Active zone components are transported differently than SV precursors and

Despite the transport defects in all three unc-104 mutants analyzed in this study, the formation of DPs in dorsal nerve cord motor neuron axons was only moderately affected. This indicates that the transport of AZ material is independent of SV precursor and DCV transport. This argument is in line with the hypothesis that AZ proteins are associated with a different organelle than SV and DCV proteins as previously suggested by Okada and colleagues (Okada et al., 1995). They showed that vesicles associated with KIF1A contained SV proteins such as synaptotagmin, synatophysin, and Rab3A but not AZ proteins like syntaxin 1A or SNAP25 (Okada et al., 1995). They additionally identified 80 nm DCVs, which associated almost exclusively with AZ components but not with SV proteins in mammalian brain lysates (Zhai et al., 2001, Shapira et al., 2003). Although the nature of AZ transport organelles remains unclear, these finding suggest that they might resemble a third class of DCVs. Evidence from photoreceptor ribbon synapses in the mouse retina supports this notion. Ribbon-associated proteins Piccolo, Bassoon, RIBEYE and RIM1 were shown to be transported in electron dense precursor spheres to nascent presynaptic sites early in synaptogenesis (Regus-Leidig et al., 2009). With the postnatal formation of mature synaptic ribbons, the number of precursor spheres declined.

In line with previous observations in the C. elegans nerve ring (Hall and Hedgecock, 1991), I found that the number of DPs is moderately reduced in unc-104 mutant motor neurons. During the collection of serial section electron micrographs I had the impression that the frequency of DPs in dorsal cord axons is lower in the two mutant alleles e1265 and ce515. They both have a stronger transport defect compared to unc-104(rh43) such that no vesicles reach the dorsal nerve cord. Of these two strains, unc-104(ce515) had the lowest frequency of DPs and exhibited significantly stronger uncoordinated (unc) movement. Adult worm body size of unc-104(ce515) mutants is reduced compared to unc-104(e1265) or wild type animals. It is

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conceivable that the complete deletion of the PH domain in allele ce515 affects additional activities of this domain, besides binding to PI(4,5)P2 in vesicle membranes. In fact, Kumar and colleagues showed that the UNC-104 PH domain preferentially binds to PI(4,5)P2, PI(4)P and brain lipids like Phosphatidylethanolamine, Phosphatidylserine, Phosphatidic acid and others. It can additionally bind phosphatidylcholine (PC) and phosphatidylinositol (PI). The lesion in the unc-104(e1265) PH domain leads to a reduced affinity to PI(4,5)P2, PI(4)P and brain lipids. However, PC and PI are still bound (Kumar et al., 2010). A complete loss of the PH domain as in unc-104(ce515) may therefore lead to an overall abolished binding to lipids and thus result in a stronger phenotype than unc-104(e1265).

Thorough quantification of fluorescently-tagged AZ markers would allow quantification of DP frequency over the whole length of motor neuron axons and might support or refute my initial impression. First confocal images the number of fluorescent puncta from AZ proteins SYD-2::GFP and RIM-1::GFP in the dorsal nerve cords did not show a clear difference between the three unc-104 mutants (Fig. VII.4).

If the number of DPs correlates with the degree of defective transport, one interpretation would be that AZ proteins are transported via UNC-104 or at least in collaboration with UNC-104. This transport should occur in organelles that bind differently to UNC-104 than DCV-like vesicles or synaptic DCVs. The AZ protein Liprin-α/SYD-2 has been shown to interact with the stalk region of KIF1A/UNC-104 in vitro and in vivo (Shin et al., 2003, Wagner et al., 2009). It is conceivable that motor proteins use SYD-2 to identify AZ transport organelles and that the Liprin-binding domain of UNC-104/KIF1A mediates this interaction. The point mutations in the unc-104 mutants analyzed in this study should not affect the stalk region with the putative Liprin-binding domain. Taking into account the mutation sites of the three unc-104 mutants analyzed, I assume that the binding site for SYD-2 is intact and AZ protein transport can still be accomplished. The reduced number of DPs could then be accredited to the reduced levels of UNC-104 as it was reported for mutants with defective cargo binding in vivo (Kumar et al., 2010). As already discussed in paragraph V., unc-104 mutants with defective Liprin-binding domain are not available for C. elegans and would have to be generated by chance via methods like Ethyl methanesulfonate (EMS) mutagenesis. However, it is not clear whether these mutants would have a visible phenotype to allow selection in a mutagenesis screen.

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Apart from that, in vitro studies could provide valuable insight into SYD-2 interaction with UNC-104.

Other explanations for the correlation between reduced DP number and defective transport in unc-104 mutants are imaginable: Muscle arms from dorsal body wall muscles have been shown to be misguided to the ventral nerve cord and to cell bodies in unc-104(rh43) and unc-104(e1265) mutants (Hall and Hedgecock, 1991;

this work). However, the contact to postsynaptic muscles cell was proposed to be necessary for successful synaptogenesis in C. elegans motor neurons (Plunkett et al., 1996). Formation of AZs in the dorsal nerve cord axons may therefore be compromised due to reduced muscle-neuron interaction in unc-104 mutants.

Also, impaired transport of proteins specifically involved in the formation and maintenance of the complex AZ network could be disturbed in unc-104 mutants. For instance, clustering of postsynaptic AMPA receptors depends on the UNC-104 binding partner SYD-2/Liprin-α (Wyszynski et al., 2002). Mislocalization of SYD-2 and its downstream targets might cause impaired synapse formation, maturation and stabilization. There is evidence from other organisms that specific proteins are needed to ensure synapse formation and maintenance (reviewed in Garner et al., 2002). Nerve-derived Agrin was found to be crucial for synapse growth and maintenance at vertebrate skeletal neuromuscular junctions (Lin et al., 2001), whereas the cell-adhesion molecule integrin is required for maturation of hippocampal synapses (Chavis and Westbrook, 2001).

The lack of transmission activity could additionally lead to the elimination of AZs (Verhage et al., 2000). Disassembly of AZs could be mediated by RSY-1, which has been proposed to eliminate ectopic synapses in HSN axons during development (Patel and Shen, 2009). The regulation of Liprin-α/SYD-2 levels via Ca²⁺ -calmodulin-dependent kinase II (CaMKII) (Hoogenraad et al., 2007) and APC/C complex (van Roessel et al., 2004) could be another mechanism to facilitate AZ removal. As discussed in paragraph V, loss of Liprin-α/SYD-2 leads to strongly reduced DP size and possibly loss of AZs.

Further investigations are necessary to understand which kinesin or other motor proteins are responsible for AZ component transport, which adaptor proteins and transport organelles are involved and how AZ cargo unloading is accomplished at nascent presynaptic sites.

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