp10A. Indeed, Klp10A overexpression alone is sufficient to inhibit dendrite pruning in ddaC neurons (Figure 8B–E), phenocopying patronin mutants (Figure 1). Overexpression of either GFP-Klp10A (n = 16, Figure 8B, D and E) or Klp10A (n = 16, Figure 8C, D and E) resulted in consistent severing pruning defects in 88% and 94% of neurons with the persistence of 662 µm and 722 µm dendrites present at 16 hr APF, respectively. Similar to patronin mutants, overexpression of GFP-Klp10A (n = 8; Figure 8B) or Klp10A (n = 8; Figure 8C) also resulted in simplified dendrite arbors at WP stage, suggesting that elevated Klp10A impairs initial dendrite arborization during growth. Moreover, overexpression of Klp10A also resulted in reduced levels of Nod-β-gal in the dendrites (n = 17; Figure 8—figure supplement 1A) and mis-localization of Kin-β-gal to the dendrites (n = 20; Figure 8—figure supplement 1B). Consistently, Klp10A overexpression also significantly increased the percentage of anterograde EB1-GFP movement in the dendrites (11%, n = 16; Figure 8G–H), suggesting a dendritic MT orientation defect. Klp10A overexpression did not caused a signification reduction in the number of EB1-GFP comets in the dendrites (Figure 8I), resembling the patronin RNAi knockdown. In addition, neither track length of EB1-GFP comets nor their speed was affected upon Klp10A overexpression (Figure 8J–K). These data imply that patronin RNAi or Klp10A overexpression might result in excessive MT depolymerization primarily at the MT minus ends, rather than affect the number of EB1-GFP comets that mark MT growth at the MT plus ends. Thus, these data strongly demonstrate that Patronin promotes dendrite pruning via antagonising Klp10A function.

Figure 8 with 3 supplements see all
Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation.

(A–C) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. ddaC neurons overexpressing the UAS control construct pruned all the dendrites (A), whereas ddaC neurons overexpressing GFP-Klp10A (B) or Klp10A (C) via two copies of ppk-Gal4 exhibited simple arbors at WP stage and dendrite pruning defects at 16 hr APF. (D) Quantification of total length of unpruned ddaC dendrites at 16 hr APF. (E) Quantification of the severing defects at 16 hr APF. Scale bar in (A) represents 50 μm. (F–G) Representative kymographs of EB1-GFP comets in UAS control or Klp10A-overexpressing ddaC dendrites. In the dendrites of the control ddaC neurons (F), EB1-GFP comets moved towards the somas. However, in ddaC neurons overexpressing Klp10A (G), EB1-GFP comets moved bidirectionally in the dendrites. Horizontal arrow indicates the direction towards the soma. Scale bar in (F) represents 10 μm, and each movie was taken for 3 min. (H) Quantification of the percentage of EB1-GFP comets moving away from soma (anterogradely) in ddaC dendrites. (I) Quantification of average numbers of EB1-GFP comets along 30 µm of dendrites within 3 min. (J) Quantification of average track length of EB1-GFP comets along 30 µm of dendrites within 3 min. (K) Quantification of average speed of EB1-GFP comets. Error bars represent SEM. The number of samples (n) in each group is shown on the bars. N.S., not significant; ***p<0.001 as assessed by one-way ANOVA test or two-tailed Student’s T test.

https://doi.org/10.7554/eLife.39964.029

In addition, we examined the functional significance of the CKK domain of Patronin in regulating dendrite pruning and dendritic MT orientation. To this end, we first confirmed that the expression of the CKK domain alone significantly rescued the dendrite pruning defects in the patronin RNAi background at 16 hr APF (n = 24; Figure 8—figure supplement 2A), similar to those in patroninc9-c5 ddaC neurons (Figure 3D, E and F). Importantly, the expression of the CKK domain, rather than the CH domain, almost fully rescued Nod-β-gal distribution (n = 15; Figure 8—figure supplement 2B) as well as retrograde EB1-GFP comets in the dendrites of patronin RNAi ddaC neurons (n = 15; Figure 8—figure supplement 2C). Moreover, the expression of the CKK domain significantly suppressed the dendrite pruning defects in Klp10A-overexpressing ddaC neurons (n = 30; Figure 8—figure supplement 2D), suggesting that the CKK domain is able to antagonize Klp10A’s function during dendrite pruning. Thus, multiple lines of evidence demonstrate that the CKK domain is important for Patronin to govern minus-end-out MT orientation in dendrites as well as dendrite pruning.

In summary, Patronin orients uniform minus-end-out MT arrays in dendrites to facilitate dendrite pruning in ddaC sensory neurons during early metamorphosis.

Discussion

During early metamorphosis, C4da or ddaC neurons undergo dendrite pruning to selectively eliminate their dendrites but keep their axons intact (Williams and Truman, 2005; Kuo et al., 2005). Dendritic MT breakdown precedes membrane scission, which leads to physical severing of proximal dendrites from the soma (Williams and Truman, 2005). In ddaC neurons, dendrites are enriched with MT minus ends and organized with predominant minus-end-out MT arrays, whereas the axons differ in MT orientation and acquire a plus-end-out pattern (Rolls et al., 2007; Zheng et al., 2008; Satoh et al., 2008). However, the involvement of MT minus-end-binding proteins in governing dendritic MT orientation and thereby dendrite pruning is completely unknown. In this study, we identified a MT minus-end-binding protein Patronin for its key role in dendrite-specific pruning of ddaC neurons. Patronin/CAMSAP/PTRN-1 were previously reported to track and stabilize MT minus ends, and regulate axon specification and dendrite morphology in mammals and worms (Richardson et al., 2014; Yau et al., 2014; Marcette et al., 2014), but their roles in neuronal MT orientation remained unknown. In this study, we provide the evidence indicating that Patronin plays an important role in governing minus-end-out orientation of dendritic MTs in Drosophila ddaC sensory neurons. Mechanistically, we show that Patronin regulates dendritic MT orientation to facilitate dendrite pruning by suppressing the Klp10A activity. Thus, we demonstrate, for the first time, that a MT minus-end-binding protein facilitates neuronal pruning by orienting proper MT arrays in neurites.

Patronin is a novel player of dendrite pruning in ddaC neurons

Patronin was first identified as a regulator of mitotic spindle (Goshima et al., 2007). Patronin recognizes and stabilizes free MT minus ends against kinesin 13-mediated MT depolymerization in cultured S2 cells (Goodwin and Vale, 2010). Recent studies have started to unravel the important roles of Patronin in neuronal development and differentiation. Mammalian CAMSAP2 stabilizes neuronal MTs in axons and dendrites by its association with noncentrosomal MT minus ends; it is required for axon specification and dendrite arborization (Yau et al., 2014). In C. elegans, PTRN-1 is required for several developmental processes, such as synapse stabilization and neurite formation (Marcette et al., 2014; Richardson et al., 2014). In this study, we provide multiple lines of in vivo evidence to demonstrate that Patronin is a novel regulator of dendrite pruning in ddaC neurons, and Patronin regulates dendrite pruning by suppressing the activity of Klp10A. First, genetic analyses with multiple RNAi lines, two patronin mutants as well as the rescue experiment unambiguously reveal that Patronin is required for dendrite pruning in a cell-autonomous manner. Second, we conducted gain-of-function studies using two independent transgenes and further demonstrated that Patronin acts in a dose-sensitive manner to govern dendrite pruning. Third, we also show Patronin’s role in initial dendrite arborization, which, however, is separable from its function in dendrite pruning. Fourth, attenuation of Klp10A function significantly rescued the patronin mutant phenotype in terms of dendrite pruning. Finally, overexpression of Klp10A inhibited dendrite pruning and resembled the patronin loss-of-function mutants. Thus, Patronin promotes dendrite pruning primarily via antagonizing the Klp10A activity.

Patronin/CAMSAPs/PTRN-1 contains a N-terminal CH domain, three CC domains, and a MT-binding CKK domain at the C-terminal region (Baines et al., 2009). Mammalian CAMSAPs recognize MT minus-ends via their CKK domains (Jiang et al., 2014). Drosophila Patronin was reported to target MT minus ends via its CC domains, whereas its CKK domain localizes along MTs and is essential for supressing MT minus-end dynamics in Drosophila S2 cells (Goodwin and Vale, 2010; Hendershott and Vale, 2014). In C. elegans neurons, the CKK domain of PTRN-1 is necessary and sufficient for its function in MT dynamics and axon regeneration (Chuang et al., 2014). Our functional domain analyses reveal that the MT-binding CKK domain is important for Patronin’s function in dendrite pruning of ddaC neurons, whereas CH and CC domains appear to be less important. Thus, our study further highlights a functional importance of the CKK domain in neuronal pruning. It is conceivable that Patronin acts via this domain to stabilize dendritic MT minus ends and thereby orient normal dendritic MTs to facilitate dendrite-specific pruning of ddaC neurons. Consistent with this idea, a recent structural analysis revealed that the CKK domain preferentially binds to a curved region of MT minus ends on the outer surface to prevent the association of kinesin-13 depolymerase with the same site via steric inhibition, thereby protecting MT minus ends from kinesin-13-mediated MT depolymerization (Atherton et al., 2017).

Patronin regulates the minus-end-out orientation of dendritic MTs in ddaC dendrites

Mammalian CAMSAP3/Nezha interacts with the spectraplakin ACF7, a cytoskeletal crosslinking protein, anchors non-centrosomal MT minus ends to actin filaments and thereby polarizes MT networks in cultured epithelial cells (Ning et al., 2016; Noordstra et al., 2016). However, it remains unknown about a role of Patronin/CAMSAPs/PTRN-1 in regulating MT polarity in differentiated neurons. Here, we demonstrate that Patronin governs uniform MT orientation in the major dendrites of ddaC sensory neurons. We first show that the MT minus-end marker Nod-β-gal was greatly reduced in the dendrites of patronin mutant neurons, concomitantly with its robust accumulation in the soma. This finding suggests a strong reduction in non-centrosomal minus ends in the dendrites. Moreover, the axon-specific marker Kin-β-gal, which marks MT plus ends, mis-localized to the dendrites in patronin mutant neurons, indicative of increased plus-end-out MT arrays in the dendrites. Consistent with these observations, we further show a significant increase in anterior EB1-GFP comets in the major dendrites of patronin neurons, suggesting impaired MT orientation. Moreover, the number of EB1-GFP comets, which reflects overall MT density and MT nucleation levels in the dendrites, was not significantly different between wild-type and patronin neurons. Finally, we demonstrate that Patronin controls MT minus-end-out orientation by suppressing the kinesin-13/Klp10A activity.

