To pass on information, the neurons that make up the nervous system connect at structures known as synapses. Chemical messengers called neurotransmitters are released from one neuron, and travel across the synapse to trigger a response in the neighbouring cell. The formation of new synapses plays an important role in learning and memory, but many aspects of this process are not well understood.

In a specific region of the synapse called the active zone, a scaffold of proteins helps to release the neurotransmitters. These proteins are made in the cell body of the neuron, and are then transported to the end of the long, thin axons that protrude from the cell body. This presents a challenge for the cell, because the components of the active zone scaffold must be correctly targeted to the synapse at the end of the axon, ensuring the active zone scaffold assembles only at its proper location.

Siebert, Böhme et al. studied how some of the proteins that are found in the active zone scaffold of the fruit fly Drosophila are transported along axons. Labelling the proteins with fluorescent markers allowed their movement to be examined under a microscope in living Drosophila larvae. The results showed that two of the proteins—known as BRP and RBP—are transported along the axons together. Further investigation revealed that a transport adaptor protein called Aplip1, which binds to RBP, is required for this movement. Siebert, Böhme et al. established the structure of the part of RBP where this interaction occurs, and found that mutating this region causes premature active zone scaffold assembly in the axonal part of the neuron. The interaction between RBP and Aplip1 is very strong, and this helps to prevent the scaffold assembling before it has reached the correct part of the neuron. Exactly how the transport adaptor and active zone protein are separated once they reach their final destination (the synapse) remains to be discovered.

The primary function of the presynaptic active zone (AZ) is to regulate the release of neurotransmitter-filled synaptic vesicles (SVs) in response to action potentials entering the synaptic bouton (Südhof, 2012). Before AZ scaffold components (e.g., ELKS family protein Bruchpilot: BRP, Rab3-interacting molecule (RIM)-binding protein: RBP) are integrated into synapses, however, they have to be transported down the often very long axons. AZ scaffold proteins are characterized by strings of interaction motifs (particularly coiled coil motifs) contributing to the avidity and tenacity of synaptic scaffolds (Tsuriel et al., 2009). Therefore they might be considered as ‘sticky cargos’ whose association status has to be precisely controlled during transport. Long-range axonal transport is conducted along polarised microtubules, using kinesin-family motor proteins for anterograde and dyneins for retrograde transport (reviewed in Maeder et al., 2014). Kinesin-1 family motor kinesin heavy chain (KHC, also known as KIF5; Sato-Yoshitake et al., 1992; Hurd and Saxton, 1996; Takamori et al., 2006) and Unc-104/Imac/KIF1 (Hall and Hedgecock, 1991; Pack-Chung et al., 2007) have been implicated in the transport of SVs, in conjunction with regulators of this process, such as Syd-1 (Hallam et al., 2002), Syd-2/Liprin-α (Serra-Pagès et al., 1998; Zhen and Jin, 1999; Miller et al., 2005; Stryker and Johnson, 2007; Wagner et al., 2009), RSY-1 (Patel and Shen, 2009), or ARL-8 (Klassen et al., 2010; Wu et al., 2013). In Caenorhabditis elegans, SV and AZ scaffold proteins exhibit extensive co-transport and undergo frequent pauses, with immobile phases promoting cargo dissociation and assembly (Wu et al., 2013). Long axons, typical for Drosophila or mammals, pose high demands for the ‘processivity’ of axonal AZ scaffold component transport. The molecular mechanisms, which provide this processivity and thus block premature assembly processes remain speculative, but might also be relevant in the context of axonal transport deficits of neurodegenerative scenarios (Millecamps and Julien, 2013). In addition, we know little concerning the composition of cargos destined for synaptic AZs.

The electron-dense AZ cytomatrix (T-bar) at the Drosophila neuromuscular junction (NMJ) is among others composed of oligomers of BRP and RBP (Kittel et al., 2006; Fouquet et al., 2009; Liu et al., 2011a; Ehmann et al., 2014). We report here that BRP and RBP, but no other tested AZ components, are co-transported in discrete transport complexes along the axon. Via a screen for RBP interaction partners, we identified the APP-like protein interacting protein 1 (Aplip1), an adaptor protein previously implicated in SV transport. Further analysis by X-ray crystallography and calorimetry showed that the second and third Src homology 3 (SH3) domain of RBP bind a specific N-terminal proline-rich (PxxP) motif of Aplip1/JIP1 with more than 10-fold higher affinity than RBP binds its synaptic ligands (Ca²⁺channels/RIM) by their cognate PxxP motifs. The integrity of this motif was essential to protect axons from forming ectopic axonal synapses, which were observed in aplip1 mutant axons by electron microscopy (EM) and super-resolution light microscopy.

In summary, we characterize a mechanism of axonal AZ protein transport through a high affinity interaction between preassembled, stoichiometric scaffold protein complexes and the transport adaptor Aplip1. This high affinity interaction is needed to allow for effective axonal transport and to protect from premature AZ assembly processes.

The molecular basis of how axonal protein transport is coupled to AZ assembly remains largely unexplored. We hypothesized that BRP might be co-transported with further AZ scaffold proteins, as transport of preformed complexes of AZ material has been suggested previously (Zhai et al., 2001; Shapira et al., 2003; Maas et al., 2012).

