Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. Here we report that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, we show that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits αPS2/βPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. Our study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.https://doi.org/10.7554/eLife.35518.001
Nerve cells or neurons can communicate with each other by releasing chemical messengers into the gap between them, the synapse. Both neurons and synapses are surrounded by a network of proteins called the extracellular matrix, which anchors, protects and supports the synapse. The matrix also helps to regulate the dynamic communication across the synapses and consequently neurons.
Little is known about the proteins of the extracellular matrix, in particular about the ones involved in structural support. This is especially important for the so-called neuromuscular junctions, where neurons stimulate muscle contraction and trigger vigorous movement. Receptor proteins on cell surfaces, such as integrins, can bind to the extracellular matrix proteins to anchor the cells and are important for all cell junctions, including synaptic junctions. But because of their many essential roles during development, it was unclear how integrins modulate the activity of the synapse.
To investigate this further, Wang et al. studied the neuromuscular junctions of fruit flies. The experiments revealed that both muscle and neurons secrete a large protein called Tenectin, which accumulates into the small space between the neuron and the muscle, the synaptic cleft. This protein can bind to integrin and is necessary to support the neuromuscular junction structurally and functionally.
Wang et al. discovered that Tenectin works by gathering integrins on the surface of the neuron and the muscle. In the neuron, Tenectin forms complexes with integrin to regulate the release of neurotransmitters. In the muscle, the complexes provide support to the synaptic structures. However, when Tenectin was experimentally removed, it only disrupted the integrins at the neuromuscular junction, without affecting integrins in other regions of the cells, such as the site where the muscle uses integrins to attach to the tendon. Moreover, without Tenectin an important intracellular scaffolding meshwork that lines up and reinforces cell membranes was no longer organized properly at the synapse.
A next step will be to identify the missing components between Tenectin/integrin complexes on the surface of neurons and the neurotransmitter release machinery inside the cells. The extracellular matrix and its receptors play fundamental roles in the development and function of the nervous system. A better knowledge of the underlying mechanisms will help us to better understand the complex interplay between the synapse and the extracellular matrix.https://doi.org/10.7554/eLife.35518.002
The extracellular matrix (ECM) and its receptors impact every aspect of neuronal development, from axon guidance and migration to formation of dendritic spines and neuromuscular junction synaptic junctions and function. The heavily glycosylated ECM proteins provide anchorage and structural support for cells, regulate the availability of extracellular signals, and mediate intercellular communications (Reichardt and Prokop, 2011). Transmembrane ECM receptors include integrins, syndecans and the dystrophin-associated glycoprotein complex (Bökel and Brown, 2002; Häcker et al., 2005; Waite et al., 2009). Integrins in particular are differentially expressed and have an extensive repertoire, controlling multiple processes during neural development. In adults, integrins regulate synaptic stability and plasticity (Morini and Becchetti, 2010; McGeachie et al., 2011). However, integrin roles in synapse development have been obscured by their essential functions throughout development. How integrins are selectively recruited at synaptic junctions and how they engage in specific functions during synapse development and homeostasis remain unclear.
One way to confer specificity to ECM/integrin activities is to deploy specialized ECM ligands for the synaptic recruitment and stabilization of selective heterodimeric integrin complexes (Reichardt and Tomaselli, 1991). For example, at the vertebrate NMJ, three laminins containing the β2 subunit (laminin 221, 421 and 521, that are heterotrimers of α2/4/5, β2 and γ1 subunits) are deposited into the synaptic cleft and basal lamina by skeletal muscle fibers and promote synaptic differentiation. However, only laminin 421 interacts directly with presynaptic integrins containing the α3 subunit and anchors a complex containing the presynaptic Cavα and cytoskeletal and active zone-associated proteins (Carlson et al., 2010). Studies with peptides containing the RGD sequence, recognized by many integrin subtypes, have implicated integrin in the morphological changes and reassembly after induction of long-term potentiation (LTP) (reviewed in [McGeachie et al., 2011]). Several integrin subunits (α3, α5, α8, β1 and β2) with distinct roles in the consolidation of LTP have been identified, but the relevant ligands remain unknown.
Drosophila neuromuscular junction (NMJ) is a powerful genetic system to examine the synaptic functions of ECM components and their receptors. In flies, a basal membrane surrounds the synaptic terminals only in late embryos; during development, the boutons ‘sink’ into the striated muscle, away from the basal membrane (Prokop et al., 1998). The synaptic cleft relies on ECM to withstand the mechanical tensions produced by the muscle contractions. The ECM proteins, including laminins, tenascins/teneurins (Ten-a and -m) and Mind-the-gap (Mtg), interact with complexes of five integrin subunits (αPS1, αPS2, αPS3, βPS, and βν) (Broadie et al., 2011). The αPS1, αPS2 and βPS subunits localize to pre- and post-synaptic compartments and have been implicated in NMJ growth (Beumer et al., 1999; Beumer et al., 2002). The αPS3 and βν are primarily presynaptic and control activity-dependent plasticity (Rohrbough et al., 2000). The only known integrin ligand at the fly NMJ is Laminin A, which is secreted from the muscle and signals through presynaptic αPS3/βν and Focal adhesion kinase 56 (Fak56) to negatively regulate the activity-dependent NMJ growth (Tsai et al., 2012). Teneurins have RGD motifs, but their receptor specificities remain unknown (Mosca et al., 2012). Mtg secreted from the motor neurons influences postsynaptic βPS accumulation (Rushton et al., 2009), but that may be indirectly due to an essential role for Mtg in the organization of the synaptic cleft and the formation of the postsynaptic fields (Rohrbough et al., 2007; Rushton et al., 2012). The large size of these proteins and the complexity of ECM-integrin interactions made it difficult to recognize relevant ligand-receptor units and genetically dissect their roles in synapse development.
Here, we report the functional analysis of Tenectin (Tnc), an integrin ligand secreted from both motor neurons and muscles; Tnc accumulates at synaptic terminals and functions in cis to differentially engage presynaptic and postsynaptic integrin. We uncovered tnc, which encodes a developmentally regulated RGD-containing integrin ligand (Fraichard et al., 2006; Fraichard et al., 2010), in a screen for ECM candidates that interact genetically with neto, a gene essential for NMJ assembly and function (Kim et al., 2012). We found that Tnc selectively recruits the αPS2/βPS integrin at synaptic locations, without affecting integrin anchoring at muscle attachment sites. Dissection of Tnc functions revealed pre- and postsynaptic biologically active cis Tnc/integrin complexes that function to regulate neurotransmitter release and postsynaptic architecture. Finally, we exploited the remarkable features of this selective integrin ligand to uncover a novel synaptic function for integrin, in engaging the spectrin-based membrane skeleton.
To search for novel ECM proteins important for NMJ development we set up a synthetic lethality screen that took advantage of the 50% lethality of an allele with suboptimal levels of Neto, neto109 (Kim et al., 2012). Neto, an obligatory subunit of ionotropic glutamate receptor (iGluR) complexes, controls the distribution and function of iGluRs as well as the assembly and organization of postsynaptic structures (Han et al., 2015; Kim et al., 2015; Ramos et al., 2015). Using this lethality screen we have previously uncovered genetic interactions between neto and several BMP pathway components (Sulkowski et al., 2014; Sulkowski et al., 2016). Lowering the dose of Mtg, an ECM protein known to organize the synaptic cleft (Rohrbough et al., 2007), induced 95% lethality (n = 286) in neto109/Y;; mtg1/+ animals, further validating our strategy. We focused on ECM candidates (Broadie et al., 2011) and identified a set of overlapping deficiencies (Df(3R)BSC-318,,–492, −494, and −655) that drastically increased the lethality of neto109 hemizygotes (from 50% for neto109/Y up to 82% for neto109/Y;; Df/+). Among the common loci disrupted by these deficiencies was tnc, a gene coding for a large mucin-type protein conserved in many insects but with no obvious mammalian homologue (Figure 1A–B) (Fraichard et al., 2006; Syed et al., 2008; Fraichard et al., 2010; Syed et al., 2012).
Tnc is a secreted molecule with five vWFC (von Willebrand factor type-C) protein interaction domains separated by two PTS-rich regions. In mucins, the PTS domains are highly O-glycosylated and form gel-like structures. Tnc has one RGD and several more RGD-like motifs that have been implicated in interaction with integrin (Fraichard et al., 2010). During development, Tnc is secreted in the lumen of several epithelial organs, including foregut, hindgut and trachea. Tnc is also expressed in the embryonic CNS. Using a polyclonal anti-Tnc antibody (Materials and methods) we found that Tnc signals are strongly enriched in the neuropile, in the proximity of the anti-Fasciclin II (FasII) positive axons (Figure 1—figure supplement 1A,B and [Fraichard et al., 2006]). The Tnc signals were absent in the CNS of a tnc mutant (tncEP- P[EPgy2]EY03355), predicted to disrupt both known tnc transcripts (Syed et al., 2012). These mutant embryos had a normal FasII pattern indicating no obvious CNS defects during late embryogenesis (Figure 1—figure supplement 1C–D). The tncEP mutant showed partial lethality (11.6% of expected homozygous progenies were viable, n = 303), which was not enhanced in heteroallelic combinations (12.2% viability for tncEP/Df, n = 392) suggesting that this mutant is equivalent to a genetic null. We also generated a small deletion mutant (tnc82) by FRT-induced recombination. These animals die as homozygous pharate adults (100% lethality, n = 221) but produce some heteroallelic escapers (17.5% viability for tnc82/Df, n = 389).
Western blot analysis revealed a Tnc-positive band of ~300 kD (the calculated MW for Tnc is 299 kD) in extracts from brains and body-wall muscles of control larvae (Figure 1C). This band was undetectable in tnc hetero-allelic combinations, tncEP/Df and tnc82/Df. A band of similar size was found in S2 cells transfected with a Tnc expression construct and was enriched in the conditioned media, indicating that Tnc is efficiently secreted in cell culture (Figure 1D, Material and methods). Neuron specific RNAi knockdown reduced Tnc levels in larval brains to 43% of the control group; this generated very strong phenotypes (below) suggesting that the residual band could reflect additional Tnc-expressing cells in the larval brain. The muscle-specific knockdown reduced the muscle Tnc levels to 19% of the control (Figure 1E).