How might Patronin regulate MT minus-end-out orientation in ddaC dendrites? Similar to its mammalian counterpart in cultured hippocampal neurons, Patronin likely associates in vivo with non-centrosomal MT minus-ends and stabilizes them against kinesin-13-mediated MT depolymerization in the ddaC dendrites. Via MT guidance or sliding by plus-end motors kinesins, growing MTs might be oriented in a minus-end-out manner in the dendrites (Yan et al., 2013; Mattie et al., 2010). In the absence of Patronin, dendritic MTs might be depolymerized into short filaments from their minus ends mediated by excessive depolymerization activity of Klp10A and/or other MT severing factors. Microtubule depolymerising or severing factors have been observed to depolymerize MTs into short filaments (McNally and Vale, 1993). Short MT filaments, which was proposed to be re-oriented in either plus-end-out or minus-end-out direction with equal probability (del Castillo et al., 2015), can serve as seeds for MT growth, resulting in a mixed MT polarity in the dendrites of patronin mutant neurons. Consistent with this speculation, we show that the MT minus-end marker Nod-β-gal robustly accumulated in the soma of patronin mutant neurons. Moreover, depletion of Klp10A in patronin mutant neurons fully restored dendritic distribution of the minus-end-marker Nod-β-gal as well as the minus-end-out MT orientation in the dendrites, supporting the idea that kinesin-13-dependent MT depolymerization at the minus ends is attributed to the impaired MT orientation in the dendrites. We further show that the number of EB1-GFP comets was not significantly different between wild-type and patronin neurons, suggesting that MT nucleation levels or plus-end growth activity appears to be unaffected by the absence of Patronin. patronin depletion might result in excessive MT depolymerization primarily at the MT minus ends. Consistent with this notion, it was reported that in Patronin-deficient Drosophila S2 cells, minus end depolymerization often halted when it reached the EB1-enriched MT plus end tips, indicating that +TIP proteins might resist continued minus-end depolymerization (Goodwin and Vale, 2010). Second, given that a small proportion of CAMSAP3 is able to bind to MT plus ends in cultured epithelial cells (Ning et al., 2016), one might envisage that Patronin might bind to dendritic MT plus ends in ddaC neurons and regulate MT orientation together with the MT plus-end-binding proteins such as kinesin-1/2, EB-1 and APC2. These proteins were previously reported to regulate dendritic MT orientation (Mattie et al., 2010; Herzmann et al., 2018; Yan et al., 2013); kinesin-1/2 and EB-1 have recently shown to be involved in dendrite pruning although the mechanism remains unknown (Herzmann et al., 2018) (Wang and Yu, unpublished data). The potential relevance between Patronin and plus-end regulators awaits further investigation in future. Finally, Patronin is recruited to the actin-rich anterior cortex by Shot, a cytoskeletal crosslinking protein, and controls the anterior-posterior MT polarity in Drosophila oocytes (Nashchekin et al., 2016). Patronin might also anchor MT minus ends to actin through Shot in ddaC sensory neurons. Given that the distal dendrites of ddaC neurons are actin-rich (Medina et al., 2006; Nagel et al., 2012), Patronin might tether free minus ends of MT filaments to the distal branches to elongate dendritic MTs with minus-end-out orientation. This possibility could be interrogated by removal of Shot or actin regulators in ddaC neurons in future studies.

A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons

Neuron highly relies on its MT cytoskeleton to support neuronal architecture as well as facilitate intracellular transport of proteins and organelles. In mammalian neurons, axonal MTs are oriented plus-end-out, whereas dendritic MTs have mixed orientations with both plus-end-out and minus-end-out patterns (Baas and Lin, 2011; Yau et al., 2016). MTs are arranged in a predominant minus-end-out orientation in major dendrites of Drosophila and C. elegans neurons (Stone et al., 2008; Goodwin et al., 2012). In Drosophila, developing da neurons, including ddaC neurons, initially exhibit a mixed orientation of MTs in dendrites and gradually mature to have a uniform minus-end-out pattern over several days (Hill et al., 2012). Mature ddaC neurons maintain the minus-end-out orientation of MTs in their dendrites before the onset of pruning (Wang and Yu, unpublished data). Emerging evidence suggests that uniform dendritic MT orientation might be a prerequisite for dendrite pruning. First, in axon-injured da neurons, dendritic MT orientation becomes mixed instead of minus-end-out (Stone et al., 2010; Song et al., 2012); concurrently, dendrite pruning is also inhibited in ddaC neurons following axon transection (Chen et al., 2012). Second, kinesin-1/2 mutant ddaC neurons show both mixed dendritic MT orientation and dendrite pruning defects (Stone et al., 2010; Herzmann et al., 2018). In this study, we further identified the MT minus-end-binding protein Patronin that is required for dendrite pruning of ddaC neurons, and loss of patronin function led to decreased Nod-β-gal levels and increased anterograde EB1-GFP comets in the dendrites. More importantly, co-depletion of the kinesin-13 MT depolymerase Klp10A restored normal Nod-β-gal levels and uniform minus-end-out MT orientation in patronin mutant dendrites and thereby rescued the dendrite pruning defects. In addition, CNN and APC1/2, two known regulators of dendritic MT polarity (Yalgin et al., 2015; Mattie et al., 2010), also appear to be required for dendrite pruning. We observed dendrite severing defects in cnnhk1 mutant ddaC neurons (52%, n = 23; Figure 8—figure supplement 3A) as well as APC1Q8, APC2N175K double mutant ddaC clones (60%, n = 10; Figure 8—figure supplement 3B). As controls, MT-associated proteins, Futsch and Tau, which regulate MT stability (Hummel et al., 2000; Herzmann et al., 2017), appear to be dispensable for dendrite pruning, as no dendrite pruning defect was observed in fustchN94 or tau RNAi ddaC neurons (Figure 8—figure supplement 3C–D). Thus, multiple lines of evidence demonstrate that dendritic MT orientation is important for dendrite pruning. Our study favors a model that Patronin stabilizes MT minus ends and maintains MT minus-end-out orientation in dendrites to facilitate the MT breakdown and thereby dendrite pruning. It is possible that uniform minus-end-out MTs may provide the tracks for localizing MT severing factors at the proximal dendrites to disassemble local MT filaments.

Materials and methods

Key resources table
Reagent typeDesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(D. melangoaster)
UAS-MicalN-terother(Terman et al., 2002)
Genetic reagent
(D. melangoaster)
SOP-flp (#42)other(Matsubara et al., 2011)
Genetic reagent
(D. melangoaster)
ppk-Gal4 on II and
III chromosome
other(Grueber et al., 2003)
Genetic reagent
(D. melangoaster)
UAS-Kin-β-galother(Clark et al., 1997)
Genetic reagent
(D. melangoaster)
UAS-EB1-GFPother(Stone et al., 2008)
Genetic reagent
(D. melangoaster)
UASp-mCherry-Patroninother(Nashchekin et al., 2016)
Genetic reagent
(D. melangoaster)
patroninc9-c5other(Nashchekin et al., 2016)
Genetic reagent
(D. melangoaster)
UAS-GFP-Patroninother(Derivery et al., 2015)
Genetic reagent
(D. melangoaster)
UASp-Arf79F-EGFPother(Shao et al., 2010)
Genetic reagent
(D. melangoaster)
UAS-Klp10Athis paper
Genetic reagent
(D. melangoaster)
UAS-GFP-Klp10Athis paper
Genetic reagent
(D. melangoaster)
UAS-Patroninthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-Patroninthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninΔCHthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninΔCKKthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninΔCHthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninΔCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCC1-3this paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCHthis paper
Genetic reagent
(D. melangoaster)
Gal4109(2)80Bloomington
Stock Center
BDSC: 8769
Genetic reagent
(D. melangoaster)
ppk-CD4-tdGFPBloomington
Stock Center
BDSC: 35843
Genetic reagent
(D. melangoaster)
GSG2295-Gal4Bloomington
Stock Center
BDSC: 40266
Genetic reagent
(D. melangoaster)
Gal44-77Bloomington
Stock Center
BDSC: 8737
Genetic reagent
(D. melangoaster)
UAS-Nod-β-galBloomington
Stock Center
BDSC: 9912
Genetic reagent
(D. melangoaster)
patronin RNAi #2Bloomington
Stock Center
BDSC: 36659
Genetic reagent
(D. melangoaster)
klp10A RNAi #2Bloomington
Stock Center
BDSC: 33963
Genetic reagent
(D. melangoaster)
Gal42-21Bloomington
Stock Center
FBal0328157
Genetic reagent
(D. melangoaster)
patronin RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v108927
Genetic reagent
(D. melangoaster)
klp10A RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v41534
Genetic reagent
(D. melangoaster)
control RNAiVienna Drosophila
RNAi Centre
VDRC: v36355
Genetic reagent
(D. melangoaster)
control RNAiVienna Drosophila
RNAi Centre
VDRC: v37288
Genetic reagent
(D. melangoaster)
patronink07433Drosophila Genetic
Resource Center
#111217
Genetic reagent
(D. melangoaster)
patronin RNAi #3National Institute
of Genetics, Japan
#18462 Ra-1
Genetic reagent
(D. melangoaster)
kat-60 RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v38368
Genetic reagent
(D. melangoaster)
kat-60 RNAi # 2Vienna Drosophila
RNAi Centre
VDRC: v106487
Genetic reagent
(D. melangoaster)
kat-60L1 RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v31599
Genetic reagent
(D. melangoaster)
kat60-L1 RNAi # 2Vienna Drosophila
RNAi Centre
VDRC: v108168
Genetic reagent
(D. melangoaster)
tau RNAi # 1Bloomington
Stock Center
BDSC: 28891
Genetic reagent
(D. melangoaster)
tau RNAi # 2Bloomington
Stock Center
BDSC: 40875
Genetic reagent
(D. melangoaster)
cnnhk21Bloomington
Stock Center
BDSC: 5039
Genetic reagent
(D. melangoaster)
FRT 82B, APC2N175K,
APC1Q8
Bloomington
Stock Center
BDSC: 7211
Genetic reagent
(D. melangoaster)
futschN94Bloomington
Stock Center
BDSC: 8805
Antibodyanti-β-galactosidasePromegaCat#: Z37831:1000
Antibodyanti-PatroninM Gonzalez-Gaitan1:500
AntibodyCy3-conjugated goat
anti-Rabbit antibody
JacksonCat#111-165-0031:500
Antibody647-conjugated goat
anti-Rabbit antibody
JacksonCat#111-605-1441:500
Antibodyanti-FutschDSHB22c101:50