RBP co-clusters with BRP in axonal aggregates of SR kinase mutants

Firstly, we chose a previously characterized mutant of a serine–arginine (SR) protein kinase at location 79D (srpk79D). The SRPK79D protein is a member of the serine–arginine protein kinase family previously shown to be involved in mRNA splicing and processing (Wang et al., 1998). Mutants of srpk79D form dramatic BRP aggregates in the axoplasm, while its endogenous substrates remain elusive (Johnson et al., 2009; Nieratschker et al., 2009). The axonal aggregations here served as a sensitive background to screen for proteins that co-accumulate together with BRP in the axon, and therefore indicate a joint transport mechanism.

In order to visualise the aggregates forming within axons of srpk79D mutant larvae, we stained with antibodies (Abs) directed against the BRP C- and N-terminus (Figure 1A, as control), and further probed for the presence of additional AZ proteins, such as Liprin-α (Figure 1B) and Syd-1 (Figure 1C), which interact with BRP at the AZ (Owald et al., 2010, 2012) and the small GTPase Rab3 that was previously shown to regulate the distribution of presynaptic components at AZs (Figure 1D; Graf et al., 2009). However, none of these AZ proteins showed co-accumulation with BRP in the aggregates (B as also described in Johnson et al., 2009). Staining with anti-RBP Abs (Liu et al., 2011a), by contrast, revealed strong co-localization of BRP and RBP in the axonal aggregates (Figure 1E). Quantification of BRP and RBP co-localization in two different srpk79D mutant null alleles (atc from Johnson et al., 2009; vn from Nieratschker et al., 2009) confirmed the impression that the axonal RBP/BRP signals were of identical size (Figure 1F; mean area of axonal spots, BRP^C-term 0.3797 ± 0.03694 µm² in srpk79D^ATC, 0.3259 ± 0.02212 µm² in srpk79D^vn; RBP^C-term 0.3892 ± 0.02097 µm² in srpk79D^ATC, 0.3696 ± 0.01645 µm² in srpk79D^vn; n = 8 nerves; mean ± SEM), and that BRP and RBP nearly always co-localized in these aggregates (Figure 1G; BRP^C-term co-localizing with RBP^C-term 93.26% ± 2.172 in srpk79D^ATC, 95.85% ± 1.302 in srpk79D^vn; RBP^C-term co-localizing with BRP^C-term 95.7% ± 0.9713 in srpk79D^ATC, 94.24% ± 1.162 in srpk79D^vn; n = 8 nerves; mean ± SEM).

Figure 1

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Thus, RBP was the only AZ protein that robustly co-accumulates with BRP in srpk79D mutant axonal aggregates. To further explore the distribution of BRP and RBP in these aggregates we used stimulated emission depletion (STED) light microscopy at a resolution of about 50 nm (Hell, 2007). Two-colour STED microscopy revealed a tight and stoichiometric association of BRP and RBP in the floating axonal aggregates of srpk79D mutants (Figure 1H), reminiscent of EM images showing T-bar super assemblies in these axons (Figure 1H; Johnson et al., 2009; Nieratschker et al., 2009). In fact, the relative distribution of RBP vs BRP^C-term was very reminiscent of the organisation at mature, synaptic AZs (Liu et al., 2011a). The tight association of BRP and RBP in these ectopic aggregates further suggested a co-transport of both AZ components. Indeed, we could identify axonal BRP spots co-positive for RBP (Figure 1I, arrows) in wild type (WT) larvae as well. Compared to srpk79D mutant axons, WT BRP/RBP co-positive aggregates were present at a lower frequency and displayed a ∼ four times smaller average diameter in control axons (Figure 1F; mean area of axonal spots, BRP^C-term 0.06895 ± 0.01 µm² in WT; RBP^C-term spots: 0.09184 ± 0.0133 in WT; n = 8 nerves; mean ± SEM).

BRP and RBP are co-transported in axons together with Aplip1

We observed active anterograde and retrograde transport of the BRP (GFP-labelled)/RBP (cherry-labelled) co-positive spots when using intravital imaging of axons of intact larvae (Rasse et al., 2005) (Figure 2A; Video 1). Thus, as our data strongly suggested that BRP and RBP are co-transported, we searched for adaptor proteins coupling them to axonal motors.

