Synaptic vesicle proteins (SVps) have a polarized distribution in neurons, and are enriched at synapses. SVps are packed into precursors of synaptic vesicles (pre-SVs), that are transported anterogradely by the Kinesin-3 family motor UNC-104, in C elegans. In
unc-104 mutants, SVps accumulate in the cell body and mislocalize to dendrites. It is known that the dendritic mis-localization of SVps in
unc-104 mutants is dependent on Dynein. We are investigating whether UNC-104 has other roles in regulating polarized distribution of SVps as motors are also known to regulate microtubule polarity and cargo sorting. We observe that in
unc-104 mutants microtubule polarity is not changed. To test whether, the loss in polarized distribution is due to reduced motor on vesicles we overexpressed PPK-1, an enzyme that makes more PI(4,5)P2, as UNC-104 is proposed to bind to vesicle membranes through PI(4,5)P2. Over-expression of PPK-1 is known to suppress cargo-binding defective alleles of UNC-104. We also observed that PPK-1 overexpression supressed the loss in polarized distribution of SVps observed in
unc-104 mutants. These data are consistent with a model where having sufficient number of UNC-104 motors on pre-SVs is required to regulate the polarized distribution of SVps. UNC-104 orthologues are proposed to be clustered in PI(4,5)P2 and cholesterol enriched lipid microdomains. To test whether clustering of lipids can mediate the polarized distribution and transport of pre-SVs, we depleted cholesterol from the growth media of C elegans. We observed that RAB-3 mislocalised to dendrites indicating that cholesterol is required for polarized distribution of SVps, although the dependence of this phenotype on PI(4,5)P2 remains to be investigated.