During larval stages, we found Tnc positive signals throughout the muscles with weak accumulation at the NMJ (Figure 1F and Figure 1—figure supplement 1F). Under the same imaging conditions, the signals were significantly reduced in tncEP/Df mutants (Figure 1G and Figure 1—figure supplement 1G, quantified in Figure 1—figure supplement 1E). Such weak NMJ immunoreactivities were previously reported for proteins secreted in the synaptic cleft (Rushton et al., 2009). To further confirm the specificity of Tnc signals, we overexpressed Tnc in motor neurons (elav-Gal4) or muscles (BG487-Gal4) and examined the NMJs (Figure 1H–J and Figure 1—figure supplement 1H–I). Paneuronal expression of Tnc induced strong accumulation of Tnc-positive signals at synaptic terminals, as well as along the motor neuron axons. At these NMJs, Tnc-labeled puncta were concentrated at the edge of anti-horseradish peroxidase (HRP)-stained boutons (Jan and Jan, 1982). Most of these signals were also observed in the absence of detergents, suggesting that Tnc is secreted in the synaptic terminal. In contrast, muscle overexpression of tnc showed increased Tnc-positive signals throughout the muscle; at synaptic terminals these signals appeared diffuse and farther away from the neuronal membrane. Thus, excess muscle Tnc may not be properly targeted and/or stabilized at synaptic terminals and may have detrimental effects on Tnc-mediated functions. We repeated these results using independent HA-tagged tnc transgenes and staining with anti-HA antibodies (Figure 1—figure supplement 2). Both tagged and untagged transgenes rescued the viability of tnc mutants (see below), indicating that Tnc functions are unaffected by addition of the tag. As above, expression of tnc-HA in neurons but not in muscles induced high accumulation of HA-positive puncta, accessible without detergent, around the synaptic boutons, indicating extracellular distribution (Figure 1—figure supplement 2A–D). Thus, Tnc is expressed in both motor neurons and muscles and appears to accumulate at the ECM surrounding synaptic terminals.
To investigate a possible role for Tnc in the function of the nervous system and/or the musculature, we examined the morphology and physiology of tnc mutants. During larval stages, both tnc mutants (tncEP/Df and tnc82/Df) had largely normal NMJ, with minimally increased bouton numbers (Figure 2—figure supplement 1A–B). A closer examination revealed smaller boutons, with less clear bouton-interbouton delimitations (details below). The few adult escapers did not fly and exhibited climbing defects (Figure 2—figure supplement 1C). Such phenotypes are consistent with previously reported flightless adults generated by RNAi-mediated Tnc knockdown (Fraichard et al., 2010).
We next recorded the evoked excitatory junction potentials (EJPs) and spontaneous miniature excitatory junction potentials (mEJPs) from muscle 6 of third instar larvae (Figure 2A–E). The mEJPs amplitude was normal in tnc mutants. However, the mean frequency of mEJPs was significantly reduced in tnc mutants compared with the control (w1118, 2.67 ± 0.14 Hz vs. tncEP/Df, 1.68 ± 0.13 Hz, p=0.0001, and tnc82/Df, 2.11 ± 0.14 Hz, p=0.0322, Figure 2D). tnc mutations caused 23% and 18% reduction in evoked EJPs amplitude of tncEP/Df and tnc82/Df animals, respectively (w1118, 26.30 ± 0.99 mV vs. tncEP/Df, 20.15 ± 1.26 mV, p=0.0042, and tnc82/Df, 21.53 ± 1.38 mV, p=0.0375, Figure 2E). Moreover, tnc mutants showed a significant decrease in quantal content (w1118, 29.05 ± 1.81 vs. tncEP/Df, 21.44 ± 1.60 and tnc82/Df, 21.53 ± 1.38, Figure 2F). Since we found no change in the resting potential and input resistance in mutant animals, the decrease in EJPs amplitude and quantal content was probably not caused by abnormal passive membrane properties in the muscle. Instead, the reduction of quantal content could be due to a decreased number of vesicle release sites or reduced probability of release. The reduced mEJP frequency is in agreement with reduced quantal content due to fewer release sites. To evaluate the vesicle release probability, we measured the paired-pulse ratio (PPR) using the EJP amplitudes evoked by two stimuli separated by duration of 50 ms (Wong et al., 2014). At 0.5 mM extracellular Ca2+ concentration, the control larvae showed mild short-term depression following paired-pulse stimulation (PPR < 1, Figure 2G–H), indicating a relatively high initial probability of vesicle release; the second stimulus, provided before the resting Ca2+ returned to baseline, lead to the exocytosis of fewer synaptic vesicles than the first stimulus. In contrast, the tnc mutant NMJs showed elevated facilitation and significantly increased PPR (w1118, 0.80 ± 0.03 vs. tncEP/Df, 1.21 ± 0.08, p=0.0231, and tnc82/Df, 1.16 ± 0.09, p=0.0295). The higher the ratio of EJP amplitudes following the first and second pulses, the lower is the probability of release. Thus, tnc mutants have significantly decreased probability of vesicle release.
Since Tnc is expressed in both pre- and post-synaptic compartments, we next examined which Tnc pool(s) is required for NMJ function. We found that tnc knockdown in motor neurons resulted in mEJPs with significantly reduced frequency as compared to the controls (tncRNAi transgene with no driver, or driver alone) (control, 2.69 ± 0.2 Hz vs. N > tncRNAi, 1.31 ± 0.10 Hz p=0.0001, and M > tncRNAi, 2.4 ± 0.17 Hz p=0.4434) (Figure 3A–C, and Figure 3—figure supplement 1). Similar results were obtained with a second RNAi line (GD14952) confirming that these phenotypes are specific to tnc depletion (Figure 3—figure supplement 1). In contrast, tnc knockdown in muscles had no effect on mEJPs frequency or amplitude. Thus, neuronal but not muscle Tnc is required for normal neurotransmitter release.
Expression of Tnc in neurons but not in muscles also rescued the mEJPs frequency and EJPs amplitude defects observed in tnc mutants (Figure 3D–F). Similar to tnc mutants, mutants carrying only a tnc transgene but no driver showed reduced mEJP frequency and EJP amplitude (compare Figure 3D–F and Figure 2). Expression of the tnc transgene in neurons, but not in muscles of tnc mutants restored the mEJPs frequency (w1118, 1.97 ± 0.27 Hz vs. N > tnc; tncEP/Df, 1.56 ± 0.18 Hz p=0.07 and M > tnc; tncEP/Df, 1.20 ± 0.16 Hz p=0.0019) and EJPs amplitude (w1118, 71.51 ± 2.39 mV vs. N > tnc; tncEP/Df, 70.1 ± 2.08 mV p=0.198 and M > tnc; tncEP/Df, 45.03 ± 3.67 mV p<0.0001) to levels that are no longer significantly different from the w1118 control. Together, these data indicate that neuron-derived Tnc regulates normal neurotransmitter release at the NMJ.
Previous studies suggest that Tnc functions as a ligand for αPS2/βPS integrin during wing morphogenesis (Fraichard et al., 2010). Drosophila integrins have been implicated in NMJ growth and synaptic function (Keshishian et al., 1996), with the βPS-containing complexes primarily in the postsynaptic compartment (Prokop et al., 1998; Beumer et al., 1999; Koper et al., 2012). If Tnc functions by recruiting integrin at the NMJ, then presynaptic but not postsynaptic βPS or αPS2 should similarly modulate the neurotransmitter release. Indeed, neuronal knockdown of myospheroid (mys), which encodes the βPS integrin subunit, significantly reduced the mEJPs frequency compared to control (mysRNAi transgene with no driver) (Figure 3G–H). In contrast, muscle knockdown of mys had no detectable effect on mEJPs frequency (control, 0.95 ± 0.14 Hz vs. N > mysRNAi, 0.47 ± 0.05 Hz p=0.0044 and M > mysRNAi, 1.00 ± 0.07 Hz p=0.9142) or amplitude. Similarly, neuronal but not muscle knockdown of inflated (if), which codes for αPS2 integrin, significantly reduced the mEJPs frequency compared to control (ifRNAi transgene with no driver) (Figure 3J–L) (control, 1.62 ± 0.51 Hz vs. N > ifRNAi, 0.99 ± 0.23 Hz p=0.0041 and M > ifRNAi, 1.67 ± 0.42 Hz p=0.9662). The ifRNAi transgene appeared stronger than mysRNAi and generated larval lethality and, occasionally, muscle attachment defects; using these transgenes we observed diminished mEJP amplitude compared to control (Figure 3L). Overall, the reduction of presynaptic αPS2/βPS mirrored the mEJP frequency deficits observed for tnc neuronal knockdown suggesting that neuron-derived Tnc functions as a ligand for presynaptic αPS2/βPS to modulate neurotransmitter release. Postsynaptic αPS2/βPS integrin does not appear to influence neurotransmitter release. Alternatively, a role for postsynaptic αPS2/βPS may be obscured by partial knockdowns and essential functions for these genes in the muscle.
If Tnc recruits integrin to modulate neurotransmitter release, then tnc and mys (or if) should interact genetically. We tested this prediction by examining the trans-heterozygote animals (mys/+;; tnc/+). Indeed, these trans-heterozygotes exhibited severe mEJP deficits that resembled tnc mutants, whereas individual heterozygote larvae (mys/+ or tnc/+) showed normal mEJPs frequency and amplitude (Figure 3M–O). This indicates that tnc and mys function together to modulate neurotransmitter release.
If Tnc recruits and/or stabilizes βPS integrin at synaptic terminals, then Tnc should co-localize with βPS and form Tnc/integrin complexes at synaptic locations and perturbations of Tnc should alter the recruitment of βPS integrin at larval NMJ. Indeed, the βPS signals were dramatically reduced at tnc mutant NMJs (Figure 4A–D). In these analyses the βPS immunoreactivities concentrated at perisynaptic locations, surrounding the control boutons, consistent with previous observations that the muscle pool constitutes the major fraction of βPS at synaptic terminals (Beumer et al., 1999). Interestingly, the βPS levels remained unchanged at the muscle attachment sites; we also observed no detachment of the muscle fibers or defects in costamere organization (Maartens and Brown, 2015). This indicates that loss of Tnc selectively impairs the recruitment of βPS integrin at synaptic terminals. The anti-Tnc antibodies marked discrete puncta at the edge of the HRP- labeled boutons in a region strongly stained by anti-βPS antibodies, but Tnc was not detectable at the muscle attachment sites (Figure 4E–F). This suggests that Tnc and βPS may directly associate at synaptic terminals. Our attempts to co-immunoprecipitate Tnc/integrin complexes from larval carcasses failed; instead, we tested for their close juxtaposition at the NMJ using a proximity ligation assay (PLA), which indicates a less than 40 nm distance between two proteins (Wang et al., 2015). As shown in Figure 4G–H, PLA signals between Tnc and βPS were detected at control but not at tnc mutant NMJs. These PLA signals were tightly packed around the synaptic boutons, unlike the Tnc or βPS immunoreactivities, which spread into the postsynaptic specializations, suggesting that Tnc and βPS form complexes in the close proximity of the synaptic terminal. Such complexes could influence the presynaptic neurotransmitter release, as described above, but could also function in the postsynaptic compartment, where most of the perisynaptic βPS resides (see below).
