Considering that RAB-5 and RAB-10 localize to discrete compartments in neuronal cell somas as well as to synapses (Brown et al., 2009; this study), a major question, which remains to be answered is, where does the RAB-5 / RAB-10 exclusion cascade occur? Given the current set of data, it is difficult to discriminate between the neuronal cell soma and the synapse. If it were to take place in the cell soma, then this cascade would likely occur in synchrony with the maturation process of iDCVs to mDCVs. In this scenario, disruption of the RAB-5 / RAB-10 cascade would lead to missorting of essential factors necessary for mDCV formation. Three such factors are: Syntaxin 6, VAMP4 and Synaptotagmin-IV. All three of these proteins are found on iDCVs and not on mDCVs in neuroendocrine PC12 cells (Tooze et al., 2001). Syntaxin 6 has been shown to be necessary for the homotypic fusion events preceding mDCV formation and is subsequently sorted to the endo-lysosomal system (Wendler et al., 2001). Synaptotagmin-IV has been shown to bind to syntaxin 6 to regulate homotypic fusion events (Ahras et al., 2006). Interestingly, recent studies in neurons has shown that certain amounts of synaptotagmin IV are also found on mDCVs and that it acts as an inhibitor of DCV release (Dean et al., 2009;
Zhang et al., 2009). These results suggest the modulation of synaptotagmin-IV levels have consequences on DCV secretion. It will be interesting to determine the localization of syntaxin 6 and synaptotagmin IV in rab-5 and rab-10 mutants. It is possible that these factors are missorted onto mDCVs. It will also be interesting to conduct a proteomic analysis of DCV content in rab-5 and rab-10 mutants to identify additional factors that may be missorted in these mutants.
In the second scenario, it is possible that the RAB-5 / RAB-10 cascade occurs at the synapse, perhaps on DCVs. A recent finding that mammalian Rab5 and Rab10 are found on purified DCVs from rat spinal cord neurons, would support this (Zhao et al., 2011). It is unlikely that these RABs are involved in DCV transport since the DCVs are able to reach the synapse in these rab-5 and rab-10 mutants. One possibility is that, once at the synapse, DCVs must form specific microdomains prior to fusion. A functional RAB-5/ RAB-10 cascade would lead to the formation of specific RAB-5 and RAB-10 microdomains. In the absence of these microdomains the DCVs may be rendered fusion incompetent. This raises the question: what is the purpose of these microdomains. A simple suggestion would be that they are necessary for efficient tethering of DCVs, prior to fusion. Previous studies have implicated
Rab10 in the insulin-triggered translocation of glucose transporter 4 (GLUT4) vesicles (GSVs) to the plasma membrane in adipocytes. Depletion of Rab10 leads to a drastic decrease in exocytosis of GSVs suggesting a role of this Rab in fusion (Sano et al., 2007; Sano et al., 2008). An appealing idea would be that the role of Rab10 in GSV fusion is analogous to its role in DCV fusion in C. elegans. Interestingly, in line with this, additional studies have implicated EHBP-1 and TBC1D4 (a mammalian Rab10 GAP) in GLUT4 translocation (Guilherme et al., 2004; Minea et al., 2005). To date, a role of Rab-5 has not been established, nor a formal RAB-5 / RAB-10 exclusion cascade described in GSV exocytosis. Additional work needs to be conducted to elucidate the potential role of these molecules to verify if the GLUT4 translocation pathway is similar to that of DCV release.
Alternatively, the requirement of the RAB-5 / RAB-10 exclusion cascade at the synapse may involve a role of the actin cytoskeleton. Presynaptic axon terminals have been shown to contain large amounts of filamentous actin (Fifkova and Dilay, 1982; Landis et al., 1988). SVs of the reserve pool were shown to be linked to the actin cytoskeleton through proteins called synapsins (Hilfiker et al., 1999). Synapsins were shown to act as negative regulators of high frequency induced SV exocytosis (Pieribone et al., 1995; Li et al., 1995). Interestingly, it has also been shown recently that synapsins act as negative regulators of DCV release in chromaffin cells (Villanueva et al., 2006). Parallel studies in chromaffin cells have also revealed that actin remodeling contributes significantly to the release of cargoes from DCVs (Felmy, 2007) and that the interaction between an actin motor protein, myosin-Va, and syntaxin-1 plays a role in DCV release (Watanabe et al., 2005). These studies suggest that DCVs are anchored on actin filaments and that exocytosis of DCVs is modulated by actin rearrangements at the synapse. Interestingly EHBP-1 contains a calponin homology (CH) domain. Since, CH domains bind to actin filaments (Korenbaum and Rivero, 2002), it is possible that the interaction of RAB-10 with EHBP-1 is necessary for a remodeling of the synaptic actin cytoskeleton. Such remodeling could be necessary to enable DCV mobility prior to fusion. In this case, the disruption of the RAB-5 / RAB-10 exclusion cascade would trap the DCVs at the synapses preventing them from fusing. Additional studies on the effect of EHBP-1 on synaptic actin dynamics would help to elucidate its role in DCV release.
In summary, we have identified through a systematic analysis of all RABs that RAB-5 and RAB-10 are novel regulators of DCV secretion in C. elegans. We have proposed that these molecules work together through a RAB exclusion cascade where active RAB-5 inactivates RAB-10 through recruiting TBC-4. This cascade is thought to occur in the neuronal cell soma or at the synapse. Furthermore, it was shown that disruption of the cascade perturbs DCV release. It is likely that this defect occurs either due to missorting of essential factors for fusion, failure to form the appropriate Rab microdomains necessary for tethering, or due to incorrect actin remodeling necessary for fusion.