The active zone

Triggered fusion of SVs with the presynaptic membrane occurs at the presynaptic AZ, a specialized area opposite to the postsynapse consisting of an elaborate protein network forming the cytomatrix at the active zone (CAZ). In electron micrographs this AZ is characterized by an electron dense projection (DP), which is thought to be involved in the spatial organization of components such as calcium channels and the SV release and retrieval machinery (reviewed in Dresbach et al., 2001)

Five protein families are known to play an important role for the functionality of the AZ (reviewed in Schoch and Gundelfinger, 2006). Protein structure with important domains and identified interactions are shown in Figure II.5 and II.6.

Fig. II.5 Protein structure and domains of CAZ proteins. Munc13-1 contains three C2-domains involved in calcium binding and a C1-domain involved in diacyl-glycerol and phorbolesters binding.

RIM1α exhibits two C2-domains, an N-terminal zinc-finger domain (Zn) and a PDZ-domain involved in numerous protein interactions. Both Bassoon and Piccolo have two zinc-finger domains and three coiled-coil (CC) domains. Piccolo additionally contains a PDZ domain, two C2-domains and

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rich sequences (Q). ELKS/CAST/ERC consists mainly of coiled-coil (CC) domains. Liprin-α is composed of N-terminal coiled coil domains (CC) and C-terminal sterile-alpha-domains (S) involved in recruiting other AZ proteins. Adapted from (Schoch and Gundelfinger, 2006).

LAR

Fig. II.6 Protein interactions in the AZ. AZ enriched proteins are shown in green, SNARE proteins in purple, GTPases in yellow and other presynaptic proteins in blue or gray. (ELKS) ERC/RAB6-interacting/CAST; (GIT) G-protein coupled receptor kinase-interactor; (LAR) LCA-related protein tyrosine phosphatase; (Rab) Ras-related in brain; (RIM) Rab interacting molecule; (BP) RIM-binding protein; (SNAP) synaptosome associated protein. Adapted from (Mittelstaedt et al., 2010).

2.2.1. Munc13 protein family

In vertebrates four Munc13 homologs (1, 2, 3 and Munc13-4) are known. Except for the splice variant ubMunc13-2 (Koch et al., 2000), they are expressed exclusively in the brain, specifically at synapses (Brose et al., 1995). The C. elegans homolog uncoordinated (unc)-13 exhibits two splice isoforms (Kohn et al., 2000). All Munc13 isoforms share a C1 domain that can bind the second messenger diacylglycerol and a central Munc homology domain flanked by two C2 domains (Fig.

II.5) (Koch et al., 2000).

Several interactions of Munc13-1 with other CAZ proteins have been demonstrated:

for example RIM1α (Betz et al., 2001) and syntaxin (Betz et al., 1997) (Fig. II.6).

Currently, Munc-13/UNC-13 is believed to play a crucial role in priming of SVs via syntaxin interaction, thereby preparing them for release (Ashery et al., 2000, Weimer et al., 2006).

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2.2.2. RIM proteins

RIM was initially identified as Rab3a interacting molecule (Wang et al., 1997). Again, several homologs and isoforms are present in vertebrates whereas only one RIM homolog (UNC-10) has been identified in C. elegans (Koushika et al., 2001). RIM proteins are localized in the plasma membrane within 100 nm of the presynaptic DPs (Weimer et al., 2006). They belong to a family of scaffolding proteins that are composed of zinc-finger, PDZ and C2 domains (Fig. II.5) and interact with various proteins besides Rab3a: Munc13-1 (Betz et al., 2001); ELKS/CAST (Ohtsuka et al., 2002); synaptotagmin, SNAP-25 and voltage-dependent calcium channels (Coppola et al., 2001) and Liprin-α (Schoch et al., 2002) (Fig. II.6). In C. elegans unc-10 mutants a reduction of docked vesicles adjacent to the DP has been proposed and was later verified by EM tomography (Weimer et al., 2006, Stigloher et al., 2011).

These finding suggesting that RIM holds SVs close to the DP and AZ membrane to facilitate docking and priming, possibly through simultaneous Rab3 and UNC-13 binding (Dulubova et al., 2005).