Since different integrin heterodimers provide spatial and temporal specificities, we next examined the distribution of integrin α-subunit(s) at tnc boutons. We found that αPS2 but not αPS1 levels were selectively reduced at tnc mutant NMJs (Figure 4I–K and Figure 4—figure supplement 1A–B), consistent with the RGD-containing Tnc being a ligand for αPS2/βPS (Fraichard et al., 2010). In addition, the levels of phosphorylated Fak were normal at tnc mutant NMJs (Figure 4—figure supplement 1C–D) suggesting that Tnc does not influence the LanA-induced αPS3/βν activation of the Fak signaling pathway (Tsai et al., 2008; Tsai et al., 2012). The synaptic abundance of βPS integrin subunit was inversely correlated with the synaptic accumulation of FasII, a homophilic adhesion molecule required for synapse stabilization and growth (Schuster et al., 1996a; Schuster et al., 1996b; Beumer et al., 2002). We found that FasII synaptic levels were increased by 40% (p<0.05, n = 28) in tnc mutants compared with the controls (Figure 4—figure supplement 1E–F). This increase resembles the elevated levels of FasII reported at mys mutant NMJs, and indicates that loss of Tnc recapitulates some of the phenotypes reported for selective mys mutants (Beumer et al., 2002).
Since secreted Tnc accumulates at synaptic terminals, both neuron- and muscle-derived Tnc could potentially recruit βPS. However, we found that tnc knockdown in muscles, but not in neurons, reduced the synaptic βPS levels (Figure 4M–O, quantified in P). In fact, tnc knockdown in neurons induced a significant increase of βPS synaptic levels, suggesting that neuron-derived Tnc limits the accumulation of predominantly postsynaptic βPS. This unexpected result prompted us to examine the distribution of Tnc itself in RNAi experiments (Figure 4—figure supplement 2). Compared to the control (tncRNAi transgene with no driver), tnc knockdown in motor neurons produced a significant increase (by 28%, p=0.0044, n = 27) of synaptic Tnc levels; this result is consistent with the apparent increase in Tnc net levels in muscle extracts from N > tncRNAi larvae (Figure 1E, right panel). Thus, neuron-derived Tnc limits the accumulation of muscle-derived Tnc at synaptic terminals. In contrast, tnc knockdown in muscle diminished the synaptic Tnc levels by 37% (p<0.0001, n = 30). This partial reduction may reflect an inefficient RNAi treatment and/or a complementary increase in the neuron-derived Tnc. Nonetheless our data indicate that muscle Tnc is required in cis for the postsynaptic recruitment of βPS and that neuron-derived Tnc limits the accumulation of both Tnc and βPS postsynaptically (see below).
To determine the function of Tnc in the muscle we first tested whether Tnc influences the assembly and organization of postsynaptic iGluR fields by examining the levels and distribution of various postsynaptic components. Drosophila NMJ utilizes two types of iGluRs, type-A and -B, which require the essential auxiliary protein Neto for their distribution and function. Lack of Tnc did not alter the intensities of GluRIIA and GluRIIB synaptic signals or the IIA/IIB ratio (Figure 5—figure supplement 1). This result is consistent with the normal mini amplitude observed at tnc mutant NMJs (Figure 2). Neto itself appeared properly recruited at Tnc-depleted synapses (Figure 5—figure supplement 1). In addition to iGluRs, Neto is critical for the recruitment of p21-activated kinase, PAK, a postsynaptic protein that stabilizes type-A receptors at PSDs (Ramos et al., 2015). We found that PAK signals are normal at Tnc-deprived NMJs, even though the βPS levels are reduced (Figure 5—figure supplement 1). This suggests that Neto controls PAK recruitment at synaptic terminals; alternatively, a very low level of βPS may suffice in recruiting/stabilizing PAK at synaptic terminals.
In the course of these experiments, we noted that tnc mutant NMJs have smaller boutons and often poorly defined bouton/interbouton boundaries. To characterize these defects, we first examined the distribution of HRP-marked neuronal membranes and Discs large (Dlg), a PDZ (PSD-95/Dlg/Zona occludens-1) domain-containing scaffolding protein (Budnik et al., 1996). Dlg localizes perisynaptically to the subsynaptic reticulum (SSR), a stack of membrane folds that surrounds the type I boutons (Guan et al., 1996). In the absence of Tnc, the type Ib boutons appeared significantly smaller and had diminished Dlg signals (Figure 5A–B, quantified in F-G). tnc knockdown in neurons induced a slight increase in the Dlg signals and no change in the bouton area, whereas tnc knockdown in muscles significantly decreased both the Dlg synaptic levels and the size of the type Ib boutons (Figure 5C–E). Similar reduction in bouton size has been reported for selective mys mutants (Beumer et al., 1999), suggesting that postsynaptic Tnc/integrin complexes control bouton size.
In electron micrographs, type Ib control boutons are surrounded by a thick SSR (Figure 5H, quantified in 5I); presynaptic T-bars and electron-dense membranes mark individual synapses. The synapses appeared to have normal organization at tnc mutant boutons, with electron-dense synaptic membranes separated by a dense synaptic cleft (Figure 5J). However, the mutant boutons had irregular shapes and were surrounded by sparse SSR, with wider spaces between the membrane layers. These ultrastructural defects are consistent with the observed morphological phenotypes and indicate that muscle Tnc regulates the SSR thickness and bouton architecture.
The tnc phenotypes may reflect increased adhesion due to elevated FasII levels (Figure 4—figure supplement 1). Alternatively, bouton architecture could be disrupted when muscles contract in the absence of a properly reinforced synaptic skeleton, including the microtubule-based and the cortical membrane skeleton. We found no microtubule defects at tnc mutant NMJs (Figure 6A–B). At the presynaptic arbor, the mature microtubule bundles that traverse the NMJ branches can be visualized with antibodies against the microtubule-associated protein Futsch. These microtubules remained organized in smooth sheaths and loops in tnc mutant larvae, similar to the control.
In contrast, loss of tnc severely disrupted the α-Spectrin accumulation at the NMJs (Figure 6C–F). The net levels of α-Spectrin were normal in extracts from tnc larval muscles, as determined by Western blot analysis, but the synaptic abundance of α-Spectrin at tnc NMJs was reduced to 37% from control levels (p<0.001, n = 21). Since α-Spectrin is essential for the integrity of the SSR (Pielage et al., 2006), loss of synaptic α-Spectrin is consistent with the sparse SSR observed at tnc mutant NMJs. Moreover, α- and β-spectrin mutant embryos have reduced neurotransmitter release and diminished EJPs amplitudes without any apparent defects in postsynaptic receptor fields (Featherstone et al., 2001); these defects are reminiscent of Tnc-deprived NMJs. Interestingly, the distorted shapes of tnc mutant boutons resemble the less individuated boutons seen in α-spectrinR22S larvae, which are impaired for spectrin tetramerization (Khanna et al., 2015). Tetramerization is required for formation of the spectrin-based membrane skeleton (SBMS) but is not required for viability in Drosophila, whereas spectrins are essential genes (Lee et al., 1993; Pielage et al., 2005; Pielage et al., 2006). A key protein involved in the organization of SBMS is Adducin (Bennett and Baines, 2001). Drosophila adducin gene encodes several isoforms, all but one detectable with the anti-Adducin antibody 1B1 (Wang et al., 2014). Using this antibody, we found that Adducin is also diminished at tnc mutant terminals (Figure 6G–I). These data suggest that Tnc is required for the proper recruitment of SBMS at the NMJ.
Similar to βPS, the accumulation of α-Spectrin at synaptic terminals appeared to be limited by neuronal Tnc and promoted by muscle-derived Tnc, as indicated by knockdown analyses (Figure 6—figure supplement 1A–D). Moreover, the changes in synaptic α-Spectrin levels followed the variations in integrin levels at synaptic terminals, since manipulations of mys/(βPS) elicited a similar profile for the synaptic α-Spectrin (Figure 6—figure supplement 1E–H). Together our data suggest that the postsynaptic Tnc/integrin complexes function to recruit the SBMS at synaptic terminals; this activity seems restricted by neuron-derived Tnc, which appears to limit the accumulation and function of postsynaptic complexes. Complete loss of Tnc triggers severe reduction of integrin and α-Spectrin at synaptic terminals, consistent with the observed disruption of bouton architecture.
Since neuron-derived Tnc recruits βPS integrin in the motor neurons to modulate neurotransmitter release (Figure 3), we next examined whether muscle Tnc functions similarly in cis to recruit postsynaptic integrin and SBMS and ensure bouton integrity. For this rescue experiment we used a wide range of Tnc levels and monitored both βPS accumulation and postsynaptic function (Ib bouton area) (Figure 7A–G). Paneuronal expression of Tnc rescued the postsynaptic βPS accumulation at tnc mutant NMJs; however, these animals had small, tnc-like boutons. Thus, neuron-derived Tnc can engage postsynaptic βPS to form non-productive complexes that cannot restore the bouton architecture. When low levels of Tnc were provided in the muscle using a weak promoter and low rearing temperatures (Figure 7D), βPS accumulation was rescued to levels exceeding the control and bouton size was fully restored. In contrast, high levels of Tnc induced massive lethality and exacerbated the loss of βPS at tnc mutant NMJs; these larvae had poorly defined boutons, with almost tubular NMJ branches (Figure 7E). Together these results indicate that only low levels of muscle Tnc could rescue the distribution and function of postsynaptic Tnc/integrin complexes, while excess muscle Tnc is toxic. On the other hand, neuron-derived Tnc can recruit and/or stabilize integrin but cannot form fully functional complexes. In support of this interpretation, we found that only low levels of muscle Tnc could fully restore the accumulation of α-Spectrin at tnc mutant NMJs (Figure 7H–M); neuronal Tnc induced only a modest increase in synaptic α-Spectrin levels.
The fact that the trans Tnc/integrin complexes cannot rescue the bouton size suggests that the cis and trans complexes have different activities. This could be due to different processing or post-translational modifications of Tnc in motor neurons vs. muscles, which have been reported to modulate the activity of ligand-integrin complexes (Reichardt and Tomaselli, 1991). Our Western blot analyses did not provide clear evidence for different post-translational modifications for neuron- and muscle-derived Tnc, although the large Tnc-specific band appeared to include multiple species only when Tnc was overexpressed in the muscle (Figure 7—figure supplement 1). Alternatively, neuron- and muscle-derived Tnc may be packaged differently and/or associate with molecules that influence their activities. Indeed, Tnc has multiple vWFC domains and RGD-like motifs that could enable a large repertoire of protein interactions.