Fly strains

UAS-MicalN-ter (Terman et al., 2002), SOP-flp (#42) (Matsubara et al., 2011), ppk-Gal4 on II and III chromosome (Grueber et al., 2003), UAS-Kin-β-gal (Clark et al., 1997), UAS-EB1-GFP (Stone et al., 2008), UASp-mCherry-Patronin, patroninc9-c5 (Nashchekin et al., 2016), UAS-GFP-Patronin (Derivery et al., 2015), UASp-Arf79F-EGFP (Shao et al., 2010), UAS-Klp10A, UAS-GFP-Klp10A, UAS-Patronin, UAS-Venus-Patronin, UASp-PatroninΔCH, UASp-PatroninΔCKK, UASp-PatroninCKK, UAS-Venus-PatroninΔCH, UAS-Venus-PatroninΔCKK, UAS-Venus-PatroninCC1-3, UAS-Venus-PatroninCKK, UAS-Venus-PatroninCH (this study).

The following stocks were obtained from Bloomington Stock Center (BSC): Gal4109(2)80, ppk-CD4-tdGFP (BL#35843), GSG2295-Gal4 (BL#40266), Gal44-77 (BL#8737), UAS-Nod-β-gal (BL#9912), patronin RNAi #2 (BL#36659), klp10A RNAi #2 (BL#33963), Gal42-21, tau RNAi #1 (BL#28891) and #2 (BL#40875), cnnhk21 (BL#5039), APC2N175K, APC1Q8 (BL#7211), futschN94 (BL#8805).

The following stocks were obtained from Vienna Drosophila RNAi Centre (VDRC): patronin RNAi # 1 (v108927), klp10A RNAi # 1 (v41534), control RNAi (v36355, v37288), kat-60 RNAi #1 (v38368) and #2 (v106487), kat-60L1 RNAi #1 (v31599) and #2 (v108168).

The following stock was obtained from Drosophila Genetic Resource Center (DGRC), Kyoto: patronink07433 (#111217).

The following stock was obtained from National Institute of Genetics, Japan: patronin RNAi #3 (#18462 Ra-1).

Generation of patronin and klp10A Transgenes

patronin and klp10A full-length cDNAs were PCR amplified from pMT-mCherry-Patronin (Addgene) and EST LD29208 (DGRC, Bloomington) into pDonor vector (Life Tech). The GATEWAY pTW, pTVW or pTGW vectors (DGRC) containing the respective fragments of the cDNAs were constructed by LR reaction (Life Tech).

The variants of patronin were generated by either PCR or site mutagenesis (Agilent Tech) using pDonor patronin as a template. The respective cDNA fragments were subcloned into pTVW or pPW vector (DGRC). The transgenic lines were established by the Bestgene Inc.

Immunohistochemistry and antibodies

The following primary antibodies were used for immunohistochemistry at the indicated dilution: mouse anti-β-galactosidase (Promega Cat#: Z3783, 1:1000), Rabbit anti-Patronin (a gift from M. Gonzalez-Gaitan, 1:500), mouse anti-Futsch (22C10, DSHB, 1:50). Cy3 or Alexa Fluor 647-conjugated secondary antibodies (Jackson Laboratories, Cat#: 111-165-003, 111-605-144) were used at 1:500 dilution. For immunostaining, pupae or larvae were dissected in PBS and fixed with 4% formaldehyde for 15 min. Mounting was performed in VectaShield mounting medium, and the samples were directly visualized by confocal microscopy.

Live imaging analysis

To image Drosophila da neurons at the wandering 3rd instar (wL3) or WP stage, larvae or pupae were first washed in PBS buffer briefly and followed by immersion with 90% glycerol. For imaging da neurons at 16 hr APF or 20 hr APF, pupal cases were carefully removed before mounted with 90% glycerol. Dendrite images were acquired on Leica TSC SP2.

MARCM analysis of da sensory neurons

MARCM analysis, dendrite imaging, and quantification were carried out as previously described. ddaC clones were selected and imaged at the WP stage according to their location and morphology. The ddaC neurons were examined for dendrite pruning defects at 16 hr APF.

RU486/mifepristone treatment for the Gene-Switch system

RU486/mifepristone treatment for the Gene-Switch system was carried out as previously described. Embryos were collected at 6 hr intervals and reared on standard food to the early 3rd instar larva stage. The larvae were transferred to the standard culture medium which contains 240 μg/ml mifepristone (Sigma Aldrich M8046). White prepupae were picked up, subject to phenotypic analysis at WP or 16 hr APF.

Quantification of ddaC dendrites

Live confocal images of ddaC neurons expressing UAS-mCD8-GFP driven by ppk-Gal4 or Gal44-77, or ppk-CD4-tdGFP were shown at WP, 16 hr APF. For wild-type or mutant ddaC neurons, the percentages of severing defect were quantified in a 275 μm x 275 μm region of the dorsal dendritic field, originating from the abdominal segments 2–5. The severing defect was defined by the presence of dendrites that remain attached to the soma at 16 hr APF (Kirilly et al., 2009; Williams and Truman, 2005; Kuo et al., 2005). Total length of unpruned dendrites was measured in a 275 μm x 275 μm region of the dorsal dendritic field using ImageJ. The number of samples (n) in each group is shown on the bars. Statistical significance was determined using either two-tailed Student’s t-test (two samples) or one-way ANOVA and Bonferroni test (multiple samples) (*p<0.05, **p<0.01, ***p<0.001, N.S., not significant). Error represent S.E.M. Dorsal is up in all images.

EB1-GFP comet imaging

Wild-type and mutant embryos were collected at 3 hr intervals. Embryos were reared on standard cornmeal food without yeast supplement until 96 hr AEL, followed by EB1-GFP imaging. EB1-GFP comet imaging was performed with Olympus FV3000 using 60X Oil lens and Zoom factor 3. ddaC neurons of 96 hr AEL larva were imaged for 3 min. 82 frames were acquired for each neuron at 2.25 s interval and analyzed using and ImageJ software.

References

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Decision letter

  1. Kang Shen
    Reviewing Editor; Stanford University, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands
  3. Shaul Yogev
    Reviewer; Yale University, United States

In the interests of transparency, eLife includes the editorial decision letter, peer reviews, and accompanying author responses.

[Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved.]

Evaluation of the final submission:

Minor issues remain unresolved. In particular, the explanation of why both loss and gain of Patronin lead to pruning defects is not definitive.

Decision letter after peer review:

Thank you for submitting your article "Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Shaul Yogev (Reviewer #1). The other two reviewers remain anonymous.

The Reviewing Editor has highlighted the concerns that require revision and/or responses, and we have included the separate reviews below for your consideration. If you have any questions, please do not hesitate to contact us.

Summary:

All three reviewers found your manuscript addressing an important question in neuronal cell biology. They also agree that this work contains many interesting data that are certainly of interest to the general readership of eLife. However, a number of important concerns were raised. I would like to ask you to address these concerns with additional experiments and discussion.

Major concerns:

1) Reviewers found that the causality between MT polarity defects and pruning is lacking. We suggest you to examine other mutants with MT polarity defects like cnn, APC to see if they have pruning phenotypes.

2) The authors hypothesized that the defects observed in Patronin knockdown are due to excessive depolymerization by klp10a. However, the number of EB1 comets is the same. It is just comet directionality that is perturbed. If the excessive depolymerization by klp10a is the cause of the phenotype, then there should be far fewer comets. It would make sense if klp10a were a cross-linking or sliding kinesin, but a depolymerising kinesin only having an effect on orientation and not number of EB1 comets seems bizarre. Why would misorientation be a result of excessive depolymerization activity? This is an important question that needs to be addressed because it gets at the fundamental question on the relationship between MT stability and polarity.

3) Genetic interaction between Patronin and Katanin kat60L should be addressed.

4) Please address whether Patronin overexpression affects dendrite pruning through the same mechanism as the loss-of-function mutant. This overexpression effect is not consistent with the model provided.

5) The CKK domain of Patronin is necessary and sufficient for pruning, which seems to be a surprising result considering that it is the minimal domain necessary to bind the MT. How can one explain this result?

We would also like to invite you to respond to the minor points that are listed in the individual reviews.

Separate reviews (please respond to each point):

Reviewer #1:

This is a very interesting paper that describes an unexpected role for Patronin in controlling dendrite MT polarity and pruning in Drosophila. The experiments are overall convincing and are properly controlled and the topic and scope are fit for eLife.