Figure 2

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Video 1

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RBP, via its second and third SH3 domain, is known to bind synaptic ligands such as Ca²⁺ channels and RIM (Liu et al., 2011a). Both the SH3 domains and the cognate PxxP motifs of the synaptic ligands are highly conserved between mammals and Drosophila (Liu et al., 2011a; Südhof, 2012; Davydova et al., 2014). However, in order to identify novel RBP interaction partners which might be relevant in the context of axonal transport, we performed a large-scale yeast two-hybrid (Y2H) screen using a construct consisting of the second and third SH3 domains of Drosophila RBP as bait (also shown in Figure 3A). As expected, several clones representing RIM and the Ca²⁺ channel α1-subunit Cacophony (Cac) were isolated (not shown). In addition, the screen recovered 14 independent fragments of Aplip1, including a full length cDNA clone (Figure 2B). Aplip1 is the Drosophila homolog of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1), a scaffolding protein that has been shown to bind kinesin light chain (KLC; Verhey et al., 2001), Alzheimer's amyloid precursor protein (APP; Taru et al., 2002), JNK pathway kinases (Horiuchi et al., 2005, 2007) and the autophagosome adaptor LC3 (Fu et al., 2014). If Aplip1 was mediating the axonal transport of RBP, moving spots co-positive for both RBP and Aplip1 should be expected. In fact, we robustly observed co-transport of RBP^cherry and Aplip1^GFP spots in both anterograde (Figure 2C, arrowhead; Video 2) and retrograde (not shown) direction at a frequency consistent with the low frequency of single Aplip1^GFP moving particles (not shown). Furthermore, we observed BRP-short^straw co-transport with Aplip1^GFP (Figure 2D; Video 3), as expected with similarly low frequencies as observed for RBP/Aplip1 co-transport (not shown), further pointing towards a co-transport of RBP and BRP in conjunction with Aplip1. We used the live imaging assay to investigate BRP transport in different aplip1 mutants to directly address whether removal of Aplip1 affects AZ scaffold protein transport. The aplip1^null allele completely and specifically removes the aplip1 gene and was generated by P-element excision (Klinedinst et al., 2013). By comparison, the aplip1^ek4 allele contains a point mutation in the C-terminal kinesin binding domain of Aplip1 that was shown to almost completely abolish the ability of Aplip1 to bind to KLC (Horiuchi et al., 2005). Anterograde and retrograde transport of BRP was drastically reduced compared to controls in both aplip1 mutant alleles (Figure 2E). Through the introduction of a genomic (gen.) construct of Aplip1 into the aplip1^null mutant background (aplip1^null, gen. rescue), however, BRP flux (spots passing through an axonal cross-section in a given time) could be restored to WT level (Figure 2E). Quantification showed that retrograde transport in the aplip1^null mutant situation was somewhat more affected (27× less compared to control) than anterograde transport (7× less). Both directions appeared equally affected (about 8× less compared to controls) in the kinesin-binding defective aplip1^ek4 mutant. It is noteworthy that the transport of SV cargo in the same mutant was reduced equally in both directions, whereas transport of mitochondria is only impaired in the retrograde direction (Horiuchi et al., 2005).

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RBP binds the transport adaptor Aplip1 via a high affinity PxxP-SH3 interaction

As our Y2H screen used the SH3-II and -III domains of RBP as bait (Figure 3A), PxxP motifs are expected to mediate the interaction with Aplip1. In fact, Aplip1 contains two PxxP motifs which were both present in most of the prey clones recovered in the Y2H screen, except for one single clone that contained only the first more N-terminal motif (Figure 2B, arrow). Using a semi-quantitative liquid Y2H assay and a set of Aplip1 constructs containing only either the first or the second PxxP motif (Figure 3B), we mapped the interaction between RBP and Aplip1 to the first of the two candidate PxxP motifs present in all clones isolated (Figure 2B). The second and third SH3 domain of RBP bound to this motif with comparable strength when measured with a semi-quantitative liquid Y2H assay (Figure 3C; mean ß-Gal4 units for: Aplip1-PxxP1/RBP SH3-II: 24.3 ± 6.6; Aplip1-PxxP1/RBPSH3-III: 29.1 ± 7.4; n = 3 independent experiments; mean ± SEM). No binding was observed between the second and third SH3 domains of RBP and Aplip1-PxxP2 (Figure 3C; mean ß-Gal4 units for: Aplip1-PxxP2/RBP SH3-II: 0.2 ± 0.0; Aplip1-PxxP2/RBPSH3-III: 0.2 ± 0.1; n = 3 independent experiments; mean ± SEM). When mutating either the PxxP1 motif of Aplip1 (P156 → A; P159 → A, giving rise to AxxA1) or introducing mutations known to interfere with PxxP ligand binding into the individual SH3 domains of RBP (SH3-II*/SH3-III*), the interaction was completely abolished (Figure 3C; mean ß-Gal4 units for: Aplip1-AxxA1/RBP SH3-II: 0.1 ± 0.1; Aplip1-AxxA1/RBP SH3-III: 0.2 ± 0.0; Aplip1-PxxP1/RBPSH3-II*: 0.1 ± 0.0; Aplip1-PxxP1/RBP SH3-III*: 0.1 ± 0.0; n = 3 independent experiments; mean ± SEM). We performed isothermal titration calorimetry (ITC) to measure the thermodynamics of the binding directly and compare Aplip1/RBP binding quantitatively to the established synaptic ligands of RBP. We used four different constructs, comprising either single RBP SH3 domains (I, II, and III) or a construct of two RBP SH3 domains (II+III) (see also Figure 3A). Whereas we could not detect any binding of the Aplip1 peptides to RBP SH3-I, we could determine K_D constants for the single SH3-II, SH3-III and the tandem SH3-II+III (Figure 3D; Figure 3—figure supplement 1) domains of RBP. Both SH3-II and SH3-III single domains showed a binding affinity to Aplip1 peptides several fold stronger compared to either Cac, RIM1 or RIM2 (Figure 3D; Figure 3—figure supplements 2–4). However, the affinity of the Aplip1 peptides to the SH3-II+III domain was the highest observed which is indicative of co-operativity between both domains in peptide binding that could increase the local concentrations of Aplip1 at RBP binding pockets (BPs).