Our rescue results may also reflect different distributions for neuron- and muscle-derived Tnc: While neuron-secreted Tnc accumulates at synaptic terminals, muscle-secreted Tnc likely distributes throughout the muscle membrane and may sequester integrin away from NMJ locations, further reducing the βPS accumulation at synaptic terminals. To test this possibility, we overexpressed Tnc in an otherwise wild-type genetic background and examined the βPS signals at perisynaptic vs. muscle attachment sites. In these experiments we observed no changes in βPS levels and distribution at muscle attachment sites or costameres (not shown). However, βPS recruitment at larval NMJ was drastically reduced when Tnc was overexpressed in either neurons or muscles (Figure 8A–E). Neuronal excess of Tnc produced a reduction of βPS synaptic levels (to 54% from control, p=0.0002, n = 23), and activities, as reflected by the reduction of bouton size compared with the control (transgene only) (Figure 8F). Excess Tnc in the muscles practically abolished the perisynaptic βPS signals. With strong muscle drivers (24B-Gal4 in Figure 8D, or G14-Gal4 in Figure 8—figure supplement 1), these larvae showed ribbon-like NMJs with no interbouton/bouton delimitations exceeding the severity of defects observed at tnc mutant NMJs (not shown). A significant number of these animals died during larval and pupal stages and only 46% (n = 211) of third instar larvae developed into adult flies.
The toxicity of excess Tnc during development prompted us to examine the distribution of βPS after a short (8 hr) pulse of Tnc expression in the muscle, using BG487-Gal4 to induce moderate, gradient muscle expression (Figure 8G–H’). This pulse triggered an increase in Tnc immunoreactivities, which distributed diffusely around the synaptic terminals. Importantly the βPS signals were also drastically diminished, particularly at postsynaptic locations. The βPS signals were no longer concentrated around the synaptic boutons, and instead appeared as thin lines inside the boutons, along the HRP-marked neuronal membrane. Thus, it appears that excess Tnc in the muscle gradually disrupted the postsynaptic βPS accumulation, revealing a small but clear pool of presynaptic βPS; further Tnc overexpression in the muscle completely disrupted both the Tnc and βPS synaptic accumulation and altered the boutons morphology. This dose dependent depletion of Tnc and βPS synaptic signals indicates that excess Tnc may form large aggregates that may be physically excluded from the intercellular space and dispersed away from the synaptic terminals. Alternatively, excess Tnc may trap integrin in the secretory compartment and/or overload a limiting step for the synaptic targeting and recruitment of Tnc/integrin complexes. We favor the former possibility because (i) Tnc itself diffused away from the synaptic terminal when in mild excess, (ii) Tnc has been previously implicated in the formation of large aggregates that fill the lumen of epithelial organs (Syed et al., 2012), and (iii) simple disruption of Tnc trafficking and targeting within the muscle cannot explain the loss of presynaptic integrin accumulation and function (Figure 8D–E and not shown).
To our knowledge this is the first example where genetic manipulations completely abolished the synaptic accumulation of βPS integrin. These larvae also exhibited drastically reduced levels of synaptic α-Spectrin and Adducin (Figure 8—figure supplement 1). Together these results indicate that optimal levels of secreted Tnc are required for proper βPS accumulation at the NMJ, which ensures normal NMJ morphology and function. These experiments also uncovered a novel function for βPS integrin in anchoring α-Spectrin at synaptic locations and coupling the ECM of the synaptic cleft (Tnc) with the spectrin-based membrane skeleton.
To examine whether Tnc directed the recruitment of integrin and spectrin complexes at the cell membrane, we took advantage of our ability to produce full-length Tnc and Tnc-HA proteins in S2 and S2R + insect cells. Secreted Tnc-HA was concentrated from S2 conditioned media, affinity coupled to Neutravidin beads of 1 μm diameter, then presented to S2R + cells, which express relatively high levels of integrin and spectrin. Unlike control beads (coated with an unrelated HA-tagged protein), Tnc-HA-coupled beads induced a local accumulation of βPS at the periphery of S2R + cells, in the close proximity of the beads (Figure 9A–B). This local recruitment of βPS was not caused by mechanical stress, since control beads, either alone or in clusters, did not trigger βPS accumulation. The βPS recruitment was dose-dependent, as beads with variable Tnc-HA levels elicited proportional βPS accumulation (not shown). The Tnc-coupled beads triggered local recruitment of αPS2 but not αPS1 integrin subunit at the surface of S2R+ cells (Figure 9C–D, and not shown). This is consistent with our NMJ observations (Figure 4) and with previous reports on small Tnc fragments mediating αPS2/βPS-dependent spreading of S2 cells via RGD and RGD-like motifs (Fraichard et al., 2010). Moreover, the Tnc-HA-coupled beads induced similar accumulation of α-Spectrin and Adducin at the bead-cell membrane interfaces (Figure 9E–J). Thus Tnc triggers the local recruitment of α-Spectrin and Adducin at the cell membrane. Whether this recruitment is mediated either directly by integrin or indirectly, via other integrin activated scaffolds, remains to be determined. Our attempts to knockdown βPS in S2R + cells, and establish a requirement for integrin for Tnc-induced α-Spectrin recruitment, were hampered by an incomplete RNAi knockdown. Nonetheless, our data establish that Tnc provides a powerful means to recruit and/or stabilize integrin and cortical skeleton components at synaptic terminals.
Although extracellular Tnc binds integrin on cell surfaces, our genetic analyses indicate that in vivo Tnc functions in cis. This suggests that Tnc positively affects the surface delivery and/or stabilization of βPS integrin. We tested this possibility by following Tnc and βPS levels and distribution in S2R + cells transiently transfected with Tnc (Figure 9—figure supplement 1). In this system, Tnc or Tnc-HA were efficiently secreted and accumulated in the media. Inside the cells, Tnc marked large aggregates, likely corresponding to secretory compartments. We found that βPS levels were significantly elevated in cells expressing Tnc; also, βPS co-localized with Tnc in large intracellular aggregates, suggesting that (a) Tnc promotes βPS secretion, and that (b) βPS and Tnc likely associate during intracellular trafficking. It is also possible that excess Tnc may trap βPS inside the secretory compartment, although Tnc appeared to be efficiently secreted and βPS levels were elevated throughout the Tnc-positive cells. Binding to Tnc may stabilize βPS at the cell surface through clustering of integrin complexes and/or by conformational changes and activation of integrins (Liddington and Ginsberg, 2002). In both cases, Tnc binding could reduce the rate of βPS integrin endocytosis as well as overall turnover (López-Ceballos et al., 2016), which may explain the elevated levels of βPS observed in Tnc-expressing cells.
The ECM proteins and their receptors have been implicated in NMJ development, but their specific roles have been difficult to assess because of their early development functions and the complexity of membrane interactions they engage. In this study, we have shown that Tnc is a selective integrin ligand that enables distinct pre- and post-synaptic integrin activities mediated at least in part through the local engagement of the spectrin-based cortical skeleton. First, Tnc depletion altered NMJ development and function and correlated with selective disruption of αPS2/βPS integrin and spectrin accumulation at synaptic terminals. Second, manipulation of Tnc and integrin in neurons demonstrated that presynaptic Tnc/integrin modulate neurotransmitter release (Figure 3); spectrin mutations showed similar disruptions of the presynaptic neurotransmitter release (Featherstone et al., 2001). Third, postsynaptic Tnc influenced the development of postsynaptic structures (bouton size and SSR complexity), similar to integrin and spectrin (Figures 3 and 4 and [Beumer et al., 1999; Pielage et al., 2006]). Fourth, presynaptic Tnc/integrin limited the accumulation and function of postsynaptic Tnc/integrin complexes (Figures 4, 5, 7 and 8). Fifth, secreted Tnc bound integrin complexes at cell membranes, but only the cis complexes were biologically active (Figures 7 and 9); trans Tnc/integrin complexes can form but cannot function at synaptic terminals and instead exhibited dominant-negative activities (Figures 7 and 8). These observations support our model that Tnc is a tightly regulated component of the synaptic ECM that functions in cis to recruit αPS2/βPS integrin and the spectrin-based membrane skeleton at synaptic terminals and together modulate the NMJ development and function.
Tnc appears to fulfill unique, complementary functions with the other known synaptic ECM proteins at the Drosophila NMJ. Unlike Mtg, which organizes the active zone matrix and the postsynaptic domains (Rohrbough et al., 2007; Rushton et al., 2009), Tnc does not influence the recruitment of iGluRs and other PSD components. LanA ensures a proper adhesion between the motor neuron terminal and muscle (Koper et al., 2012) and also acts retrogradely to suppress the crawling activity-dependent NMJ growth (Tsai et al., 2012). The latter function requires the presynaptic βν integrin subunit and phosphorylation of Fak56 via a pathway that appears to be completely independent of Tnc. Several more classes of trans-synaptic adhesion molecules have been implicated in either the formation of normal size synapses, for example Neurexin/Neuroligin, or in bridging the pre- and post-synaptic microtubule-based cytoskeleton, such as Teneurins (Banovic et al., 2010; Mosca et al., 2012). However, genetic manipulation of Tnc did not perturb synapse assembly or microtubule organization, indicating that Tnc functions independently from these adhesion molecules. Instead, Tnc appears to promote expression and stabilization of αPS2/βPS complexes, which in turn engage the spectrin-based membrane skeleton (SBMS) at synaptic terminals. On the presynaptic side these complexes modulate neurotransmitter release. On the postsynaptic side, the Tnc-mediated integrin and spectrin recruitment modulates bouton morphology. A similar role for integrin and spectrin in maintaining tissue architecture has been reported during oogenesis; egg chambers with follicle cells mutant for either integrin or spectrin produce rounder eggs (Bateman et al., 2001; Ng et al., 2016).
Our data are consistent with a local function for the Tnc/βPS-recruited SBMS at synaptic terminals; this is distinct from the role of spectrin in endomembrane trafficking and synapse organization (Kizhatil et al., 2007; Lorenzo et al., 2010; Tjota et al., 2011). Embryos mutant for spectrins have reduced neurotransmitter release (Featherstone et al., 2001), a phenotype shared by larvae lacking presynaptic Tnc or βPS integrin (Figures 3 and 4). However, Tnc perturbations did not induce synapse retraction and axonal transport defects as seen in larvae with paneuronal α- or β- spectrin knockdown (not shown) (Pielage et al., 2005). Spectrins interact with ankyrins and form a lattice-like structure lining neuronal membranes in axonal and interbouton regions (Koch et al., 2008; Pielage et al., 2008; Goellner and Aberle, 2012). We found that Tnc manipulations did not affect the distribution of Ankyrin two isoforms (Ank2-L and Ank2-XL) in axons or at the NMJ (not shown); also loss of ankyrins generally induces boutons swelling, whereas Tnc perturbations shrink the boutons and erode bouton-interbouton boundaries. Like tnc, loss of spectrins in the striated muscle shows severe defects in SSR structure ([Pielage et al., 2006] and Figure 5). Lack of spectrins also disrupts synapse assembly and the recruitment of glutamate receptors (Pielage et al., 2005; Pielage et al., 2006). In contrast, manipulations of tnc had no effect on PSD size and composition (Figure 5—figure supplement 1). Instead, tnc perturbations in the muscle led to boutons with altered size and individualization and resembled the morphological defects seen in spectrin tetramerization mutants, spectrinR22S (Khanna et al., 2015). spectrinR22S mutants have more subtle defects than tnc, probably because spectrin is properly recruited at NMJs but fails to crosslink and form a cortical network. Spectrins are also recruited to synaptic locations by Teneurins, a pair of transmembrane molecule that form trans-synaptic bridges and influence NMJ organization and function (Mosca et al., 2012). Drosophila Ten-m has an RGD motif; we found that βPS levels were decreased by 35% at ten-mMB mutant NMJs (not shown). Thus, Ten-m may also contribute to the recruitment of integrin and SBMS at the NMJ, a function likely obscured by the predominant role both play in cytoskeleton organization.
