An example of this application is shown by the removal of Rab proteins from synaptic vesi-cles by GDI. A hypothesis that Rab3 plays an important role in synaptic vesicle recruitment rather than docking has emerged over the years [258]. Although Rab3 have a suggested role in regulating dense core vesicle docking [16, 15], the precise function of Rab3 in synaptic
trans-mission is still not fully understood. Rab3 directly interacts with the active zone scaffolding protein Rim [75] providing a physical link between synaptic vesicles and the presynaptic active zone [68]. Additionally, a similar reduction of docked vesicles in Rab3-, Rim- or Rab3/Rim-double mutants in C. elegans were observed, assuming that this interaction might be crucial for the tethering of synaptic vesicles [79]. On the other hand, a recent study showed that Rab3 directly associates with the tail of the actin motor protein myosin5a. supporting a role of Rab3 in the transport of neuronal vesicles rather than the attachment to the plasma membrane [205].
However, as outlined in the introduction 1.1.1, docking is mainly defined as a morphological observation of vesicles located in close proximity to the plasma membrane. This only provides a static picture at a given time point and does not reflect the dynamic actions of docked vesicles that constantly undergo association and dissociation from the plasma membrane [259]. This is further supported by the relative high amount of synaptic vesicles observed in the docked vesicle fraction compared to the free vesicle fraction. Such a high number of vesicles does not reflect the morphologically observed low number of vesicles attached to the plasma membrane.
Here, alterations in the docked vesicle fraction were analyzed as a consequence of a GDI induced removal of Rab proteins from synaptic vesicles. The effect of GDI treatment on the attachment of SVs as well as global changes in the presynaptic proteome as a consequence of GDI incubation were examined using the iTRAQ based quantitation. In this study, addition of recombinant GDI only led to a significant decrease in the amount of Rab proteins in the presence of excess GDP, but synaptic vesicles remained attached to the plasma membrane.
This provides direct evidence that Rab3 is not required for attaching synaptic vesicles to the plasma membrane after docking had taken place. However, docking might not be determined by a single protein-protein interaction. In fact, deletion of a large number of proteins (Munc18, Munc13, Rim [260], synaptotagmin [47], syntaxin [2]) result in changes in the amount of vesicles attached to the plasma membrane, indicating that docking is possibly mediated by a series of protein-protein interactions. Thus, Rab3 might only contribute to docking as a transient contact between vesicle and active zone, that is followed by additional factors that determine the attachment of vesicles to the plasma membrane.
• Munc18 has been shown to promote docking in vivo [4, 7, 6], possibly mediated by binding to the "closed" conformation of synatxin 1 [8, 9]. But at this stage it remains elusive if the attached vesicles are only docked or already primed. Therefore, it is also possible that Munc18 binds to the "open" synatxin 1, stabilizing the SNARE acceptor complex [261] and thus facilitating SNARE assembly [262, 263]. On the vesicular side synaptotagmin seems to be the prime candidate for docking aside from its function as the neuronal calcium sensor. In chromaffin cells, synaptotagmin has been shown bind
to the SNARE acceptor complex, anchoring vesicles and promoting SNARE assembly [261, 47]. However, docking mechanisms in chromaffin cells might be different from neurons, as these cells do not contain many of the active zone proteins.
• The SNARE complex is very stable [264]. Assuming that SNARE proteins are already assembled at this stage (possibly as a consequence of Munc18), this interaction itself might be sufficient to keep vesicles attached.
• Cytoskeletal components, in particular the F-actin network, might entrap synaptic vesi-cles in the subplasmalemmal cytoskeleton. Actin dynamics play an important role in in the presynaptic nerve terminal [265, 266] and interact with the vesicle protein synapsin [125]. Considering the large number of cytoskeletal components identified in this study, I assume that the cytoskeletal network persists in the isolated docked vesicle fraction.
Apart from a possible direct interaction between the cytoskeleton and synaptic vesicle, de-attachment of vesicles might just simply be abolished because these whole organelles get physically entangled in these network.
• It cannot be completely ruled out that the Rab3 mediated effect on docking is sufficiently compensated by Rab27b. These Rab proteins share common GEFs [267, 268] and appear to have overlapping functions in synaptic vesicle exocytosis [21]. Similar to Rab3, Rab27 has also been shown to influence docking [17, 18], but in contrast to Rab3, Rab27 is resistent to GDI-membrane retrieval [21]. Instead, inactive GDP-bound Rab27b has been suggested to persist on membrane as an inactive homodimer [269].
Strikingly, except for the reduction of Rab proteins, the molecular composition of the active zone remains constant. Precisely, the amount of more than 500 proteins that were identified were unchanged. Surprisingly, this included the Rab3 interacting protein Rim. Rim proteins are large multi-domain molecules that are proposed to function as central organizers interact-ing with multiple proteins [87, 67, 85]. However, the Rab3-bindinteract-ing site is localized to the N-terminus, whereas other parts of Rim mediate different functions. For example, the central PDZ domain interacts with calcium channels, localizing them to the active zone [84]. Impor-tantly, these domains appear to act autonomously[270]. Thus, a disruption of the Rab3-Rim-interaction does not interfere with Rim function in tethering calcium channels to release sites.
This possibly accounts for Rim localization at the active zone. Taken together I hypothesize that Rab3 is possibly involved in the first contact of synaptic vesicles with the presynaptic ac-tive zone via its interaction with Rim and thus might initiate the attachment of vesicles to the plasma membrane, but it does not restrict the diffusion of these vesicles after docking.