My main concern is that although this paper makes a very interesting connection between microtubule stability, polarity and pruning, it is not clear from the experiments how stabilization of MTs favors minus-end-out orientation, and whether it is polarity or stability that is important for pruning. This latter point could be addressed by examining some of the other mutants that affect MT polarity (cnn, APC, EB or others) or stability (Futch, Tau or others). by using genetic interactions the authors could place the Patronin-KLP10 module in the context of what we already know about MT regulation in this system.

My second reservation is that I am not convinced that Patronin overexpression affects dendrite pruning through the same mechanism as the loss-of-function as the paper seems to imply. This is because OE of the CKK domain has no effect and because loss of KLP10 (which should be equivalent to Patronin overexpression) has no phenotype. The authors should clearly discuss the possible distinct mechanisms of Patronin loss and gain of function.

Minor Comments:

– Please show more representative kymographs for the EB movies (i.e that show that most of the time the phenotype is not a complete reversal), and move the quantification from the supplementary figure to main figure.

– Please quantify MT growth speed and run length for EB movies. This could help to understand whether there are different properties to the minus-end-out versus plus-end-out nucleation.

– Is there any effect on axonal MT polarity (EB movies) in Patronin loss of function or KLP10 OE?

– Please add tubulin staining to support the conclusion that overall MT levels are not changed.

– What happens at later time-points? Do the mutant dendrites eventually "catch-up"?

– I was confused by the fact that the Patronin phenotype is 100% penetrant with the overexpressed motor fusions but only partial with the EB comets. Does this mean that these motor constructs "read" something besides MT polarity? Please comment on this.

Reviewer #2:

In their manuscript, Wang, Rui and Yu investigate the role of the microtubule (MT) minus-end binding protein, Patronin, in Drosophila dendritic arborization neurons. The open questions the authors sought to address were: 1) the role of Patronin in neuronal development and pruning, and 2) the contribution of the minus-end out MT orientation to dendrite pruning. Both knockdown and overexpression of Patronin inhibits the normal timecourse of dendritic pruning in the Drosophila pupal dendritic arborization neurons. They find that Patronin is necessary for the maintenance of MT polarity in dendrites. Instead of microtubules being predominantly minus-end out, in Patronin knockdown or mutant neurons, there is a mixture of minus- and plus-end out MTs. Inhibiting the MT depolymerizing kinesein, klp10A (kinesin-13), in this background restores the polarity of the MTs, indicating that in the absence of patronin, klp10A somehow contributes to this mixed MT polarity. Inhibiting klp10a also rescues the dendritic pruning defects observed in the absence of Patronin. The overall message of the paper is that Patronin antagonizes klp10a activity normally to maintain uniform MT minus-end out polarity during dendritic maintenance and pruning. In the absence of patronin, klp10A somehow functions in disrupting the uniform polarity of microtubules in the dendrites, which in turn disrupts dendritic pruning. The data are very nice and the results are clear; however, the model is incomplete. The effect of Patronin and klp10a on MTs are correlative with the pruning defects, not causative, and there can be a wide variety of other MAPs (stabilizers, severing proteins, etc.) whose functions are directly or indirectly disrupted upon Patronin or klp10a knockdown. These other possibilities are completely ignored, making the model disjointed and speculative.

The authors do not mention the direct interaction between Patronin and the MT severing enzyme, katanin (Jiang et al., 2014 and Jiang et al., 2018, the latter of which was not even cited). The authors do mention that katanin itself does not have a role in pruning, but katanin p60-like does based on prior work. It seems important to look at the genetic interaction between Patronin and Kat60L1 as well, before constructing an entire model around Patronin and klp10a.

The authors state many times that Patronin may stabilize uniform minus-end out MTs in the dendrites, but do not explain why and are unable to get at a mechanism in their paper. If the authors think that Patronin anchors MT minus ends in some way in the dendrites, then the CKK results do not make sense. The CKK domain of Patronin is necessary and sufficient for pruning. There is no elaboration as to the functional significance of this domain. Presumably, the CKK domain is not able to anchor the MTs in any way, since it is the minimal domain necessary to bind the MT. So, how does this fit into their model?

The authors claim that the defects observed in patronin knockdown are due to excessive depolymerization by klp10a. However, the number of EB1 comets is the same. It is just comet directionality that is perturbed. This does not make sense with their hypothesis. If it is excessive depolymerization by klp10a, then there should be far fewer comets. It would make sense if klp10a were a cross-linking or sliding kinesin, but a depolymerising kinesin only having an effect on orientation and not number of EB1 comets seems bizarre. Why would misorientation be a result of excessive depolymerization activity?

The authors state: "As a result, dendritic MTs may collapses into short and unstable filaments, which no longer anchor within the dendrites, instead fall into the soma." How does a filament fall into the soma? Likely, the MT would just depolymerize entirely, or depolymerize to a point where another stabilizing MAP is located and regrow. The concentration of tubulin in most cells favors new MT growth, and perhaps this new growth could be oriented incorrectly and this is why there is mixed MT polarity. Maybe the absence of Patronin affects another MAP that is necessary for the orientation of the MTs. As the authors note, many proteins result in a mixed MT polarity upon knockdown. There are many different MAPs that could be perturbed upon Patronin knockdown, and therefore this may not be a direct effect of Patronin function, but an indirect effect via other MAP functions.

Finally, why would uniform MT orientation facilitate MT breakdown? This also seem correlative, and the papers they cite provide nothing but speculation as to why the relationship between MT orientation and the progress of pruning. In order to say uniform MT orientation is necessary for MT breakdown and subsequent pruning, the authors would need to test a number of MAPs that have varying effects on MT orientation and assess their effects on the pruning timecourse.

Reviewer #3:

In this manuscript by Wang et al., the authors report a role for the microtubule minus end protein Patronin/CAMSAP/PTRN-1 in dendrite pruning in the dda dendrites in Drosophila. In addition, they find a role for Patronin in maintaining minus end out microtubule polarity in these dendrites as well. The CKK domain of Patronin is necessary and sufficient for its role in pruning. Both of these functions appear to operate through the antagonism of the depolymerizing kinesin Klp10A as the phenotypes can be suppressed upon removal of Klp10A function. Overall the data are well presented and the conclusions well supported by the data.

Two big questions I am left with from this work are:

1) Why does the maintenance of minus end out microtubules in the dendrite allow for productive pruning? The authors speculate a bit about this in the Discussion, but a clearer connection would elevate the paper. In particular all of the phenotypic observations are made in steady state dendrites. The authors assume the two phenotype- maintenance of minus end out MTs and efficient dendritic pruning -but this is speculation. Would it be possible to better characterize MT polarity in both control and patronin mutants during the pruning process, i.e. between WP and 16h APF?

2) I am confused by the Patronin gain of function phenotype. If the model is "that Patronin promotes dendritic pruning via antagonizing KLP10A function", then my expectation would be that Patronin overexpression would not lead to pruning defects. This model is at least supported by the MT polarity phenotype upon Patronin overexpression (i.e. that minus end out MT polarity is perturbed), but again this does not fit so neatly into the model presented.

In some cases, the data presented suggest precocious pruning in some mutant conditions. For example, Figure 7G and H: In the Klp10A OE, there appears to be fewer branches at the WP stage as compared to controls. Is this the case?

Where does Patronin localize in the neurons at 1) steady state, and 2) during pruning?

Minor Points:

Figure 4 legend is out of order in its description of the panels, making it hard to follow

Figure 4F-H: where are the dendrites here? Aren't they supposed to be labelled with curly brackets as is indicated in the legend? Also, the Kin-B-gal is very hard to see. What are the arrowheads pointing to?

In many places problems with the writing make it difficult to interpret what the authors are saying. For example, the description of Figure 5 in subsection “Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons” is particularly confusing. The data presented in the figure are clear, but the wording muddies the authors' intent.

In the same section: "over 80% of anterograde branches moved away from the soma"-isn't this the definition of anterograde?

Minor corrections:

Results section, first paragraph: mutant clones.

Various points throughout the Results section: On average.

Subsection “Overexpression of Patronin causes dendrite pruning defects in ddaC neurons”, first paragraph, final sentence: as a.

Subsection “Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotype in MT orientation in ddaC dendrites”: Previous studies or A previous study.

Discussion section, first paragraph: PTRN-1.

Figure 4D and H axis label "moving"

Figure 4: What is GFP labeling here? No explanation for this in figure legend or text.

[Editors' note: further revisions were suggested, as described below.]

Thank you for resubmitting your work entitled "Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila" for further consideration at eLife. Your revised article has been favorably evaluated by Anna Akhmanova (Senior Editor), a Reviewing Editor, and two reviewers.

The manuscript has been improved but there are some remaining, as outlined below:

Please address the remaining comments by including the katanin knockdown data if you have it. Please also expand discussions on the issue raised by reviewer 1. Upon receiving your revision, I will make a decision without sending it to the reviewers.

Reviewer #1:

The revisions have strengthened several points, but other key questions remain open. My overall impression is still that the manuscript describes a very interesting observation, although the underlying mechanism is still unclear.

– The connection between MT polarity (rather than stability) and pruning is now stronger. However, how polarity actually affects pruning is not explored.

– I couldn't find the phenotype of the Katanin single knock down, which makes interpreting the double knock down with Patronin impossible. I think that this is a technical point that has to be addressed or this experiment should be removed.

– The conclusion that Patronin loss and gain of function lead to pruning defects in the same way has been strengthened by the revisions. It is still unclear to me how this actually fits into the proposed mechanism (see below).

– The revisions haven't clarified the mechanism through which Patronin affects polarity and not stability. There is some disagreement between the reduced Futch staining and the normal number of plus-end comets. The authors' model about Klp10A affecting only minus ends does not explain why it would stop depolymerizing before overtaking the entire polymer, or how it distinguishes the minus ends of minus-end-out vs those of plus-end-out MTs. As a minor point, it is possible that short MTs would turn around in the axon or dendrite as they suggest, but the reference cited (del Castillo et al) describes MT sliding by dynein and kinesin-1 and it is not clear why the authors chose it to support their model.

https://doi.org/10.7554/eLife.39964.040

Author response

Major concerns:

1) Reviewers found that the causality between MT polarity defects and pruning is lacking. We suggest you to examine other mutants with MT polarity defects like cnn, APC to see if they have pruning phenotypes.