We have previously reported that α-Spectrin is severely disrupted at NMJs with suboptimal levels of Neto, such as neto109- a hypomorph with 50% lethality (Kim et al., 2012). These mutants also had sparse SSR, reduced neurotransmitter release, as well as reduced levels of synaptic βPS ([Kim et al., 2012] and not shown). In this genetic background, lowering the dose of tnc should further decrease the capacity to accumulate integrin and spectrin at synaptic terminals and enhance the lethality. This may explain the increased synthetic lethality detected in our genetic screen.
In flies or vertebrates, the ECM proteins that comprise the synaptic cleft at the NMJ are not fully present when motor neurons first arrive at target muscles. Shortly thereafter, the neurons, muscles and glia begin to synthesize, secrete and deposit ECM proteins. At the vertebrate NMJ, deposition of the ECM proteins forms a synaptic basal lamina that surrounds each skeletal myofiber and creates a ~ 50 nm synaptic cleft. In flies, basal membrane contacts the motor terminal in late embryos, but is some distance away from the synaptic boutons during larval stages (Prokop et al., 1998). Nonetheless, the NMJ must withstand the mechanical tensions produced by muscle contractions. Our data suggest that Tnc is an ideal candidate to perform the space filling, pressure inducing functions required to engage integrin (Pines et al., 2012) and establish a dynamic ECM-cell membrane network at synaptic terminals. First, Tnc is a large mucin with extended PTS domains that become highly O-glycosylated, bind water and form gel-like complexes that can extend and induce effects similar to hydrostatic pressure (Syed et al., 2008). In fact, Tnc fills the lumen of several epithelial tubes and forms a dense matrix that acts in a dose-dependent manner to drive diameter growth. Second, the RGD and RGD-like motifs of Tnc have been directly implicated in αPS2/βPS-dependent spreading of S2 cells (Fraichard et al., 2010). Third, secreted Tnc appears to act close to the source (Syed et al., 2012), presumably because of its size and multiple interactions. In addition to the RGD motifs, Tnc also contains five complete and one partial vWFC domains, that mediate protein interactions and oligomerization in several ECM proteins including mucins, collagens, and thrombospondins (Bork, 1991). The vWFC domains are also found in growth factor binding proteins and signaling modulators such as Crossveinless-2 and Kielin/Chordin (Lin et al., 2005; Wharton and Serpe, 2013) suggesting that Tnc could also influence the availability of extracellular signals. Importantly, Tnc expression is hormonally regulated during development by ecdysone (Fraichard et al., 2010). Tnc does not influence integrin responsiveness to axon guidance cues during late embryogenesis; unlike integrins, the tnc mutant embryos have normal longitudinal axon tracks (Figure 1—figure supplement 1 and [Stevens and Jacobs, 2002]). Instead, Tnc synthesis and secretion coincide with the NMJ expansion and formation of new bouton structures during larval stages. Recent studies have reported several mucin-type O-glycosyltransferases that modulate integrin signaling and intercellular adhesion in neuronal and non-neuronal tissues, including the Drosophila NMJ (Tran and Ten Hagen, 2013; Dani et al., 2014). Tnc is likely a substrate for these enzymes that may further regulate Tnc activities.
In flies as in vertebrates, integrins play essential roles in almost all aspects of synaptic development. Early in development, integrins have been implicated in axonal outgrowth, pathfinding and growth cone target selection (Myers et al., 2011). In adult flies, loss of αPS3 integrin activity is associated with the impairment of short-term olfactory memory (Grotewiel et al., 1998). In vertebrates, integrin mediates structural changes involving actin polymerization and spine enlargement to accommodate new AMPAR during LTP, and ‘lock in’ these morphological changes conferring longevity for LTP (McGeachie et al., 2011). Thus far, integrin functions at synapses have been derived from compound phenotypes elicited by use of integrin mutants, RGD peptides, or enzymes that modify multiple ECM molecules (Rohrbough et al., 2000; McGeachie et al., 2011; Dani et al., 2014). Such studies have been complicated by multiple targets for modifying enzymes and RGD peptides and by the essential functions of integrin in cell adhesion and tissue development.
In contrast, manipulations of Tnc, which affects the selective recruitment of αPS2/βPS integrin at synaptic terminals, have uncovered novel functions for integrin and clarified previous proposals. We demonstrate that βPS integrin is dispensable for the recruitment of iGluRs at synaptic sites and for PSD maintenance. We reveal an unprecedented role for integrin in connecting the ECM of the synaptic cleft with spectrin, in particular to the spectrin-based membrane skeleton. These Tnc/integrin/spectrin complexes are crucial for the integrity and function of synaptic structures. Our studies uncover the ECM component Tnc as a novel modulator for NMJ development and function; these studies also illustrate how manipulation of a selective integrin ligand could be utilized to reveal novel integrin functions and parse the many roles of integrins at synaptic junctions.
To generate the tnc82 allele, transposons PBac[RB]e01245 and PBac[WH]f00323 were mobilized by FRT-induced recombination as previously described (Parks et al., 2004; Thibault et al., 2004). The UAS-tnc lines were generated by insertion of the tnc cDNA in pUAST vector and germline transformation (BestGene, Inc.). The following fly lines were obtained from the Bloomington Stock Center: the P-element lines tncEP (P[EPgy2]EY03355) and ten-m mutant Mi[ET1]Ten-mMB10734 (Bellen et al., 2004); the deficiency lines Df(3R)BSC-318,,–492, −494, and −655 (Parks et al., 2004); and the TRIP lines UAS-tncRNAi (P[TRIP.HMC05051] attP40), and UAS-mysRNAi (P[TRIP.JF02819] attP2), and UAS-if RNAi (P[TRiP.HMS01872]attP40). Additional tnc, mys and if RNAi lines showed similar phenotypes, but were not reported here because of mild effects (such as for UAS-tncRNAi line from Vienna Drosophila RNAi Center, P[GD14952]v42326), or aberrant muscle development (in the case of mys and if knockdown). Other fly stocks used in this study were previously described: neto109 (Kim et al., 2012); UAS-Dlg (Budnik et al., 1996); 24B-Gal4, elav-Gal4, G14-Gal4, BG487-Gal4.
Full-length cDNA for tnc was assembled in pRM-Tlr plasmid (Serpe and O'Connor, 2006) from the following elements: (1) a synthetic PinAI/SalI fragment joining the Tolloid-related signal peptide with the 5’ of tnc CDS; (2) SalI 7 kb fragment isolated from BACPAC CH322-177A22 (Venken et al., 2009); (3) SalI/XhoI PCR product covering the 3’ of tnc CDS. We used this plasmid to introduce two HA tags in two steps: (1) replace the SalI 7 kb fragment with a pair of primers that introduced 2xHA tags separated by a SalI site; (2) reintroduce SalI kb fragment. The primers used here were:
For-5’-Phos- TCGAGCTATCCCTATGACGTCCCGGACTATGCACAGTCGACTACCCGTACGATGTGCCCGATTACGCAC and Rev: 5’-Phos-
TCGAGTGCGTAATCGGGCACATCGTACGGGTAGTCGACTGTGCATAGTCCGGGACGTCATAGGGATAGC. All constructs were verified by DNA sequencing. UAS-tnc lines were generated by insertion of the tnc cDNA in pUAST and germline transformation (BestGene, Inc.).
To measure the climbing ability, a custom device to fractionate populations based on negative geotaxis was used (Benzer, 1967). The flies were placed in the first tube (numbered 0) and left to recover for 2 hr. The fractionation consisted of sequential cycles of tapping down the flies, and moving the flies that climbed above the threshold in 15 s to the next tube. The climbing index was calculated by the formula (1xN1 + 2xN2 + 3xN3 + 4xN4 + 5xN5)/N, where Nr is the number of flies in fraction r, and N is the total number of flies.
Drosophila S2 and S2R+ cells were used for expression of recombinant proteins and immunohistochemistry as described previously (Serpe et al., 2008). For protein analysis, wandering third instar larvae were dissected, and brains or the body wall (muscle and cuticle) were isolated. The tissues were mechanically disrupted and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, protease inhibitor cocktail (Roche) for 30 min on ice. The lysates were separated by SDS-PAGE on 4–12% NuPAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). The rabbit polyclonal anti-Tnc antibodies were generated as previously described (Fraichard et al., 2006) against a synthetic peptide (APVQEYTEIQQYSEGC) (Pacific Immunology Corp.) and affinity purified. Primary antibodies were used at the following dilutions: rabbit anti-Tnc 1:1,000; anti-Tubulin (Sigma-Aldrich), 1:1000; mouse anti-α-Spectrin (3A9), 1:50. Immune complexes were visualized using secondary antibodies coupled with IR-Dye 700 or IR-Dye 800 followed by scanning with the Odyssey infrared imaging system (LI-COR® Biosciences).
Neutravidin beads of 1 μm diameter (Life Technologies) were washed in PBS and preloaded for 30 min at room temperature (RT) with biotin conjugated anti-HA antibody (clone 3F10, Roche). An unrelated HA-tagged protein, Tolloid (Tld), was used as a control (Serpe and O'Connor, 2006). Tld-HA and Tnc-HA were concentrated from S2 conditioned media using Amicon Ultra filters (10 kDa) and bound to the preloaded beads overnight. The beads were washed with PBS and presented for 1.5 hr to S2R + cells cultured on chambered coverglass (Thermo Fisher) at 26°C. Cells were next fixed with 4% PFA for 20 min and stained using standard procedures as described below.
