Proper localization and maturation of synaptic components is required for neuronal function. We have previously characterized the unc-16 gene for its role in regulating synaptic vesicle localization. UNC-16 is a member of the JIP3 protein family that includes Drosophila Sunday driver and mammalian JIP3 and has been shown to physically interact with the kinesin-I complex(Sakamoto et al, 2005). However, transport of synaptic vesicles also requires the UNC-104 kinesin; synaptic vesicles(SVs) are retained in the cell bodies of unc-104 mutants(Hall and Hedgecock,1991). We have previously reported that unc-16 mutations, but not unc-116, partially suppress the SV retention defect of unc-104 mutants. This implies that UNC-16 has functions independent of UNC-116(Byrd et al 2001). We report here our recent progress on understanding this kinesin-independent function of UNC-16.Using a set of GFP markers, we first addressed where in the secretory pathway UNC-16 and two kinesins, UNC-116 and UNC-104 function. UNC-16, kinesin-I and UNC-104 all affected the localization of SNB-1::GFP as well as a Golgi marker(MansII; Rolls et al, 2002). Like SVs and SNB-1::GFP, the Golgi marker was retained in the cell bodies of unc-104 mutants. In unc-104;unc-16 double mutants, the retention of the Golgi marker was partially suppressed. Moreover, by serial reconstruction analysis of electron micrographs, we observed that the Golgi size is altered in unc-16 single and unc-104;unc-16 double mutants. These results are consistent with the idea that UNC-16 is influencing membrane dynamics in the secretory pathway, possibly by regulating vesicular transfer. By ultrastructural analysis we detected an overall reduction of morphologically normal synaptic vesicles at synapses in unc-16 mutants. However, in unc-16 mutant adults the fluorescent puncta from SNB-1::GFP are larger than normal by quantitative confocal analysis. We interpret these observations to mean that SNB-1::GFP may be retained in an intermediate vesicular compartment; and such compartments likely contribute to the generation of morphologically mature synaptic vesicles. We reason, a key to this interpretation is to determine the vesicular compartment containing UNC-16. Towards this goal, we have begun a series of localization studies between UNC-16 and various markers to membrane components of the secretory pathway in collaboration with Barth Grants lab. Results from these cell biological and genetic analyses will be presented at the meeting.

Affiliation:
- MCD Biology, UC Santa Cruz, Santa Cruz, CA.