We have now examined the possible role of cnn and APC1/2 (known regulators of dendritic MT polarity) in dendrite pruning. We observed dendrite severing defects in 52% of cnnhk21 mutant ddaC neurons (Figure 8—figure supplement 3A). Likewise, APC1Q8, APC2N175K double mutant ddaC neurons also exhibited dendrite severing defects (60%, Figure 8—figure supplement 3B). Thus, our new data further support the conclusion that proper dendritic MT polarity is important for dendrite pruning. We have now included these new data in the revised Figure 8—figure supplement 3A-B and text (subsection “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

2) The authors hypothesized that the defects observed in Patronin knockdown are due to excessive depolymerization by klp10a. However, the number of EB1 comets is the same. It is just comet directionality that is perturbed. If the excessive depolymerization by klp10a is the cause of the phenotype, then there should be far fewer comets. It would make sense if klp10a were a cross-linking or sliding kinesin, but a depolymerising kinesin only having an effect on orientation and not number of EB1 comets seems bizarre.

It was indeed unexpected that the number of EB1 comets remained unchanged in dendrites upon patronin RNAi knockdown. Two co-authors of this manuscript have repeated the experiment and found the same result (revised Figure 5D). Moreover, Klp10A overexpression did not cause a signification reduction in the number of EB1-GFP comets in the dendrites (Figure 8I) but caused the MT polarity defect in the dendrites (Figure 8G-H) (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”), resembling the patronin RNAi knockdown (Figure 5D). These data imply that patronin RNAi or Klp10A overexpression might result in excessive MT depolymerization primarily at the MT minus ends, rather than affect the number of EB1-GFP comets that mark MT growth at the MT plus ends. Consistent with this notion, it was reported that in Patronin-deficient Drosophila S2 cells, minus end depolymerization often halted when it reached the EB1-enriched MT plus end tips, indicating that +TIP proteins including EB1 might resist continued minus-end depolymerization (Goodwin and Vale, 2010). On the other hand, in worm touch neurons, depletion of the patronin homolog leads to an increase in the number of EB1 comets (Chuang et al., 2014). We have now included the new data in the revised Figure 8F-K and text (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”).

Why would misorientation be a result of excessive depolymerization activity? This is an important question that needs to be addressed because it gets at the fundamental question on the relationship between MT stability and polarity.

In the absence of Patronin, dendritic MTs might be depolymerized into short filaments from their minus ends mediated by excessive depolymerization activity of Klp10A and/or other MT severing factors. Microtubule depolymerising or severing factors have been observed to depolymerize MTs into short filaments (McNally F. et al., Cell 1993). Short MT filaments, which was proposed to be re-oriented in either plus-end-out or minus-end-out direction with equal probability (del Castillo et al., 2015), can serve as seeds for MT growth, resulting in a mixed MT polarity in the dendrites of patronin mutant neurons. We have now included this discussion in the revised manuscript (subsection “Patronin regulates the minus-end-out orientation of dendritic MTs in ddaC dendrites”).

3) Genetic interaction between Patronin and Katanin kat60L should be addressed.

A previous study reported that the putative MT severing factor Kat-60L1 is required for dendrite pruning (Lee et al., 2009). We have now conducted genetic interactions between patronin and kat-60L1. When Kat-60L1 was depleted via two RNAi lines in the patronin RNAi background, we did not observe any enhancement or suppression between patronin and kat-60L1 in dendrite pruning. Thus, this new data indicates no genetic interaction between patronin and kat-60L1. We have now included the new data in the revised Figure 7—figure supplement 1C) and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

4) Please address whether Patronin overexpression affects dendrite pruning through the same mechanism as the loss-of-function mutant. This overexpression effect is not consistent with the model provided.

To address whether Patronin overexpression, like the loss-of-function mutant, also affects dendrite pruning through excessive Klp10A activity, we have knocked down klp10A in Patronin-overexpressing ddaC neurons. Knockdown of klp10A, via two independent RNAi lines, significantly suppressed the dendrite pruning defects in Patronin-overexpressing ddaC neurons (Figure 7F-J). Moreover, knockdown of klp10A also significantly suppressed the MT orientation defect in the dendrites of Patronin-overexpressing ddaC neurons (Figure 7K-N). Therefore, these results suggest that both gain of patronin function and loss of patronin function affects dendrite pruning at least partially by upregulation of Klp10A function. We have now included the new data in the revised Figure 7F-N and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

5) The CKK domain of Patronin is necessary and sufficient for pruning, which seems to be a surprising result considering that it is the minimal domain necessary to bind the MT. How can one explain this result?

In the original manuscript, we show that CKK-deleted Patronin variant (PatroninΔCKK) failed to rescue the dendrite pruning defects in patroninc9-c5 ddaC neurons, whereas the expression of the CKK domain significantly rescued patroninc9-c5-associated dendrite pruning defects, suggesting that the CKK domain is necessary and sufficient for Patronin’s function during dendrite pruning. We have now conducted additional experiments to elaborate the functional significance of the CKK domain. First, we confirmed that the expression of the CKK domain alone significantly rescued the dendrite pruning defects in the patronin RNAi background (Figure 8—figure supplement 2A). Second, the expression of the CKK domain almost fully rescued Nod-lacZ distribution as well as retrograde EB1-GFP comets in the dendrites of patronin RNAi ddaC neurons (Figure 8—figure supplement 2B-C). Third, the expression of the CKK domain significantly suppressed the dendrite pruning defects in Klp10A-overexpressing ddaC neurons (Figure 8—figure supplement 2D), suggesting that the CKK domain is able to antagonize Klp10A’s function during dendrite pruning. Thus, multiple lines of evidence demonstrate that the CKK domain is important for Patronin to govern minus-end-out MT orientation in dendrites as well as dendrite pruning. Thus, we propose the model in which via its CKK domain, Patronin is essential for the establishment of minus-end-out MTs in the dendrites of ddaC neurons. We have now included these new data in the revised manuscript (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”).

We have also tuned down the conclusion as “The CKK domain of Patronin is important for Patronin to govern minus-end-out MT orientation in dendrites as well as dendrite pruning” in the revised manuscript.

We would also like to invite you to respond to the minor points that are listed in the individual reviews.

Separate reviews (please respond to each point):

Reviewer #1:

This is a very interesting paper that describes an unexpected role for Patronin in controlling dendrite MT polarity and pruning in Drosophila. The experiments are overall convincing and are properly controlled and the topic and scope are fit for eLife.

My main concern is that although this paper makes a very interesting connection between microtubule stability, polarity and pruning, it is not clear from the experiments how stabilization of MTs favors minus-end-out orientation, and whether it is polarity or stability that is important for pruning. This latter point could be addressed by examining some of the other mutants that affect MT polarity (cnn, APC, EB or others) or stability (Futch, Tau or others). by using genetic interactions the authors could place the Patronin-KLP10 module in the context of what we already know about MT regulation in this system.

We have now examined the possible role of cnn and APC1/2 (regulators of dendritic MT polarity) as well as futsch and tau (regulators of MT stability) in dendrite pruning. We observed dendrite severing defects in 52% of cnnhk21 mutant ddaC neurons (Figure 8—figure supplement 3A). Likewise, APC1Q8, APC2N175K double mutant ddaC neurons also exhibited dendrite severing defects (60%, Figure 8—figure supplement 3B). In contrast, no dendrite pruning defect was observed in fustchN94 or tau RNAi ddaC neurons (Figure 8—figure supplement 3C-D). These new data, together with our patronin results, further indicate that proper dendritic MT polarity is important for dendrite pruning. We have now included the new data in the revised Figure 8—figure supplement 3 and text (subsection “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

Previous studies reported that the putative MT severing factor Kat-60L1 and the MT-associated protein Tau are involved in dendrite pruning (Lee et al., 2009; Herzmann et al., 2017). We have now conducted genetic interactions between patronin and kat-60L1/tau. When either Kat-60L1 or Tau was knocked down via two RNAi lines in the patronin RNAi background, we did not observe any enhancement or suppression between patronin and kat-60L1/tau in dendrite pruning (Figure 7—figure supplement 1C-D). Thus, these results indicate that MT polarity but not MT stability is important for dendrite pruning. These new results, together with our original result that the patronin mutant phenotypes were fully rescued by klp10A RNAi knockdown, support the conclusion that the Patronin-Klp10A module is a novel regulatory pathway that governs MT polarity as well as dendrite pruning. We have now included the new data in the revised Figure 7—figure supplement 1C-D and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

We propose that in wild-type ddaC neurons, Patronin likely associates with non-centrosomal MT minus-ends and stabilizes them against kinesin-13-mediated MT depolymerization in the dendrites. Via MT guidance or sliding by plus-end motors kinesins, growing MTs might be oriented in a minus-end-out manner in the dendrites (Yan et al., 2013; Mattie et al., 2010). In the absence of Patronin, dendritic MTs might be depolymerized into short filaments from their minus ends mediated by excessive depolymerization activity of Klp10A and/or other MT severing factors. Microtubule depolymerising or severing factors have been observed to depolymerize MTs into short filaments (McNally et al., Cell 1993). Short MT filaments, which was proposed to be re-oriented in either plus-end-out or minus-end-out direction with equal probability (del Castillo et al., 2015), can serve as seeds for MT growth, resulting in a mixed MT polarity in the dendrites of patronin mutant neurons. We have now included this discussion in the revised manuscript (subsection “Patronin regulates the minus-end-out orientation of dendritic MTs in ddaC dendrites”).

My second reservation is that I am not convinced that Patronin overexpression affects dendrite pruning through the same mechanism as the loss-of-function as the paper seems to imply. This is because OE of the CKK domain has no effect and because loss of KLP10 (which should be equivalent to Patronin overexpression) has no phenotype. The authors should clearly discuss the possible distinct mechanisms of Patronin loss and gain of function.