Wandering third instar larvae were dissected as described previously in ice-cooled Ca2+-free HL-3 solution (Stewart et al., 1994; Budnik et al., 2006). Embryos were collected and fixed using standard procedures (Patel, 1994). Primary antibodies from Developmental Studies Hybridoma Bank were used at the following dilutions: mouse anti-GluRIIA (8B4D2), 1:100; mouse anti-Dlg (4F3), 1:1000; mouse anti-Brp (Nc82), 1:200; mouse anti-α-Spectrin (3A9), 1:50; mouse anti-FasII (1D4), 1:10; mouse anti-Futsch (22C10), 1:100; mouse anti-Adducin (1B1), 1:50; mouse anti-βPS integrin (CF.6G11) 1:10; mouse anti-αPS1 integrin (DK.1A4) 1:10; mouse anti-αPS2 integrin (CF.2C7) 1:10. Other primary antibodies were utilized as follow: rabbit anti-Tnc 1:100; rat anti-HA (3F10) (Lu and Roche, 2012) 1:500; rabbit anti-FAK (phospho Y397) (Abcam, ab39967), 1:100; rabbit anti-GluRIIB, 1:2000; rabbit anti-GluRIIC, 1:1000 (Ramos et al., 2015); rabbit anti-PAK, 1:5000 (a gift from Nicholas Harden) (Conder et al., 2004); 1:1000; rat anti-Neto, 1:1000 (Kim et al., 2012); and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.). Alexa Fluor (AF) 405-, AF488-, AF568-, and AF647- conjugated secondary antibodies (Molecular Probes) were used at 1:200. All samples were mounted in ProLong Gold (Invitrogen).
Samples of different genotypes were processed simultaneously and imaged under identical confocal settings in the same imaging session with a laser scanning confocal microscope (CarlZeiss LSM780). All images were collected as 0.2 μm optical sections and the z-stacks were analyzed with Imaris software (Bitplane). To quantify fluorescence intensities synaptic ROI areas surrounding anti-HRP immunoreactivities were selected and the signals measured individually at NMJs (muscle 4, segment A3) from ten or more different larvae for each genotype (number of samples is indicated in the graph bar). The signal intensities were calculated relative to HRP volume and subsequently normalized to control. Boutons were counted in preparations double labeled with anti-HRP and anti-Dlg. Bouton size was estimated by using the ImageJ software. All quantifications were performed while blinded to genotype. The numbers of samples analyzed are indicated inside the bars. Statistical analyses were performed using the Student t-test with a two-tailed distribution and a two-sample unequal variance. Error bars in all graphs indicate standard deviation ±SEM. ***p<0.001, **p<0.005, *p<0.05, ns- p>0.05.
PLA was performed following published protocols (Wang et al., 2015). In brief, wandering third instar larvae were dissected, fixed and incubated with primary antibodies (anti-Tnc and anti-βPS integrin) overnight at 4°C. To detect the markers, the samples were incubated with AF-conjugated secondary antibodies (anti-rabbit- AF488, anti-mouse-AF405 and anti-HRP-AF647) for 1 hr in the dark. For PLA, the samples were washed with Wash Buffer A (DUO 92101 Kit, Sigma), incubated first with PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS (1:5 dilution) for 2 hr at 37°C, then with 200 μl Ligation solution for 1 hr at 37°C, and finally with Amplification solution for 2 hr at 37°C. After washes in Wash Buffer B, the samples were mounted in ProLong Gold (Invitrogen) and examined by confocal imaging.
The standard larval body wall muscle preparation first developed by Jan and Jan (1976) (Jan and Jan, 1976) was used for electrophysiological recordings (Zhang and Stewart, 2010). Wandering third instar larvae were dissected in physiological saline HL-3 saline (Stewart et al., 1994), washed, and immersed in HL-3 containing 0.5 or 0.8 mM Ca2+ using a custom microscope stage system (Ide, 2013). The nerve roots were cut near the exiting site of the ventral nerve cord so that the motor nerve could be picked up by a suction electrode. Intracellular recordings were made from muscle 6, abdominal segment 3 and 4. Data were used when the input resistance of the muscle was >5 MΩ and the resting membrane potential was between −60 mV and −70 mV. The input resistance of the recording microelectrode (backfilled with 3 M KCl) ranged from 20 to 25 MΩ. Muscle synaptic potentials were recorded using Multiclamp 700B amplifiers (Molecular Devices) and Axon Clamp 2B amplifier (Axon Instruments) and analyzed using pClamp 10 software. Following motor nerve stimulation with a suction electrode (200 μsec, 1.9 V), evoked EJPs were recorded. Four to six EJPs evoked by low frequency of stimulation (0.1 Hz) were averaged. For mini recordings, TTX (1 μM) was added to prevent evoked release (Stewart et al., 1994). To calculate mEJP mean amplitudes, 100 events from each 10 or more NMJs (only one NMJ per animal was used) were measured and averaged using the Mini Analysis program (Synaptosoft). Minis with a slow rise and falling time arising from neighboring electrically coupled muscle cells were excluded from analysis (Gho, 1994; Zhang et al., 1998). Paired stimuli (200 μsec, 1.9 V, 0.05 Hz) were applied with a suction electrode at 50 ms inter-stimulus intervals. The amplitude of the eEJP was determined as an average from 5 to 8 steady consecutive sweeps. The paired-pulse ratio (PPR) was expressed as the amplitude ratio of the second synaptic response to the first synaptic response (Zhang and Stewart, 2010). Quantal content was calculated by dividing the mean EJP by the mean mEJP after correction of EJP amplitude for nonlinear summation according to previously described methods (Stevens, 1976; Feeney et al., 1998). Corrected EJP amplitude = E[Ln[E/(E - recorded EJP)]], where E is the difference between reversal potential and resting potential. The reversal potential used in this correction was 0 mV (Feeney et al., 1998; Lagow et al., 2007). Statistical analysis was performed with KaleidaGraph 4.5 (Synergy Software) using ANOVA followed by a Tukey post hoc test. Differences were considered significant at p<0.05. Data are presented as mean ±SEM.
Wandering third instar larvae were dissected in physiological saline HL-3 saline and fixed for 30 min on dissection plate in fixation buffer (0.1 M Sodium Cacodylate buffer, pH7.2; 2 mM MgCl2; 1% glutaraldehyde; 4% paraformaldehyde). The samples were trimmed to include only the abdominal segments A2 and A3, transferred in a 1.5 mL Eppendorf tube, fixed overnight at 4 ˚C, then washed extensively with 0.1 M Sodium Cacodylate buffer with 132 mM Sucrose, pH 7.2. The samples were further processed and analyzed according to published protocols (Ramachandran and Budnik, 2010) at the Microscopy and Imaging Facility, NICHD.
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Talila VolkReviewing Editor; Weizmann Institute of Science, Israel
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Tenectin recruits integrin to stabilize boutons and regulate vesicle release at the Drosophila neuromuscular junction" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andreas Prokop (Reviewer #2).
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
All three reviewers (see below their comments) expressed major criticism regarding the manuscript conclusions. The major comment is that the results are confusing and some time contradicting regarding the muscle, versus neuron-dependent function of Tenectin in recruiting integrins to the NMJ. In addition a direct versus indirect effect of Tenectin on the levels of Spectrins at the NMJ should be further validated. Many controls are also missing.
We hope you find our comments useful. If you address these comments and would like to make a fresh submission at eLife, rather than elsewhere, we will be happy to examine this afresh.
This paper describes the expression and function of Tenectin at the larval NMJ. While there is a lot of information in the paper, it will primarily be of interest to specialists in fly neuromuscular system development.
There are also some problematical aspects to the data, as follows:
1) Maybe I am not understanding something here, but there seems to be an unresolved contradiction about cis vs. trans action of Tnc. They show that muscle, but not neuronal Tnc knockdown reduces βPS expression (Figure 4M) and from this (subsection “Muscle Tnc recruits postsynaptic integrin”, second paragraph) they conclude that it muscle Tnc acts in cis to recruit βPS, which is primarily expressed in muscles.
However, in Figure 6 they show that in a tnc mutant the loss of expression of βPS is restored by neuronal expression of Tnc, indicating that it is acting in trans (Figure 6D). When they express Tnc in muscles they lose βPS expression. The results in Figure 4 would predict that neuronal expression of Tnc in the mutant should not rescue, since neuronal knockdown does not affect βPS, and that muscle expression of Tnc should rescue, since muscle knockdown decreases βPS. Perhaps this is a Tnc overexpression phenotype. To test this, they should use weaker muscle drivers and reduce the temperature to reduce GAL4 activity, or make a direct muscle promoter>Tnc fusion. Unless I am missing something here, if they can express Tnc at physiological levels in the muscles in a tnc mutant, this should rescue βPS expression, not eliminate it.
2) The data in Figure 8 are not convincing. Looking at the high-mag views of the HA beads and placing them into the low-mag views of the cells, one can see that there is no accumulation of βPS, αPS2, or spectrin around the beads relative to their levels elsewhere in the cells. In fact the beads are in low-expression areas for all of these markers. Given this, one cannot believe that the levels of these markers are lower around the control beads. No high-mag views of the control beads are shown, and you can't even see these beads in the fluorescent images since they are not labeled. To do this experiment correctly, they need to use a different HA-tagged protein on the control beads, so that they can be seen in the fluorescent images, and show equivalent high-mag views of the beads so that it can be determined if there is really more integrin around the beads. This should also be quantitated by measuring fluorescence intensity in the bead regions in Tnc-HA vs. Control protein-HA beads.
3) Figure 8—figure supplement 1 is also unconvincing and incomplete. There is clearly a lot of βPS within the cell in Tnc-HA expressing cells, but levels at the cell surface are not clearly increased (at least the images shown do not demonstrate this). They could address this by staining live cells with anti-βPS without detergent. Maybe Tnc-HA is expressed at very high levels and sequesters βPS within a secretory compartment where it cannot be degraded, thus increasing its intracellular levels. This does not show that Tnc-HA acts in cis to increase integrin surface localization.
4) If they want to evaluate whether Tnc-HA induces βPS, they should make a stable S2 line expressing Tnc-HA, which is easily done using stable transformation vectors. If Tnc-HA expression is toxic they can drive it from a conditional promoter. Similarly, they can use stable transfection to knock down βPS with RNAi if the transient RNAi knockdown is inadequate (subsection “Tnc engages integrin and spectrin at the cell membrane”, first paragraph).
This work by Wang and colleagues has some clear merits and high potential, but I do not recommend publication, but rather rejection with the option to re-submit. The main reason for this suggestion is the lack of clarity across the paper how experiments link together into new understanding. In my view there are seemingly contradicting results in this work that suggest a very intricate role and regulation of tnc, but have to be resolved before this manuscript can be considered for publication. The contradictions mainly consist in obscure effects observed upon pre- versus post-synaptic manipulations that require a far more refined experimental approach and logic dissection. Instead, data are being uncritically reported and the reader is left alone with a highly confusing message, if any message at all. There is potential in many of the data, but the authors failed to turn them into a readable and informative manuscript. Apart from these shortcomings there are a number of further issues that need clarification. See my detailed comments below.