2.2.3. ELKS/CAST/ERC proteins

Members of this protein family consist mostly of coiled coil domains (Fig. II.5) and were identified in several independent screens, hence the various names. ELKS are glutamate (E), leucine (L), lysine (K) and serine (S) rich proteins and were first described in papillary thyroid carcinoma to activate a cytoplasmic tyrosine kinase (Nakata et al., 1999, Nakata et al., 2002). CAST (CAZ-associatedstructural protein) was isolated from rat brain membrane fractions and found to localize at synapses by immuno-histochemistry and immuno-EM (Ohtsuka et al., 2002). ERC (ELKS/Rab6-interacting protein/CAST) was identified in a yeast-two-hybrid screen for proteins interacting with the PDZ domain of RIM (Wang et al., 2002).

The N-terminus of the Drosophila AZ protein Bruchpilot is partially homologous to human and C. elegans ELKS (Wagh et al., 2006) and was recently identified as major structural component of the Drosophila NMJ and photoreceptor dense projection (T-bar) (Kittel et al., 2006, Wagh et al., 2006, Fouquet et al., 2009). At synaptic sites, loss of BRP leads to impaired calcium channel clustering in the AZ, loss of T-bars and reduced evoked response (Kittel et al., 2006). Like all CAZ proteins, also ELKS interacts with other AZ proteins: RIM1, Munc13-1 (Ohtsuka et

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al., 2002) and Liprin-α (Ko et al., 2003b) (Fig. II.6). To date, only one ELKS homolog is known in C. elegans (Ohtsuka et al., 2002). Although no synaptic defects could be distinguished in C. elegans elks-1 loss-of-function mutants, ELKS-1 does play a crucial role in regulating functionality of SYD-2 (Dai et al., 2006), a scaffolding protein implicated in AZ assembly (see 2.2.5 and III).

2.2.4. Piccolo and Bassoon

Piccolo and Bassoon are structurally related proteins (Fenster et al., 2000) and the largest of the known CAZ proteins (Cases-Langhoff et al., 1996, tom Dieck et al., 1998). Through their zinc fingers and coiled coil domains (Fig. II.5), binding to other proteins like ELKS (Takao-Rikitsu et al., 2004) and dual prenylated Rab3A and synaptobrevin2/VAMP2 (Fenster et al., 2000) is mediated. Piccolo additionally has a PDZ domain interacting with cAMPGEFII (Fujimoto et al., 2002). The C2 domains bind to L-type voltage-dependent calcium channels (Shibasaki et al., 2004) and forms calcium-dependent homo- and heterodimers with Rim2 (Fujimoto et al., 2002) suggesting a regulatory function in synaptic transmission as calcium sensor (Fig.

II.6).

Mouse Bassoon mutants encounter free floating ribbons, disturbed localization of AZ components and impaired synaptic transmission in retina and cochlear hair cells (Dick et al., 2003, Khimich et al., 2005, Frank et al., 2010), suggesting a function in synapse assembly in vertebrates. To date, no homologs were identified in C. elegans and Drosophila.

2.2.5. Liprin-α proteins

Liprin-α proteins are scaffolding proteins with N-terminal coiled coil domains and three C-terminal sterile alpha motif (SAM) domains (Fig. II.5) originally identified as LAR Interaction Protein (LIP.1). Vertebrate Liprin-α2 and -α3 are brain-specific, while Liprin-α1 and -α4 are expressed also outside the nervous system (Serra-Pages et al., 1998). With their C-terminal coiled coil domains, they interact with RIM (Schoch et al., 2002), ELKS (Ko et al., 2003b) and G protein-coupled receptor kinase-interacting protein (GIT) (Ko et al., 2003a) (Fig. II.6). They additionally bind to the SV motor

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protein KIF1A/UNC-104 (Shin et al., 2003) and the postsynaptic glutamate receptor interacting protein (GRIP) (Wyszynski et al., 2002).

The invertebrates Drosophila and C. elegans carry only one liprin-α gene: Dliprin-α and syd-2, respectively. Dliprin-α was shown to be required for normal presynaptic AZ morphology and proper synaptic transmission (Kaufmann et al., 2002). The C.

elegans homolog SYD-2 (synapse defective 2) was isolated in a genetic screen for mutations affecting SV localization in C. elegans motor neurons (Zhen and Jin, 1999). SYD-2 has been implicated in the recruitment of several synaptic proteins to presynaptic DPs suggesting it to be a key regulator of DP assembly (Zhen and Jin, 1999; Patel et al., 2006).

Although many AZ proteins have been identified and various genetic and biochemical interactions were demonstrated, the mechanism by which these proteins form the highly complex CAZ is largely unknown. In fact, most proteins localize independently of each other to synaptic sites (Koushika et al., 2001, Deken et al., 2005).