To address whether Patronin overexpression, like the loss-of-function mutant, also affects dendrite pruning through excessive Klp10A activity, we have knocked down klp10A in Patronin-overexpressing ddaC neurons. Knockdown of klp10A, via two independent RNAi lines, significantly suppressed the dendrite pruning defects in Patronin-overexpressing ddaC neurons (Figure 7F-J). Moreover, knockdown of klp10A also significantly suppressed the MT orientation defect in the dendrites of Patronin-overexpressing ddaC neurons (Figure 7K-N). Therefore, these results suggest that both gain of patronin function and loss of patronin function affects dendrite pruning at least partially by upregulation of Klp10A function. We have now included the new data in the revised Figure 7F-N and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

Minor Comments:

– Please show more representative kymographs for the EB movies (i.e that show that most of the time the phenotype is not a complete reversal), and move the quantification from the supplementary figure to main figure.

We have now showed the representative kymographs for EB1 movies and moved the quantification from the supplemental Figure 5 to the main Figure 5M in the revised manuscript. Please note that in the revised Figures 5-6, the new images were acquired at different experimental conditions, compared with images shown in our initial manuscript. We previously reared the larva in the grape-juice food to obtain brighter EB1-GFP signals. Due to the poor nutrition, those larvae appeared less healthy with reduced eclosion rate, likely due to cellular stress. To improve the growth condition, we have now reared the larvae on normal cornmeal food without yeast supplement (live yeast often generates auto-fluorescent signals). In the new condition, the larvae were much healthier and survived to the adulthood. Their ddaC neurons also showed detectable EB1-GFP comets, however, with fewer comet number than those reared in the grape-juice food. Importantly, two co-authors of this manuscript independently obtained similar results on EB1 comet movement and MT orientation from both patronin RNAi and Patronin-overexpressing neurons in larvae on the new growth condition. Therefore, we took this opportunity to replace the previous EB1 images (the original Figures 5-6) with the new images that are representative and obtained from larvae with an improved growth condition. We have included this information in the revised Materials and methods.

– Please quantify MT growth speed and run length for EB movies. This could help to understand whether there are different properties to the minus-end-out versus plus-end-out nucleation.

We have now included the quantification results of MT growth speed and tracking length for EB movies in the revised Figures 5-6 as well as text (subsection “Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons”).

– Is there any effect on axonal MT polarity (EB movies) in Patronin loss of function or KLP10 OE?

We have now examined EB1 movement in the axons of patronin RNAi ddaC neurons. Compared with 4% of retrograde EB1-GFP comets (predominantly in a plus-end-out orientation) in the axons of wild-type neurons, patronin knockdown led to 41% of retrograde EB1-GFP comets in the axons (Figure 5—figure supplement 1C), suggesting a severe defect in the plus-end-out orientation of their axonal MTs. Thus, patronin plays an important role in both dendritic and axonal MT polarity. We have now included the new data in the revised Figure 5—figure supplement 1C and text (subsection “Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons”).

– Please add tubulin staining to support the conclusion that overall MT levels are not changed.

In the original manuscript, we did not conduct tubulin staining and did not claim that overall MT levels are not changed. We have now detected the overall polymerized MT levels in the dendrites of patronin RNAi ddaC neurons using the antibody 22C10 against Futsch, a microtubule-associated protein. Our quantification data indicate that the intensity of overall MT levels was significantly reduced in the dendrites of patronin RNAi ddaC neurons, compared with the controls (Figure 5—figure supplement 1B). This finding is consistent with the previous reports that MT density is reduced upon the depletion of Patronin or its mammalian homologs (Goodwin and Vale, 2010; Yau et al., 2014). Thus, our new data indicate that Patronin regulates both MT polarity and levels in the dendrites. We have now included the new data in the revised Figure 5—figure supplement 1B and text (subsection “Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons”).

– What happens at later time-points? Do the mutant dendrites eventually "catch-up"?

We have now examined the dendrite pruning process at two late time points, 24 h APF and 32 h APF. The larval dendrites of patronin RNAi ddaC neurons were largely removed by 32 h APF (Figure 1—figure supplement 1B), presumably due to large-scale apoptosis and migration of the dorsal abdominal epidermis, on which ddaC neurons arborize their larval dendrites (William and Truman, 2005a). This is similar to the final pruning of EcRDN-overexpressing ddaC/D/E neurons at 30 h APF (Williams and Truman, 2005; Kirilly et al., 2009; Kirilly et al., 2011). We have now included the new data in the revised Figure 1—figure supplement 1B and text (subsection “Patronin is required for dendrite pruning and arborization in ddaC neurons”).

– I was confused by the fact that the Patronin phenotype is 100% penetrant with the overexpressed motor fusions but only partial with the EB comets. Does this mean that these motor constructs "read" something besides MT polarity? Please comment on this.

The penetrance difference was caused by the different quantification methods. For the Nod-β-Gal phenotype, we quantified the percentage of neurons with aberrant Nod-β-Gal distribution, whereas for the EB1-GFP phenotype, we quantified the percentage of comets with anterograde movement. If we quantify the percentage of neurons with anterograde EB1-GFP movement, we have now observed 93% of patronin RNAi neurons showing abnormal EB1-GFP movement direction in the dendrites of patronin RNAi neurons. This penetrance of EB1-GFP phenotype (93%) is comparable with Nod-β-lacZ penetrance (100%). In addition, EB1-GFP was expressed at a low level under the control of the weaker Gal4 driver Gal44-77 in order to visualize its comets, whereas Nod-lacZ was driven by the strong driver ppk-Gal4. Thus, the efficiency of patronin RNAi knockdown using Gal44-77 is likely lower than that using ppk-Gal4, leading to the partial EB1-GFP phenotype.

Reviewer #2:

In their manuscript, Wang, Rui and Yu investigate the role of the microtubule (MT) minus-end binding protein, Patronin, in Drosophila dendritic arborization neurons. The open questions the authors sought to address were: 1) the role of Patronin in neuronal development and pruning, and 2) the contribution of the minus-end out MT orientation to dendrite pruning. Both knockdown and overexpression of Patronin inhibits the normal timecourse of dendritic pruning in the Drosophila pupal dendritic arborization neurons. They find that Patronin is necessary for the maintenance of MT polarity in dendrites. Instead of microtubules being predominantly minus-end out, in Patronin knockdown or mutant neurons, there is a mixture of minus- and plus-end out MTs. Inhibiting the MT depolymerizing kinesein, klp10A (kinesin-13), in this background restores the polarity of the MTs, indicating that in the absence of patronin, klp10A somehow contributes to this mixed MT polarity. Inhibiting klp10a also rescues the dendritic pruning defects observed in the absence of Patronin. The overall message of the paper is that Patronin antagonizes klp10a activity normally to maintain uniform MT minus-end out polarity during dendritic maintenance and pruning. In the absence of patronin, klp10A somehow functions in disrupting the uniform polarity of microtubules in the dendrites, which in turn disrupts dendritic pruning. The data are very nice and the results are clear; however, the model is incomplete. The effect of Patronin and klp10a on MTs are correlative with the pruning defects, not causative, and there can be a wide variety of other MAPs (stabilizers, severing proteins, etc.) whose functions are directly or indirectly disrupted upon Patronin or klp10a knockdown. These other possibilities are completely ignored, making the model disjointed and speculative.

We have now examined the possible role of cnn and APC1/2 (regulators of dendritic MT polarity) as well as futsch and tau (MT stabilizers) in dendrite pruning. We observed dendrite severing defects in 52% of cnnhk21 mutant ddaC neurons (Figure 8—figure supplement 3A). Likewise, APC1Q8, APC2N175K double mutant ddaC neurons also exhibited dendrite severing defects (60%, Figure 8—figure supplement 3B). In contrast, no dendrite pruning defect was observed in fustchN94 or tau RNAi ddaC neurons (Figure 8—figure supplement 3C-D). These new data, together with our patronin results, further indicate that proper dendritic MT polarity is important for dendrite pruning. We have now included the new data in the revised Figure 8—figure supplement 3 and text (subsection “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

Previous studies reported that the putative MT severing factor Kat-60L1 and the MT-associated protein Tau are involved in dendrite pruning (Lee et al., 2009; Herzmann et al., 2017). We have now conducted genetic interactions between patronin and kat-60L1/tau. When either Kat-60L1 or Tau was knocked down via two RNAi lines in the patronin RNAi background, we did not observe any enhancement or suppression between patronin and kat-60L1/tau in dendrite pruning. Thus, these data suggest that Tau or Kat-60L1 functions are unlikely altered upon Patronin RNAi knockdown. We have now included the new data in the revised Figure 7—figure supplement 1C-D and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in

dendrites”).

The authors do not mention the direct interaction between Patronin and the MT severing enzyme, katanin (Jiang et al., 2014 and Jiang et al., 2018, the latter of which was not even cited).

We have now cited Jiang et al., 2018, in the revised manuscript (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

The authors do mention that katanin itself does not have a role in pruning, but katanin p60-like does based on prior work. It seems important to look at the genetic interaction between Patronin and Kat60L1 as well, before constructing an entire model around Patronin and klp10a.

Given the direct interaction between mammalian Patronin (CAMSAPs) and katanin (Jiang et al., 2014 and 2018), we have now examined the potential genetic interactions between patronin and kat-60 in dendrite pruning. RNAi Knockdown of kat-60, via two distinct RNAi lines, did not enhance or suppress the pruning defects in patronin RNAi ddaC neurons (Figure 7—figure supplement 1B). Likewise, although Kat-60L1 was reported to be required for dendrite pruning (Lee et al., 2009), neither did double knockdown of kat-60L1 and patronin exhibit any enhancement or suppression in the dendrite pruning defects (Figure 7—figure supplement 1C). Thus, these data indicate no genetic interaction between patronin and kat-60/kat-60L1. We have now included the new data in the revised Figure 7—figure supplement 1B-C and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

The authors state many times that Patronin may stabilize uniform minus-end out MTs in the dendrites, but do not explain why and are unable to get at a mechanism in their paper. If the authors think that Patronin anchors MT minus ends in some way in the dendrites, then the CKK results do not make sense. The CKK domain of Patronin is necessary and sufficient for pruning. There is no elaboration as to the functional significance of this domain. Presumably, the CKK domain is not able to anchor the MTs in any way, since it is the minimal domain necessary to bind the MT. So, how does this fit into their model?