The Introduction reads well and provides a sound overview of ECM at mammalian and Drosophila synapses. It would be helpful to mention the obvious gaps of understanding in the field and how this work addresses these gaps. A tip: do the authors know this review: Singhal, N., Martin, P. T. (2011). Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction. Dev Neurobiol 71, 982-1005 – https://doi.org/10.1002/dneu.20953
Introduction or start of Results: It needs to be clearly stated that tnc has no mammalian homologue.
“Using a polyclonal anti-Tnc antibody (Materials and methods) we found that Tnc signals are concentrated during late embryogenesis in the ventral cord, near the pioneer, anti-Fasciclin II (FasII) positive axons (Figure 1—figure supplement 1A, B and (Fraichard et al., 2006)): For this statement, the resolution of analyses is far too low, and I do not think that this statement is important. Delete and stay with the statement that staining it is strongly enriched in the neuropile.
“In contrast, the Tnc CNS signals were absent in a tnc mutant (tncEP – P[EPgy2]EY03355), predicted to disrupt both known tnc transcripts (Syed et al., 2012)”: Point out that Fas2 pattern is not disturbed in the mutant CNS. Obviously, there is not interaction with reported functions of integrins in this context:
Stevens and Jacobs, 2002; Broadie, Baumgartner and Prokop, 2011. Drosophila
Subsection “Tnc localizes at the synaptic cleft”, second paragraph: indicate the two tnc transcripts in Figure 1A.
Subsection “Tnc localizes at the synaptic cleft”, second paragraph and thereafter: the correct term is "hetero-allelic": if tncDf is a true deficiency you need to talk of a "hemizygous" condition.
Subsection “Tnc localizes at the synaptic cleft”, third paragraph”: At this early point, the pre-postsynaptic statement cannot be made. Tnc expression in the neuropile is in patches which might reflect staining around dendrites. Therefore, the expression/localisation in motorneuons may well be postsynaptic (equivalent to muscle expression in the periphery).
Subsection “Tnc localizes at the synaptic cleft”, third paragraph”: Does complete knock-down in the CNS get rid of all expression or is tnc only expressed in motorneurons but not very efficiently knocked down? Along these lines, given potentially expected different driver strengths in CNS and muscles, the conclusions about where tnc is strongest expressed cannot be drawn from the knock-down data.
Subsection “Tnc localizes at the synaptic cleft”, last paragraph”: the staining does not look like being around nuclei, but seems suprisingly nuclear. If it were in Golgi or ER, it would have a broader expression.
Subsection “Tnc localizes at the synaptic cleft”, last paragraph”: the reduced staining is not very convincing. This needs to be quantified.
Subsection “Tnc localizes at the synaptic cleft”, last paragraph: either show and quantify the tnc over-expression data or take them out.
Figure 1—figure supplement 2 is not well described in its legend and is confusing. What nuclei are shown on the right? I guess it is muscle nuclei? But why is there no expression in those when driving tnc with 24B (bottom right image). Please, clarify, because at the moment this does not make sense. Expression data must be quantified across these experiments.
Subsection “tnc mutants have impaired NMJ physiology”, first paragraph: either show larval turning assay results or take the statement out.
Why were white flies chosen for climbing assays? At what age of flies were these experiments performed? An independent experiment with knock-down of tnc would be helpful.
Figure 2: indicate the statistical test used. Indicate in your sample number also how many events were included in the statistics: e.g. 200/13 (200 events from 13 NMJs).
Subsection “tnc mutants have impaired NMJ physiology”, last paragraph: mention that reduced mEJC frequency is also in agreement with reduced quantal content due to less release sites.
Subsection “tnc mutants have impaired NMJ physiology”, last paragraph: "as expected", provide references for this statement.
Subsection “tnc mutants have impaired NMJ physiology”, last paragraph: what interval length was used? This is not indicated in text or figure. Ideally, a plot with increasing length should be shown to illustrate that this is true short-term depression. In the graph indicate that you show the ratio of 1st/2nd pulse
Subsection “tnc mutants have impaired NMJ physiology”, last paragraph: It needs a clear explanation as to why short-term facilitation explains decreased probability. I can come up with my own speculations, but it is the task of the authors to make this clear.
“Both pre- and postsynaptic components modulate neurotransmitter release”: where does this statement come from? It is confusing without further explanation, especially since it is followed by posing this problem and since it opposes the title. Should this be the title of the section?
Subsection “Neuronal Tnc modulates neurotransmitter release”, first paragraph: why "interestingly"? Is this not expected if presynaptic expression leads to release at NMJs and mutants show reduced frequency? Please, build a logic and solid argumentation.
In all graphs, indicate absence of tested significance with a symbol, such as "ns". In this way it is clear which bars were assessed that the asterisks have not simply been forgotten to insert.
Subsection “Neuronal Tnc modulates neurotransmitter release”, last paragraph: the term "recruits" is not supported by the data; at this point you can merely state that tnc seems to act as a ligand for presynaptic integrin.
Figure 3G. I wonder whether only one set of experiments is sufficient here. Reproducing the data through knock-down of talin or inflated would clearly strengthen these observations.
Subsection “Muscle Tnc recruits postsynaptic integrin”, first paragraph: the co-localisation is not convincing and the authors must refine their argumentation. There is too much expression of integrin to make such a statement. Any correlation analyses of tnc would likely show overlap, but there is much expression of integrin that does not correlate. Even more, these data (double-labelling with HRP) clearly confirm previous observations that integrin localises also strongly to SSR, i.e. postsynaptic membrane. Most of the apparent overlap occurs therefore in areas that are irrelevant for the presynaptic function, as demonstrated by lack of effect upon postsynaptic integrin or tnc knock-down.
Figure 4: to address the presynaptic localisation of integrins, it would be very helpful to knock down integrins postsynaptically and see that staining remains in a sharp line only around boutons – which should then vanish in tnc mutant background. Please, combine G and H into one graph (both are normalised anyway)
Subsection “Muscle Tnc recruits postsynaptic integrin”, second paragraph: "secreted into the synaptic cleft"?
Subsection “Muscle Tnc recruits postsynaptic integrin”, second paragraph: loss of βPS upon muscle but not neuronal knock-down of tnc is in stark contrast to data in Figure 1—figure supplement 2 where only neuronal tnc sheds into the synaptic cleft. The stabilisation theory based on ligand binding does therefore not work.
Subsection “Muscle Tnc recruits postsynaptic integrin”, second paragraph: How can the authors suddenly favour postsynaptically derived Tnc, although all their data from before show a physiological relevance only for neuronal Tnc? I am completely lost at this point.
Subsection “Muscle Tnc recruits postsynaptic integrin”, last paragraph: "is required for the recruitment of PAK" – the statement as is, suggests a direct mechanism.
Subsection “Muscle Tnc recruits postsynaptic integrin”: the last sentence is too condensed.
Figure 5: I do not believe the EM data. How many muscles from how many independent animals were analysed? Were larvae processed in the same vial? Image quality is very poor and there is a clear osmolarity effect; from anti-FAK staining one gets the impression that SSR is smaller, yet here it is blown up in mutants. Analyses have to be performed in whole mounts with SSR markers (for example anti-DLG) and EM analyses have to be made more transparent to the reader and properly quantified. The odd shape of boutons looks not more than the section being almost tangential to the bouton. Shape statements are far easier made from whole mount stainings.
Subsection “tnc mutants have disrupted spectrin-based membrane skeleton”, third paragraph: there are no analyses of bouton sizes in the paper, which must be provided for mutant and upon pre- and post-synaptic knockdown of tnc.
Subsection “tnc mutants have disrupted spectrin-based membrane skeleton”: to my knowledge, loss of spectrin causes a cell-autonomous defect in neuronal terminals, i.e. the change in boutons is not caused by loss of spectrin in muscles. This does not fit with the argumentation of the authors.
Subsection “Overexpression of Tnc disrupts postsynaptic βPS integrin and spectrin”: the manuscript gets utterly confusing now, and the authors make no effort to shed light into this. It becomes close to unreadable. There seems to be a constant contradiction between pre- and postsynaptic requirements which are not resolved.
Subsection “Overexpression of Tnc disrupts postsynaptic βPS integrin and spectrin”, second paragraph: please, explain what the point of these experiments is? What do we learn? This is an artificial situation that does not tell us much.
Discussion, first paragraph: integrins are very well known to be locally activated through ligands, so I do not understand this claim.
The manuscript "Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction" represents an interesting investigation of the role of a secreted EMC protein at the NMJ. The manuscript provides novel information on the role of Tnc in recruiting the integrin complex (αPS2/βPS) to the NMJ along with spectrin and adducin. Overall the manuscript presents interesting and novel insights into the role of Tnc and integrin complexes at the NMJ. Much of the data and the quantitation was convincing and supported the conclusions. However there are a number of points that need to be addressed with the explanation of the data, some of the major conclusions drawn, a need for discussion of alternative hypotheses and the confirmation of the protein complex in vivo. These points are outlined below:
For the RNAi experiments only one tncRNAi line and one mysRNAi was used throughout. Neither of these lines have been previously published to be specific to Tnc or mys and thus this either needs to be proven or alternative previously verified lines used to ensure that there are not any off target effects. Specifically the experiments need at least two RNAi lines used for the Tnc and mys knockdown to ensure no off target effects or RNAi lines verified by others to be specific. The mysRNAi for instance is presumably a weaker one given that muscle knockdown of mys if strong would result in embryonic lethality.
In Figure 3 comparing mEJP Frequency for the tncEP/Dfwas less strongly affected compared to the N>tncRNAi in Figure 3E versus 3B. It is surprising that the RNAi would have a strong effect that the EP/Df combination especially given that in Figure 1 the protein levels were substantively reduced in the EP/Df but not the N>tncRNAi. This disconnect between levels of protein versus phenotypes need to be addressed in the text.
With the residual Tnc immunolabeling present in the tncEP/Df, it is logical to conclude that there is significantly more Tnc present in the N>tncRNAi NMJ. However the degree of reduction of Tnc at the NMJ is not shown with either N>tncRNAi or M>tncRNAi. This should be included or if not, explained. This becomes very relevant in Figure 4 as the effect of M>tncRNAi on the recruitment of αPS2βPS to the NMJ had a stronger effect than N>tncRNAi.
The degree of Tnc loss in the tncEP/Df combination in Figure 1G (and Figure 1—figure supplement 1D) was surprisingly not complete given that the allele seems to be a null. The immunolabeling in the image does not match the quantitation by Western seen in Figure 1C. The phrase "much reduced" in tncEP/Df could be quantified using the approaches utilized throughout the manuscript for all the other markers. The text should also address if this is truly the presence of residual Tnc at the NMJ (and the implications of this) or is this background?