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Finally, in order to get a deeper atomic insight into the structural basis of the binding of RBP towards Aplip1 in comparison to its synaptic ligands, we crystallized the Drosophila RBP SH3-II domain together with both an Aplip1 (Figure 3E; Tables 1, 2, 3) and a Cac peptide (Figure 3F; Tables 1, 3, 4), and RBP SH3-III with a Cac peptide (Figure 3—figure supplement 5; Tables 1, 3). Drosophila RBP SH3-II and -III share 49.2% sequence identity and adopt the canonical fold of SH3 domains (Figure 3E,F; Figure 3—figure supplement 5). Both domains superimpose with a root mean deviation of 0.8 Å for 64 pairs of Cα-atoms. Both peptides sequences harbor the canonical class I interaction motif +xΨPxxP (+, positively charged; x, any amino acid; Ψ hydrophobic amino acid, see Figure 3D for sequence) and are bound into the respective SH3 domain in ‘plus’ direction. We observed the classical poly-proline helix that allows for mainly hydrophobic protein-peptide interaction in all three structures. We detected the same hydrogen pattern between the protein side chains and peptide backbone in the structure of SH3-II with Aplip1 and Cac. The major difference is the side chain orientation of R1687 of Cac that π-stacks with its guanidinium function with Y1372, except for one copy, where it forms a salt-bridge to E1341. The equivalent residue to R1687 of Cac is R153 of the Aplip1 peptide, which forms, by contrast, a bidentate salt-bridge to D1336 (Table 3). A second major difference is induced by the two consecutive proline residues in the Cac peptide. Consequently, the peptide has a more polyproline type II conformation that brings T1692 closer to the protein surface and allows P1693 to deeper point in a hydrophobic pocket of the SH3-II domain. Whereas the C-terminal portion of the Aplip1 peptide is folded in a short 3₁₀ helix, the N-terminus of the Aplip1 peptide adopts a random coil conformation with hydrophobic interactions to the surface of SH3-II. The Cac-derived peptide bound to SH3-III is fully defined in the electron density. However, the peptide main chain interaction with the SH3 domains is conserved. The side chain orientation of Cac R1687 is again different if bound to SH3-II or SH3-III. In complex with SH3-III, R1687 forms a bidentate hydrogen bond to SH3-III D1463 and E1648. A π-stacking interaction is not possible since Y1372 of SH3-II is replaced by SH3-III L1499. The central PxxP motifs of Aplip1 superimpose well in both structures if bound to SH3-II and SH3-III. Towards its C-terminus, the Aplip1-PxxP1 peptide adopts a slightly different random coil conformation compared to the structure when bound to SH3-II caused by two additional hydrogen bonds from T1692 and K1695 to the SH3-II domain (Table 3).

Table 1

Data collection
Structure	RBP SH3-II	RBP SH3-II	RBP SH3-III
	Aplip1	Cac	Cac
PDB entry	4Z88	4Z89	4Z8A
Space group	C2	P21	I222
Wavelength (Å)	0.91841	0.91841	0.91841
Unit cell
a; b; c (Å)	108.3; 62.4; 163.6	58.3; 122.2; 68.5	52.1; 54.3; 73.6
α; β; γ (°)	90.0; 90.3; 90.0	90.0; 113.2; 90.0	90.0; 90.0; 90.0
Resolution (Å)*	50.00–2.09	50.00–2.64	50.00–1.75
	(2.19–2.09)	(2.74–2.64)	(1.86–1.75)
Unique reflections	64,269 (7760)	25,229 (2591)	10,690 (1579)
Completeness*	98.9 (92.4)	96.9 (95.0)	98.7 (92.6)
<I/σ(I)>*	7.7 (2.6)	8.0 (2.1)	14.2 (2.2)
Rmeas*, †	0.127 (0.533)	0.157 (0.726)	0.127 (0.663)
CC1/2*	99.1 (68.0)	98.9 (81.2)	99.7 (76.5)
Redundancy*	3.7 (3.7)	3.5 (3.2)	5.6 (3.1)
Refinement
Non-hydrogen atoms	7564	6239	850
Rwork*, ‡	0.210 (0.314)	0.255 (0.367)	0.159 (0.233)
Rfree*, §	0.236 (0.396)	0.312 (0.490)	0.208 (0.332)
Average B-factor (Å2)	40.8	52.10	18.8
No. of complexes	24	10	1
Protein residues	6484/41.0	663/51.1	74/17.6
Peptide residues	861/42.7	92/63.6	15/15.9
Buffer molecules	11/40.2	1/46.3	–
Water molecules	57/29.6	134/30.3	110/28.6
r.m.s.d.#
bond length (Å)	0.007	0.005	0.010
bond angles (°)	1.224	1.140	1.210
Ramachandran outliers (%)	0.1	0.56	0
Ramachandran favoured (%)	98.4	98.0	100

1. *

   values in parentheses refer to the highest resolution shell.

2. †

   R_meas = Σ_h [n/(n − 1)]^1/2 Σ_i|I_h − I_h,i|/Σ_hΣ_iI_h,i where I_h is the mean intensity of symmetry-equivalent reflections and n is the redundancy.

3. ‡

   R_work = Σ_h|F_o − F_c|/ΣF_o (working set, no σ cut-off applied).

4. §

   R_free is the same as R_work, but calculated on 5% of the data excluded from refinement.

5. #

   Root-mean-square deviation (r.m.s.d.) from target geometries.