In the original manuscript, we show that CKK-deleted Patronin variant (PatroninΔCKK) failed to rescue the dendrite pruning defects in patroninc9-c5 ddaC neurons, whereas the expression of the CKK domain significantly rescued patroninc9-c5-associated dendrite pruning defects, suggesting that the CKK domain is necessary and sufficient for Patronin’s function during dendrite pruning. We have now conducted additional experiments to elaborate the functional significance of the CKK domain. First, we confirmed that the expression of the CKK domain alone significantly rescued the dendrite pruning defects in the patronin RNAi background (Figure 8—figure supplement 2A). Second, the expression of the CKK domain almost fully rescued Nod-lacZ distribution as well as retrograde EB1-GFP comets in the dendrites of patronin RNAi ddaC neurons (Figure 8—figure supplement 2B-C). Third, the expression of the CKK domain significantly suppressed the dendrite pruning defects in Klp10A-overexpressing ddaC neurons (Figure 8—figure supplement 2D), suggesting that the CKK domain is able to antagonize Klp10A’s function during dendrite pruning. Thus, multiple lines of evidence demonstrate that the CKK domain is important for Patronin to govern minus-end-out MT orientation in dendrites as well as dendrite pruning. Thus, we propose the model in which via its CKK domain, Patronin is essential for the establishment of minus-end-out MTs in the dendrites. We have now included these new data in the revised Figure 8—figure supplement 2 and text (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”).

We have also tuned down the conclusion as “The CKK domain of Patronin is important for Patronin to govern minus-end-out MT orientation in dendrites as well as dendrite pruning” in the revised manuscript. This revised conclusion fits into the model better.

The authors claim that the defects observed in patronin knockdown are due to excessive depolymerization by klp10a. However, the number of EB1 comets is the same. It is just comet directionality that is perturbed. This does not make sense with their hypothesis. If it is excessive depolymerization by klp10a, then there should be far fewer comets. It would make sense if klp10a were a cross-linking or sliding kinesin, but a depolymerising kinesin only having an effect on orientation and not number of EB1 comets seems bizarre.

It was indeed unexpected that the number of EB1 comets remained unchanged in dendrites upon patronin RNAi knockdown. Two co-authors of this manuscript have repeated the experiment and found the same result (revised Figure 5D). Moreover, Klp10A overexpression did not cause a signification reduction in the number of EB1-GFP comets in the dendrites (Figure 8I) but caused the MT polarity defect in the dendrites (Figure 8G-H) (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”), resembling the patronin RNAi knockdown (Figure 5D). These data imply that patronin RNAi or Klp10A overexpression might result in excessive MT depolymerization primarily at the MT minus ends, rather than affect the number of EB1-GFP comets that mark MT growth at the MT plus ends. Consistent with this notion, it was reported that in Patronin-deficient Drosophila S2 cells, minus end depolymerization often halted when it reached the EB1-enriched MT plus end tips, indicating that +TIP proteins including EB1 might resist continued minus-end depolymerization (Goodwin and Vale, 2010). On the other hand, in worm touch neurons, depletion of the patronin homolog leads to an increase in the number of EB comets (Chuang, et al., 2014). We have now included the new data in the revised Figure 8F-K and text (subsection “Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation”).

Why would misorientation be a result of excessive depolymerization activity?

In the absence of Patronin, dendritic MTs might be depolymerized into short filaments from their minus ends mediated by excessive depolymerization activity of Klp10A and/or other MT severing factors. Microtubule depolymerising or severing factors have been observed to depolymerize MTs into short filaments (McNally F. et al., Cell 1993). Short MT filaments, which was proposed to be re-oriented in either plus-end-out or minus-end-out direction with equal probability (del Castillo et al., 2015), can serve as seeds for MT growth, resulting in a mixed MT polarity in the dendrites of patronin mutant neurons. We have now included this discussion in the revised manuscript (subsection “Patronin regulates the minus-end-out orientation of dendritic MTs in ddaC dendrites”).

The authors state: "As a result, dendritic MTs may collapses into short and unstable filaments, which no longer anchor within the dendrites, instead fall into the soma." How does a filament fall into the soma? Likely, the MT would just depolymerize entirely, or depolymerize to a point where another stabilizing MAP is located and regrow. The concentration of tubulin in most cells favors new MT growth, and perhaps this new growth could be oriented incorrectly and this is why there is mixed MT polarity.

We thank the reviewer for the helpful comment on our hypothesis. We have rephrased the sentences as “In the absence of Patronin, dendritic MTs might be depolymerized into short filaments from their minus ends mediated by excessive depolymerization activity of Klp10A and/or other MT severing factors. Microtubule depolymerising or severing factors have been observed to depolymerize MTs into short filaments (McNally F. et al., Cell 1993). Short MT filaments, which was proposed to be re-oriented in either plus-end-out or minus-end-out direction with equal probability (del Castillo et al., 2015), can serve as seeds for MT growth, resulting in a mixed MT polarity in the dendrites of patronin mutant neurons.” in the revised Discussion (subsection “Patronin regulates the minus-end-out orientation of dendritic MTs in ddaC dendrites”).

Maybe the absence of Patronin affects another MAP that is necessary for the orientation of the MTs. As the authors note, many proteins result in a mixed MT polarity upon knockdown. There are many different MAPs that could be perturbed upon Patronin knockdown, and therefore this may not be a direct effect of Patronin function, but an indirect effect via other MAP functions.

We have now examined the possible role of two important MAPs, namely Futsch and Tau in dendrite pruning. No dendrite pruning defect was observed in fustchN94 or tau RNAi ddaC neurons (Figure 8—figure supplement 3C-D). Moreover, we have also conducted genetic interactions between patronin and kat-60L1/tau. When either Kat-60L1 or Tau was knocked down via two RNAi lines in the patronin RNAi background, we did not observe any enhancement or suppression between patronin and kat-60L1/tau in dendrite pruning (Figure 7—figure supplement 1C-D). Formally, we cannot exclude the possibility that the MT orientation phenotypes in patronin mutant neurons might be caused by impaired functions of multiple MAPs. In our current study, we focus mainly on the Patronin-Klp10A module in which Patronin regulates dendritic MT orientation at least partially by antagonizing the MT depolymerizing kinesin Klp10A. We have now included these new data in the revised figures (Figure 7—figure supplement 1C-D, Figure 8—figure supplement 3C-D) and text (subsections “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites” and “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

Finally, why would uniform MT orientation facilitate MT breakdown? This also seem correlative, and the papers they cite provide nothing but speculation as to why the relationship between MT orientation and the progress of pruning. In order to say uniform MT orientation is necessary for MT breakdown and subsequent pruning, the authors would need to test a number of MAPs that have varying effects on MT orientation and assess their effects on the pruning timecourse.

We have now examined the possible role of cnn and APC1/2 (known regulators of dendritic MT polarity) in dendrite pruning. We observed dendrite severing defects in 52% of cnnhk21 mutant ddaC neurons (Figure 8—figure supplement 3A). Likewise, APC1Q8, APC2N175K double mutant ddaC neurons also exhibited dendrite severing defects (60%, Figure 8—figure supplement 3B). In addition, both kinesin-1 and kinesin-2 mutant ddaC neurons with mixed dendritic MT orientations also exhibited dendrite pruning defects (Mattie et al., 2010; Herzmann et al., 2018). Thus, multiple lines of evidence demonstrate that uniform dendritic MT orientation is important for dendrite pruning. We have now included the new data in the revised Figure 8—figure supplement 3A-B and text (subsection “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

Reviewer #3:

In this manuscript by Wang et al., the authors report a role for the microtubule minus end protein Patronin/CAMSAP/PTRN-1 in dendrite pruning in the dda dendrites in Drosophila. In addition, they find a role for Patronin in maintaining minus end out microtubule polarity in these dendrites as well. The CKK domain of Patronin is necessary and sufficient for its role in pruning. Both of these functions appear to operate through the antagonism of the depolymerizing kinesin Klp10A as the phenotypes can be suppressed upon removal of Klp10A function. Overall the data are well presented and the conclusions well supported by the data.

Two big questions I am left with from this work are:

1) Why does the maintenance of minus end out microtubules in the dendrite allow for productive pruning? The authors speculate a bit about this in the Discussion, but a clearer connection would elevate the paper. In particular all of the phenotypic observations are made in steady state dendrites. The authors assume the two phenotype- maintenance of minus end out MTs and efficient dendritic pruning -but this is speculation. Would it be possible to better characterize MT polarity in both control and patronin mutants during the pruning process, i.e. between WP and 16h APF?

To further substantiate the link between microtubule polarity and dendrite pruning, we have now examined the possible role of cnn and APC1/2 (known regulators of dendritic MT polarity) in dendrite pruning. We observed dendrite severing defects in 52% of cnnhk21 mutant ddaC neurons (Figure 8—figure supplement 3A). Likewise, APC1Q8, APC2N175K double mutant ddaC neurons also exhibited dendrite severing defects (60%, Figure 8—figure supplement 3B). In addition, both kinesin-1 and kinesin-2 mutant ddaC neurons with mixed dendritic MT orientations also exhibited dendrite pruning defects (Mattie et al., 2010; Herzmann et al., 2018). Thus, multiple lines of evidence demonstrate that uniform dendritic MT orientation is important for dendrite pruning. We have now included the new data in the revised Figure 8—figure supplement 3A-B and text (subsection “A high correlation between MT orientation and dendrite-specific pruning in ddaC sensory neurons”).