The results from this manuscript suggest that αPS2βPS and Tnc may directly interact. The S2 cell experiment was a strong approach to understanding the associations of Tnc, the integrin complex, spectrin and adducin. A stronger and a necessary approach to investigating the association of these proteins was to prove that these occur in vivo using a proximity ligation assay (PLA) to determine where in the NMJ these associations happen. The advantage of this approach is that the tncEP/Df mutant would serve as a good control as would the different RNAi and overexpression approaches. The PLA experiment would also be able to address if the M>tnc expression was able to recruit the αPS2βPS complex to ectopic sites away from the NMJ. This would go a long way to support many of the conclusions and how a clear link at the NMJ between these protein complexes.
Further to this point, the link between integrins to spectrin and adducin is the key point of interest but other than the S2 cells there is little in the way to support a physical link between these components. It is equally possible that the loss of spectrin or adducin itself may lead to the loss of αPS2βPS from the NMJ. What happens to spectrin or adducin when αPS2 is reduced and vice versa?
For Figure 5 it is mentioned that "In the course of these experiments we noted that tnc mutant NMJs showed aberrant morphology with poorly defined bouton/interbouton boundaries and more tubular branches, particularly in the proximal region." Given that this is the first mention of these different phenotypes, an inclusion of some examples is warranted as prior indications from Figure 2—figure supplement 1 was there were little morphological changes.
For the TEM analysis comments such as "However, the mutant boutons were drastically distorted and no longer maintained normal round/oval shapes" and "This phenotype was highly penetrant and affected all type 1B neurons" should be supported by the number of boutons that were analyzed and the number of synapses/larvae analyzed in the text itself.
The distribution of Dlg should be shown (data not shown throughout the manuscript) and the quantitation provided, especially given the effects on adducin on Dlg at the NMJ. This later point should be addressed in the text as well.
The M>tncRNAi data with α-spectrin showed no decrease compared to the UAS-tncRNAi control. While the N>tncRNAishowed an upregulation. Yet the tncEP/Df displayed a reduction in α-spectrin. The manuscript needs to address these differences and provide a model to help guide through the different interpretations of these effects.
The manuscript makes the statement "Since neuron-derived Tnc recruits βPS integrin in the motor neurons to modulate neurotransmitter release (Figure 3…)" – this is a very strong statement given that N>tncRNAishowed a mild increase of the distribution of βPS to the NMJ (Figure 4L, 4N) and the effect on αPS2 is not shown. Figure 3 provides no evidence that the changes observed with N>tncRNAi are due to the loss of βPS. This would require a rescue experiment to provide a direct link.
The interpretations and conclusions for the role of neuronal or muscle driven Tnc need to be tempered. N>tnc rescued the degree of βPS present at the NMJ in the tncEP/Df mutant while M>tnc did not and this lead to the conclusion that neuronal Tnc is key to the recruitment of βPS. Yet N>tnc alone decreases the βPS at the NMJ (Figure 7C, 7E) compared to controls while N>tncRNAi knock down (Figure 4L, 4N) shows slight more βPS at the NMJ compared to control. This suggests that neuronal derived Tnc blocks integrin accumulation at the NMJ. On the muscle side of the equation M>tnc cannot rescue the tncEP/Df which isn't too surprising given that M>tnc removes all the tnc from the NMJ. Thus these experiment don't prove that neuronal derived Tnc is sufficient and necessary to recruit βPS. Especially given that Tnc knockdown in muscles but not in neurons reduced the amount of synaptic βPS.
Along these lines the tnc mutants with muscle overexpressed tnc had a dramatic reduction in βPS as well as the M>tnc in a wild type background. The panels from Figure 1J, Figure 1—figure supplement 1F and Figure 1—figure supplement 2F would suggest that overexpression of tnc may be significantly deleterious to the entire NMJ not just the distribution of βPS.
The authors state that expression of N>tnc in the tncEP/Df mutant was able to rescue the reduction in α-spectrin however the degree of increase in α-spectrin does not appear to be strong. For the quantitation (Figure 6J) it was not clear what was being compared –.…each experimental to control which would suggest that the N>tnc was significantly different from control (and didn't rescue to a great extent) –.…or each experimental to each other which would suggest that all three experimental were significant different from each other which didn't appear to be the case. For this type of multiple experiment analysis it would also be more relevant to carry out a One Way Anova with a post hoc multi-comparison test rather than a students' t-test.
It was also intriguing that the degree of rescue of Adducin and βPS immunolabeling at the NMJ was so much better than that for α-spectrin with the N>tnc. Given the strength of the statements made in the text is it possible the wrong data was included in Figure 6J?
The major conclusion from this section should be tempered to reflect that neuronal expression of Tnc can rescue the tncEP/Df spectrin, βPS and adducin levels. Whether M>Tnc is able or not will require a more measured approach to ensure that muscle expressed Tnc is present at the NMJ, that the muscles themselves are not deleteriously affected. This might be case given the stronger effects on βPS by the increased expression of Tnc in wild type muscles compared to the tncEP/Df muscles. These effects may simply by an increase the deleterious effects of Tnc expression and thus lead to a great disruption of NMJ morphology rather than an specific effect on "ligand redundancy and/or other mechanisms may allow for partial βPS and/or α-Spectrin synaptic accumulation". Would the increased expression of Tnc or any ECM component lead to disruption of muscles as a result from ER stress?
The statement "Unlike βPS, which was completely lost at these NMJs" is not supported by the images presented in Figure 4E, Figure 6B and 6E: βPS is not completely lost at these NMJs. The authors need to be more careful about making this type of absolute statement.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for resubmitting your work entitled "Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila NMJ" for further consideration at eLife. Your revised article has been favorably evaluated by K VijayRaghavan (Senior Editor), a Reviewing Editor, and three reviewers.
The manuscript has been improved and the reviewers were impressed by the quality and amount of data. However, there are some remaining issues that need to be addressed before acceptance, as outlined below:
Two of the reviewers requested to add controls, including additional RNAi lines for tenectin, and for the GAL4 used. This can be done speedily. Also, the genetic interaction between βPS and tenectin mutants would further support the results indicated by reviewer 1. Please see if you can do this.
Below, please find the specific comments of three reviewers.
Wang et al. report an interesting finding on distinct pre-synaptic and post-synaptic roles for tenectin (Tnc), a selective integrin ligand, at the Drosophila neuromuscular junction (NMJ). Tnc in the neuron interacts with βPS/αPS2 integrin to control neurotransmitter release, whereas Tnc in the muscle interacts with post-synaptic αPS2/βPS integrin to regulate bouton morphology. By manipulating Tnc, they uncovered a novel role for integrin in recruiting the spectrin based membrane skeleton at the NMJ.
This paper will be of broad interest to the readership of eLife because it advances our understanding of NMJ development and synaptic physiology, as well as integrin/extracellular matrix biology. Although Tnc has been studied at the Drosophila NMJ, Wang et al. is among the first to reveal that Tnc has differential function in neurons and muscle. The data presented in this paper is thorough, with good controls and overall high-quality figures. However, I have two major concerns that should be addressed before publication. First, the inclusion of only one RNAi line targeting Tnc is insufficient, especially given the fact that the neuronal RNAi line only knocks down Tnc levels to 43% of control. The inclusion of at least two RNAi lines showing the same phenotype would both confirm and strengthen the author's conclusions. This is not a detail and needs to be addressed.
Second, the conclusion that αPS2, βPS, and Tnc function together to modulate neurotransmitter release (Figure 2) should be strengthened by genetic interaction experiments between βPS and Tnc indicating that either a) double mutants do not have a more severe phenotype, or b) trans-heterozygotes show enhancement of phenotype.
Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila NMJ' Wang et al.
In this manuscript the authors identify the mucin class protein tenectin as an ECM component of the Drosophila NMJ synapse from an interaction screen with Neto. The description of tenectin mutants is comprehensive, the technical quality of the data is high and the authors do identify interesting interactions with integrins. However ultimately little new insight is gained into the role of the ECM in synapse development or function beyond the identification of a new component. As such, the manuscript seems better suited to a more specialised neuroscience journal.
The manuscript by Wang et al. reports a novel function of tenectin (tnc) at the neuromuscular junction (NMJ) in Drosophila. The authors have identified Tnc, a mucin-like protein in a genetic screen for regulators of Neto, that the Serpe lab has previously shown to be required at the NMJ. In this elegant study, the authors use a combination of physiology, immunostainings and genetic approaches to probe the pre- and post-synaptic roles of tnc at the NMJ. Their results show that tnc mutants have reduced mini frequency, EJPs and quantal content. Paired pulse facilitation experiments show that tnc mutants have lower release probability. Next, the authors use pre- and post-synaptic specific manipulations (knock-down and rescue) to show that tnc is required in neurons but not muscles for normal neurotransmitter release. They further show that presynaptic RNAi knock-down of α and β integrin genes phenocopy the tnc phenotypes and suggest that tnc may act as an integrin ligand. Next, the authors examine the effects of altering tnc levels pre- and post-synaptically on integrins and synaptic morphology at the NMJ and find that the source and dose of tnc are critical determinants of bouton architecture and synaptic function. S2 cell experiments nicely show the ability of Tnc to cluster integrins as also suggested by the PLA assay.
Overall, this is an interesting study that sheds key novel insights into the role of extracellular matrix ligands and receptors in shaping the NMJ architecture and function. Although in my opinion the spectrin connection does not add much to the story and the precise mechanism remains to be established (it may have to do with post-translational modifications, as suggested) this study is well executed, novel and of broad interest.
My only criticism is the absence of driver controls for phenotypic studies. While the authors use the UAS lines as background, which is fine, it is rather standard and important to control for any potential contributions of the GAL4 drivers to the phenotypes evaluated here.https://doi.org/10.7554/eLife.35518.025
- Qi Wang
- Tae Hee Han
- Peter Nguyen
- Mihaela Serpe
- Qi Wang
- Tae Hee Han
- Peter Nguyen
- Mihaela Serpe
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
This work was supported by the Intramural Research Program at the NICHD, NIH. We thank Bing Zhang, Graham Thomas, Herman Aberle, Kelly Ten Hagen, Ed Giniger, and Alan Hinnebusch for helpful discussions and suggestions. We are grateful to Nicholas Harden for antibodies. We thank Tom Brody, Rosario Vicidomini and Lindsey Friend for comments on this manuscript. Electron microscopy was in part performed at the Microscopy and Imaging Core of NICHD, NIH with the assistance of Chip Dye. We also thank the Bloomington Stock Center at the University of Indiana for fly stocks and the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies.
- Talila Volk, Reviewing Editor, Weizmann Institute of Science, Israel
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.