6. CC, coiled coil.

Table 2

RBP SH3-II	Range	Aplip1	Range
chain A	1318–1382	chain M	153–163
chain B	x1318–1382	chain N	155–159
chain C	x1318–1381	chain O	154–163
chain D	x1318–1382	chain P	153–159
chain E	1319–1381	chain Q	151–163
chain F	x1318–1380	chain R	153–159
chain G	x1318–1381	chain S	151–163
chain H	x1318–1382	chain T	152–156
chain I	x1318–1382	chain U	152–163
chain J	x1318–1381	chain V	152–158
chain K	x1318–1381	chain W	152–163
chain L	x1318–1381	chain X	152–158

1. Completeness of the model given for the 12 complexes of RBP SH3-II bound to the Aplip1 peptide ¹⁴⁹TRRRRKLPEIPKNKK¹⁶³. Superscript ‘x’ indicates additional N-terminal residues of RBP SH3-II originating from the linker region between the protease cleavage site and the N-terminus.

Table 3

Aplip1	SH3-II	Distance
Arg153N	Asp1359OD2	2.4
Arg153NH2	Asp1336OD1	3.0
Arg153NH2	Asp1336OD2	2.6
Lys154N	Asn1334OD1	2.9
Lys154O	Asn1334ND2	3.0
Pro156O	Asn1376ND2	2.8

Cac	SH3-II	Distance
Gly1686N	Asp1359OD2	2.7
Arg1687N	Asp1359OD2	2.8
Arg1688N	Asn1334OD1	3.0
Arg1688O	Asn1334ND2	2.9
Pro1690O	Asn1376ND2	2.8

Cac	SH3-III	Distance
Arg1687NH1	Asp1463OD1	2.9
Arg1687NH1	Glu1488OE2	3.0
Arg1687NH2	Glu1488OE2	3.1
Arg1688N	Asn1461OD1	2.8
Arg1688O	Asn1461ND2	3.0
Pro1690O	Asn1376ND2	2.9
Thr1692OG	Asn1376ND2	2.9
Lys1695O	Tyr1451OH	2.8
Ser1697OG	Leu1450O	2.7

1. Hydrogen bonding interaction of RBP SH3-II with Aplip1 and Cac, as well as RBP SH3-III in complex with Cac. Distance ≤3.2 Å are given in Å.

Table 4

RBP SH3-II	Range	Cac	Range
chain A	1318–1381	chain a	1686–1697
chain B	x1318–1381	chain b	1686–1695
chain C	x1318–1382	chain c	1686–1697
chain D	x1318–1381	chain d	1686–1697
chain E	1318–1382	chain e	1685–1694
chain F	x1318–1382	chain f	1685–1693
chain G	x1318–1382	chain g	1686–1693
chain H	1318–1381	chain h	1686–1693
chain I	x1318–1381	chain i	1686–1693
chain J	x1318–1382	chain j	1686–1697

1. Completeness of the model given for the six complexes of RBP SH3-II and the bound Cac peptide ¹⁶⁸⁵IGRRLPPTPSKPSTL¹⁶⁹⁹. Superscript ‘x’ indicates additional N-terminal residues of RBP SH3-II originating from the linker region between the protease cleavage site and the N-terminus.

The Aplip1-PxxP1 motif is needed for effective axonal RBP/BRP transport

Consistent with the idea that Aplip1 is mediating RBP transport, we found axonal aggregates consisting of both RBP and BRP in the aplip1^ek4, as well as the aplip1^null allele (Figure 4B,C). This ectopic RBP/BRP accumulation was rescued after introducing a genomic construct of Aplip1 into the aplip1^null mutant background (aplip1^null, gen. rescue; Figure 4D). Pan-neuronal expression of an Aplip1 cDNA equally rescued the axonal RBP/BRP accumulations (Figure 4I, quantification in K, L). Importantly, however, the expression of an Aplip-AxxA1 cDNA construct (integrated at the same chromosomal integration site as the control construct; expression and axonal presence confirmed with a newly generated Aplip1 Ab; not shown) could no longer rescue the RBP/BRP accumulation phenotype (Figure 4J, quantification in Figure 4K,L). Thus, we conclude that Aplip1 is involved in the transport of RBP/BRP to the AZ, whereby its functionality in this context largely depends on the integrity of its N-terminal PxxP1 motif.

Figure 4

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Aplip1 promotes BRP transport in absence of RBP

As indicated above, BRP accumulated in the axons of aplip1 mutants as well. Thus, BRP could be transported through Aplip1 via binding to RBP, other yet undetected co-transported AZ proteins, or BRP could bind Aplip1 independently of RBP. We therefore created aplip1/rbp and aplip1/brp double mutants to investigate the functional relation of RBP and BRP with regard to Aplip1-dependent transport. While removing BRP in srpk79D mutants also abolished the axonal RBP spots (Figure 5—figure supplement 1D), removing BRP in aplip1 mutants had no apparent effect on axonal RBP accumulations (Figure 5B; control in Figure 5A). On the other hand, genetic elimination of RBP did not interfere with the accumulation of BRP in aplip1 mutant axons (Figure 5E; controls in Figure 5C,D). Thus, BRP transport also ‘suffers’ from the absence of the Aplip1 adaptor when RBP is removed in parallel. Hence, Aplip1 promotes BRP transport even in the absence of RBP. To address a putative molecular basis of this relationship, we performed a Y2H assay to test for direct interaction between five different BRP constructs and a full length Aplip1 construct (see Figure 3B for domain structure). Despite these efforts, robust interactions between Aplip1 and BRP fragments could not be detected (data not shown). Nonetheless, both RBP but also BRP were easily detected in anti-GFP immunoprecipitations (IPs) from a synaptic membrane preparation (Figure 5F; Figure 5—figure supplement 2) derived from Drosophila head extracts of pan-neuronal driven Aplip1-GFP cDNA construct (Depner et al., 2014). Of note, within axons of rbp^null mutant larvae, ectopic BRP accumulations could not be observed (not shown). Thus, we provide evidence for an RBP-independent but Aplip1-dependent transport component for BRP, whose mechanistic details have still to be deciphered. Taken together, our results imply that though BRP and RBP are co-transported in the WT situation, their Aplip1-dependent transport can be genetically uncoupled.