We have also attempted to examine dendritic MT polarity in both control and patronin RNAi ddaC neurons at 6 h APF before the onset of dendrite pruning. Unfortunately, due to their opaque brown cases, the pupae showed undetectable EB1-GFP signals in ddaC neurons at this prepupal stage. Those pupal case cannot be peeled at this stage. To date, no lab in the field has successfully reported EB1-GFP imaging during the pruning process due to these technical limitations.

2) I am confused by the Patronin gain of function phenotype. If the model is "that Patronin promotes dendritic pruning via antagonizing KLP10A function", then my expectation would be that Patronin overexpression would not lead to pruning defects. This model is at least supported by the MT polarity phenotype upon Patronin overexpression (i.e. that minus end out MT polarity is perturbed), but again this does not fit so neatly into the model presented.

In the original version, our data suggest that Patronin overexpression may behave as a dominant negative, as it phenocopied patronin mutants in terms of dendrite pruning and dendritic MT orientation. To further substantiate this point, we have now knocked down klp10A in Patronin-overexpressing ddaC neurons. knockdown of klp10A, via two independent RNAi lines, significantly suppressed the dendrite pruning defects in Patronin-overexpressing ddaC neurons (Figure 7F-J). Moreover, knockdown of klp10A also significantly suppressed the MT orientation defect in the dendrites of Patronin-overexpressing ddaC neurons (Figure 7K-N). These results suggest that both gain of patronin function and loss of patronin function affects dendrite pruning at least partially by upregulation of Klp10A function. We have now included the new data in the revised Figure 7F-N and text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”).

In some cases, the data presented suggest precocious pruning in some mutant conditions. For example, Figure 7G and H: In the Klp10A OE, there appears to be fewer branches at the WP stage as compared to controls. Is this the case?

Klp10A overexpression led to simplified dendrite arbors at the WP stage, which is the dendrite arborization defect. These simplified dendrite arbors persisted and remained attached to the soma at 16 h APF, indicative of dendrite pruning defects, but not the precocious dendrite pruning.

Where does Patronin localize in the neurons at 1) steady state, and 2) during pruning?

We have now conducted immuno-staining using anti-Patronin antibody at the larval (96 h AEL) and white pupal stages. Patronin localized uniformly to dendrites, axons and soma at both larval and pupal stages.We have now included the new data in the revised Figure 4—figure supplement 1B and text (subsection “Patronin is required for proper distribution of dendritic and axonal MT markers”).

Minor Points:

Figure 4 legend is out of order in its description of the panels, making it hard to follow

We have now corrected the order in the description of the panels in the revised legends.

Figure 4F-H: where are the dendrites here? Aren't they supposed to be labeld with curly brackets as is indicated in the legend? Also, the Kin-B-gal is very hard to see. What are the arrowheads pointing to?

We have now added the curly brackets to highlight the dendrites in the revised Figure 4. We have also included the description of the arrowheads as follows, “Arrowheads point to ectopic Kin-β-gal aggregates in the dendrites” in the revised figure legends.

In many places problems with the writing make it difficult to interpret what the authors are saying. For example, the description of Figure 5 in subsection “Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons” is particularly confusing. The data presented in the figure are clear, but the wording muddies the authors' intent.

We have now rephrased the description of Figure 5 and made it more concise in the revised manuscript.

In the same section: "over 80% of anterograde branches moved away from the soma"-isn't this the definition of anterograde?

We have now deleted “anterograde” and modified the sentence as “In these mutant dendrite branches, over 80% of EB1-GFP comets moved away from the soma, suggesting a predominant plus-end-out MT pattern” in the revised text.

Minor corrections:

Results section, first paragraph: mutant clones.

We have now made the change in the revised text.

Various points throughout the Results section: On average.

We have now made the corrections in the revised manuscript accordingly.

Subsection “Overexpression of Patronin causes dendrite pruning defects in ddaC neurons”, first paragraph, final sentence: as a

We have now made the correction in the revised text.

Subsection “Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotype in MT orientation in ddaC dendrites”: Previous studies or A previous study.

We have made the change in the revised manuscript.

Discussion section, first paragraph: PTRN-1.

We have now made the correction in the revised manuscript.

Figure 4D and H axis label "moving"

We have now changed to “moving” in the revised figures.

Figure 4: What is GFP labeling here? No explanation for this in figure legend or text

GFP labelling indicates the mCD8-GFP fusion protein. We have modified the original sentence to “Confocal images of ddaC neurons expressing mCD8-GFP, Nod-lacZ or Kin-lacZ and immunostained for β-galactosidase at wL3 stages.” in the legends of the revised Figure 4 as well as all other relevant figures.

[Editors' note: further revisions were suggested, as described below.]

Please address the remaining comments by including the katanin knockdown data if you have it. Please also expand discussions on the issue raised by reviewer 1. Upon receiving your revision, I will make a decision without sending it to the reviewers.

Reviewer #1:

The revisions have strengthened several points, but other key questions remain open. My overall impression is still that the manuscript describes a very interesting observation, although the underlying mechanism is still unclear.

– The connection between MT polarity (rather than stability) and pruning is now stronger. However, how polarity actually affects pruning is not explored.

We thank the reviewer for the endorsement on our revised manuscript. In the current manuscript, we have, for the first time, demonstrated that the MT minus-end-bind protein Patronin plays an important role in regulating MT minus-end-out orientation in dendrites and thereby promoting dendrite pruning of sensory neurons. Thus, this study provides a firm basis for future investigations into several interesting questions, for example, how MT polarity affects pruning.

– I couldn't find the phenotype of the Katanin single knock down, which makes interpreting the double knock down with Patronin impossible. I think that this is a technical point that has to be addressed or this experiment should be removed.

Knockdown of katanin (kat-60), via two distinct RNAi lines, did not affect normal dendrite pruning in ddaC neurons. We have now included these new data in the revised text (subsection “Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites”, paragraph two) and Figure 7—figure supplement 1B.

– The conclusion that Patronin loss and gain of function lead to pruning defects in the same way has been strengthened by the revisions. It is still unclear to me how this actually fits into the proposed mechanism (see below).

– The revisions haven't clarified the mechanism through which Patronin affects polarity and not stability. There is some disagreement between the reduced Futch staining and the normal number of plus-end comets. The authors' model about Klp10A affecting only minus ends does not explain why it would stop depolymerizing before overtaking the entire polymer, or how it distinguishes the minus ends of minus-end-out vs those of plus-end-out MTs.

We have now included the following discussion in the revised Discussion part “patronin depletion might result in excessive MT depolymerization primarily at the MT minus ends. Consistent with this notion, it was reported that in Patronin-deficient Drosophila S2 cells, minus end depolymerization often halted when it reached the EB1-enriched MT plus end tips, indicating that +TIP proteins might resist continued minus-end depolymerization (Goodwin and Vale, 2010).”

As a minor point, it is possible that short MTs would turn around in the axon or dendrite as they suggest, but the reference cited (del Castillo et al) describes MT sliding by dynein and kinesin-1 and it is not clear why the authors chose it to support their model.

In the discussion part of the cited paper (del Castillo et al., 2015), the authors proposed and discussed the possibility that short MT fragments might be able to turn around in the axons or dendrites, although the main point of the paper focuses on MT sliding by dynein and kinesin-1.

We greatly appreciate the editors for kind support and advice. Following the editors’ advice, in the revised manuscript we have included the katanin knockdown data and also expanded the discussion on the issue raised by the reviewer #1.

https://doi.org/10.7554/eLife.39964.041

Article and author information

Author details

  1. Yan Wang

    1. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore
    2. NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, National University of Singapore, Singapore, Singapore
    Contribution
    Resources, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft
    Contributed equally with
    Menglong Rui
    Competing interests
    No competing interests declared
  2. Menglong Rui

    Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore
    Contribution
    Resources, Data curation, Formal analysis, Investigation, Visualization, Methodology
    Contributed equally with
    Yan Wang
    Competing interests
    No competing interests declared
  3. Quan Tang

    1. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore
    2. Department of Biological Sciences, National University of Singapore, Singapore, Singapore
    Contribution
    Resources, Data curation, Software, Formal analysis, Visualization, Methodology
    Competing interests
    No competing interests declared
  4. Shufeng Bu

    1. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore
    2. Department of Biological Sciences, National University of Singapore, Singapore, Singapore
    Contribution
    Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Fengwei Yu

    1. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore
    2. NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, National University of Singapore, Singapore, Singapore
    3. Department of Biological Sciences, National University of Singapore, Singapore, Singapore
    4. Neuroscience and Behavioral Disorder Program, Duke-NUS Graduate Medical School, National University of Singapore, Singapore, Singapore
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    fengwei@tll.org.sg
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0268-199X

Funding

Temasek Life Sciences Laboratory, Singapore (TLL-2040)

  • Fengwei Yu

National Research Foundation, Singapore (SBP-P3, SBP-P8)

  • Fengwei Yu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank M Gonzalez-Gaitan, TJ Harris, YN Jan, D St Johnston, AL Kolodkin, MM Rolls, T Uemura, the Bloomington Stock Center (BSC), VDRC (Austria), Drosophila Genetic Resource Center (Japan), National Institute of Genetics (Japan) for generously providing antibodies and fly stocks. We are grateful to MM Rolls for providing the technical advice on EB1-GFP imaging. We thank members of the FY laboratory for helpful assistance. We also thank the TLL microscopy facility for technical assistance. YW was recipients of the NGS postgraduate scholarship, Singapore. This work was supported by Temasek Life Sciences Laboratory (TLL), Singapore (to FY).

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Kang Shen, Stanford University, United States

Reviewer

  1. Shaul Yogev, Yale University, United States

Publication history

  1. Received: July 12, 2018
  2. Accepted: March 8, 2019
  3. Version of Record published: March 28, 2019 (version 1)

Copyright

© 2019, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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