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RBP and BRP form ectopic AZs at the axonal plasma membrane of aplip1 mutants

The BRP flux in axons of aplip1 mutants was severely diminished, but not completely abolished (Figure 2E). At the same time, AZ localization of both BRP and RBP at synaptic terminals of aplip1 mutants was still observed in both aplip1 alleles (not shown), although slightly reduced (not shown). This indicates that alternative transport mechanisms and adaptors exist which operate in parallel to Aplip1, as the synaptic phenotype is relatively weak. In fact, axonal accumulations of BRP have already been described for Acyl-CoA long-chain Synthetase (Acsl, Liu et al., 2011b) as well as for Unc-51 (Atg1) mutants (Wairkar et al., 2009). In our experiments, we found RBP to invariably co-cluster with BRP in the mutants mentioned (Figure 6B,C; control in Figure 6A), and equally in mutants of the Drosophila ß-amyloid protein precursor-like (Appl; Torroja et al., 1999a, 1999b; Figure 6D) and Unc-76 (Gindhart et al., 2003; Figure 6E). The fact that RBP and BRP tightly co-accumulated in axonal aggregates of all these transport mutants strengthens the probability that BRP is always co-transported with RBP.

Figure 6

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To gain a deeper insight into the substructure of the BRP/RBP accumulations in aplip1 mutant axons, we again used two-colour STED microscopy. In contrast to the srpk79D aggregates, however, STED images of axonal BRB/RBP accumulations were reminiscent of mature synaptic AZs (Liu et al. 2011a), with BRP^C-term signal surrounding the RBP signal, which, in turn, is oriented closer towards the axonal plasma membrane (Figure 7A, arrow head; plasma membrane indicated by dashed line). Interestingly, in contrast to the floating T-bar super-aggregates in srpk79D mutants (Johnson et al., 2009; Nieratschker et al., 2009), these axonal BRP spots in aplip1 mutants were positive for Syd-1 (compare Figures 1C, 7B). Intriguingly, floating T-bars have been observed in synaptic boutons in syd-1 mutants (Owald et al., 2010). Together, this is suggestive of a role of Syd-1 in the membrane-anchoring of AZ proteins.

Figure 7 with 1 supplement see all

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Furthermore, we asked whether BRP/RBP aggregates identified in aplip1 mutants represent ectopic AZs forming at the axonal plasma membrane. In fact, EM analysis easily revealed T-bar structures, typical for synaptic terminals (Figure 7C, arrow heads, magnification in E), at axonal plasma membranes of aplip1 mutants (Figure 7D, arrow heads, magnification in F), but never in controls (not shown). We found these ectopic axonal T-bars surrounded by SV profiles (Figure 7D, arrows), very similar to ‘normally positioned’ T-bars at the presynaptic terminal (Figure 7C, arrows). Consistently, the SV marker Synaptotagmin-1 (Syt-1) was found to be associated with BRP/RBP accumulations in aplip1^null mutants (Figure 7H, quantification in Figure 7K). This phenotype could be rescued by the expression of an Aplip1 WT cDNA construct (Figure 7I, quantification in Figure 7K) but not by the expression of the Aplip1-AxxA1 construct (Figure 7J; quantification in Figure 7K). Thus, a point-like interaction surface of Aplip1 which binds RBP with high affinity is important to block a whole sequence of assembly events at the axonal plasma membrane, including AZ scaffold (‘T-bar’) formation and the accumulation of SVs.

To further support the importance of adaptor protein—cargo interaction in blocking ectopic AZ assembly we downregulated the expression of motor proteins. This also leads to transport defects and ectopic axonal AZ protein accumulations but in principle leaving the adaptor protein—cargo interaction intact. Interestingly, motoneuronal driven Imac-RNAi led to only few axonal BRP/RBP accumulations although with no preference concerning their direction in relation to the axonal plasma membrane (Figure 7—figure supplement 1B; arrow heads). In contrast motoneuronal driven KHC-RNAi showed prominent axonal aggregates consistent of BRP/RBP but most of the time showing an irregular, elongated shape (Figure 7—figure supplement 1C; arrow heads). As mentioned above, proper T-bars were identified in aplip1 mutant axons with ease. In contrast, systematic EM analysis of khc mutant axons revealed just one electron dense material that showed a T-bar-like appearance (Figure 7—figure supplement 1D; arrow head, magnifications in E, F) but never in control (ctrl) or motoneuronal driven Imac-RNAi.

In summary, we find that the SH3-II and -III interaction surface of RBP serves as a multi-functional platform for differential protein interaction with either other AZ components or the transport adaptor and therefore, motor-cargo linkage. Thus, interaction surfaces of RBP/BRP ‘cargo complexes’ might be shielded and blocked from undergoing premature assembly by interactions with transport adaptors, while genetically induced loss of these adaptors might provoke premature AZ assembly.

Large multi-domain scaffold proteins such as BRP/RBP are ultimately destined to form stable scaffolds, characterized by remarkable tenacity and a low turnover, likely due to stabilization by multiple homo- and heterotypic interactions simultaneously (Sigrist and Schmitz, 2011). How these large and ‘sticky’ AZ scaffold components engage into axonal transport processes to ensure their ‘safe’ arrival at the synaptic terminal remains to be addressed. We find here that the AZ scaffold protein RBP binds the transport adaptor Aplip1 using a ‘classic’ PxxP/SH3 interaction. Notably, the same RBP SH3 domain (II and III) interaction surfaces are used for binding the synaptic AZ ligands of RBP, that is, RIM and the voltage gated Ca²⁺ channel (Wang et al., 2002; Kaeser et al., 2011; Liu et al., 2011a; Davydova et al., 2014), though with clearly lower affinity than for Aplip1. A point mutation which disrupts the Aplip1-RBP interaction provoked a ‘premature’ capture of RBP and the co-transported BRP at the axonal membrane, thus forming ectopic but, concerning T-bar shape and BRP/RBP arrangement, WT-like AZ scaffolds. The Aplip1 orthologue Jip1 has been shown to homo-dimerize via interaction of its SH3 domain (Kristensen et al., 2006). Thus, the multiplicity of interactions, with Aplip1 dimers binding to two SH3 domains of RBP as well as to KLC, might form transport complexes of sufficient avidity to ensure tight adaptor–cargo interaction and prevent premature capture of the scaffold components.

Our intravital imaging experiments showed that within axons RBP and BRP are co-transport in shared complexes together with Aplip1, whereas we, despite efforts, were unable to detect any co-transport of other AZ scaffold components, that is, Syd-1 or Liprin-α with BRP/RBP (not shown). In addition, STED analysis of axonal aggregates in srpk79D mutants showed BRP/RBP in stoichiometric amounts, but also failed to detect other AZ scaffold components. Moreover, BRP and RBP co-aggregated in the axoplasm of several other transport mutants we tested (acsl, unc-51, appl, unc-76), consistent with both proteins entering synaptic AZ assembly from a common transport complex. Of note, during AZ assembly at the NMJ, BRP incorporation is invariably delayed compared to the ‘early assembly’ phase which is driven by the accumulation of Syd-1/Liprin-α scaffolds (Fouquet et al., 2009; Owald et al., 2010, 2012). As the early assembly phase is, per se, still reversible (Owald et al., 2010), the transport of ‘stoichiometric RBP/BRP complexes’ delivering building blocks for the ‘mature scaffold’ might drive AZ assembly into a mature, irreversible state (Owald et al., 2010), and seems mechanistically distinct from early scaffold assembly mechanisms.

Previous work suggested that AZ scaffold components (Piccolo, Bassoon, Munc-13 and ELKS) in rodent neurons are transported to assembling synapses as ‘preformed complexes’, so-called Piccolo-Bassoon-Transport Vesicles (PTVs; Zhai et al., 2001; Shapira et al., 2003; Maas et al., 2012). The PTVs are thought to be co-transported with SV precursors (Ahmari et al., 2000; Tao-Cheng, 2007; Bury and Sabo, 2011) anterogradely mediated via a KHC(KIF5B)/Syntabuli/Syntaxin-1 complex (Cai et al., 2007) and retrogradely via a direct interaction between Dynein light chain and Bassoon (Fejtova et al., 2009). Since their initial description, however, further investigations of PTVs have been hampered by the apparent relative scarcity of PTVs, and by the lack of genetic or biochemical options for specifically interfering with their transport or final incorporation into AZs.

Despite efforts we were not able to detect a direct interaction of Aplip1 and BRP although their common transport can be uncoupled from the presence of RBP. One possible explanation could be a direct interaction of Aplip1 to other AZ proteins that are co-transported together with BRP and RBP. It is interesting that the very C-terminus of BRP is essential for SV clustering around the BRP-based AZ cytomatrix (Hallerman et al., 2010). Thus, it is tempting to speculate that adaptor/transport complex binding might block premature AZ protein/SV interactions before AZ assembly, but further analysis will have to await more atomic details as we could gain for the RBP::Aplip1 interaction.

The down-regulation of the motor protein KHC also provoked severe axonal co-accumulations of BRP and RBP but per se should leave the adaptor protein-AZ cargo interaction intact. In contrast to aplip1, the axonal aggregations in khc mutants adapted irregular shapes most of the time, likely not representing T-bar-like structures. Thus, our data suggest a mechanistic difference when comparing the consequences between eliminating adaptor cargo interactions with a direct impairment of motor functions. Still, we cannot exclude that trafficking of AZ complexes naturally antagonizes their ability to assemble into T-bars.

The idea that proteins/molecules are held in an inactive state till they reach their final target has been observed in many other cell types. For example, in the context of local translation control, mRNAs are shielded or hidden in messenger ribonucleoprotein particles during transport so that they are withheld from cellular processing events such as translation and degradation. Shielding is thought to operate through proteins that bind to the mRNA and alter its conformation while at the correct time or place the masking protein is influenced by a signal that alleviates its shielding effect (Spirin, 1996; Johnstone and Lasko, 2001). As another example, hydrolytic enzymes, for example, lysosomes, are transported as proteolytically inactive precursors that become matured by proteolytic processing only within late endosomes or lysosomes (Ishidoh and Kominami, 2002). Particularly relevant in the context of AZ proteins involved in exocytosis, the H_abc domain of Syntaxin-1 folds back on the central helix of the SNARE motif to generate a closed and inactive conformation which might prevent the interaction of Syntaxin-1 with other AZ proteins during diffusion (Dulubova et al., 1999; Ribrault et al., 2011).

Previously, genetic analysis of C. elegans axons forming en passant synapses suggested a tight balance between capture and dissociation of protein transport complexes to ensure proper positioning of presynaptic AZs. In this study, overexpression of the kinesin motor Unc-104/KIF1A reduced the capture rate and could suppress the premature axonal accumulations of AZ/SV proteins in mutants of the small, ARF-family G-protein Arl-8. Interestingly, large axonal accumulations in arl-8 mutants displayed a particularly high capture rate (Klassen et al., 2010; Wu et al., 2013). Similarly, both aplip1 alleles exhibited enlarged axonal BRP/RBP accumulations. Thus, the capture/dissociation balance for AZ components might be shifted towards ‘capture’ in these mutants, consistent with the ectopic axonal T-bar formation. It is tempting to speculate that loss of Aplip1-dependent scaffolding and/or kinesin binding provokes the exposure of critical ‘sticky’ patches of scaffold components such as RBP and BRP. Such opening of interaction surfaces might increase ‘premature’ interactions of cargo proteins actually destined for AZ assembly, thus increase overall size of the cargo complexes by oligomerization between AZ proteins and, finally, promote premature capture and ultimately ectopic AZ-like assembly. On the other hand, the need for the system to unload the AZ cargo at places of physiological assembly (i.e., presynaptic AZ) might pose a limit to the ‘wrapping’ of AZ components and ask for a fine-tuned capture/dissociation balance.

Several mechanisms for motor/cargo separation such as (i) conformational changes induced by guanosine-5′-triphosphate hydrolysis, (ii) posttranslational modification as de/phosphorylation, or (iii) acetylation affecting motor-tubulin affinity, have been suggested for cargo unloading (Hirokawa et al., 2010). Notably, Aplip1 also functions as a scaffold for JNK pathway kinases, whose activity causes motor-cargo dissociation. JNK probably converges with a mitogen-activated protein kinase (MAPK) cascade (MAPK kinase kinase Wallenda phosphorylating MAPK kinase Hemipterous) in the phosphorylation of Aplip1, thereby dissociating Aplip1 from KLC. Thus, JNK signaling, co-ordinated by the Aplip1 scaffold, provides an attractive candidate mechanism for local unloading of SVs (Horiuchi et al., 2007) and, as shown here, AZ cargo at synaptic boutons. Our study further emphasises the role of the Aplip1 adaptor, whose direct scaffolding role through binding AZ proteins might well be integrated with upstream controls via JNK and MAP kinases. Intravital imaging in combination with genetics of newly assembling NMJ synapses should be ideally suited to further dissect the obviously delicate interplay between local cues mediating capturing and axonal transport with motor-cargo dissociation.

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Author details

1. Matthias Siebert

   Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

   Contribution

   MS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Mathias A Böhme

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-1739-5825

2. Mathias A Böhme

   1. Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany
   2. NeuroCure, Charité-Universitätsmedizin Berlin, Berlin, Germany

   Contribution

   MAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Matthias Siebert

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-0947-9172

3. Jan H Driller

   Institute of Chemistry and Biochemisty/Structural Biochemistry, Freie Universität Berlin, Berlin, Germany

   Contribution

   JHD, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Husam Babikir

   Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

   Contribution

   HB, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Malou M Mampell

   Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

   Contribution

   MMM, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Ulises Rey

   1. Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany
   2. Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

   Contribution

   UR, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Niraja Ramesh

   Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

   Contribution

   NR, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2867-7131

8. Tanja Matkovic

   Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

   Contribution

   TM, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

9. Nicole Holton

   Institute of Chemistry and Biochemisty/Structural Biochemistry, Freie Universität Berlin, Berlin, Germany

   Contribution

   NH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

10. Suneel Reddy-Alla

    Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

    Contribution

    SR-A, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

11. Fabian Göttfert

    Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Contribution

    FG, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

12. Dirk Kamin

    Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Contribution

    DK, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

13. Christine Quentin

    Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

    Contribution

    CQ, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

14. Susan Klinedinst

    Department of Molecular Cellular and Developmental Biology, University of Michigan, Ann Arbor, United States

    Contribution

    SK, Acquisition of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

15. Till FM Andlauer

    Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

    Contribution

    TFMA, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-2917-5889

16. Stefan W Hell

    Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Contribution

    SWH, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

17. Catherine A Collins

    Department of Molecular Cellular and Developmental Biology, University of Michigan, Ann Arbor, United States

    Contribution

    CAC, Conception and design, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

18. Markus C Wahl

    Institute of Chemistry and Biochemisty/Structural Biochemistry, Freie Universität Berlin, Berlin, Germany

    Contribution

    MCW, Conception and design, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

19. Bernhard Loll

    Institute of Chemistry and Biochemisty/Structural Biochemistry, Freie Universität Berlin, Berlin, Germany

    Contribution

    BL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0001-7928-4488

20. Stephan J Sigrist

    1. Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany
    2. NeuroCure, Charité-Universitätsmedizin Berlin, Berlin, Germany

    Contribution

    SJS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    stephan.sigrist@fu-berlin.de

    Competing interests

    The authors declare that no competing interests exist.

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