The synapse is a specialized site of cell-cell contact and functions to transmit signals between neurons and their targets. The presynaptic site consists of an electron dense active zone surrounded by synaptic vesicles. It is well established that vesicular biogenesis at the Golgi and subsequent transport by microtubule-based motor proteins known as kinesins is essential for the accumulation of synaptic vesicles. Extensive biochemical, morphological and physiological studies, however, have shown that the chemical composition, the size and the shape of newly synthesized, actively transported synaptic vesicles differ from those that are present at functional synapses. These observations thus imply that synaptic vesicles require a 'maturation' step to be accumulated at synapses and to carry out their function. Compared to what we have learned about kinesin-based synaptic vesicle transport, little is known about how synaptic vesicle precursors mature. At least two pathways are implicated in vesicle maturation; precursor vesicles may mature following an initial fusion with the plasma membrane or through an intermediate endosomal compartment. Using a SNB-1::GFP marker to visualize presynaptically localized synaptic vesicles (Nonet, 1999), 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. Our studies, together with others, demonstrate an evolutionarily conserved function for the JIP3 family of proteins acting as a molecular tether for conventional kinesin (Byrd et al, 2001; Verhey et al, 2001; Bowman et al, 2000). In worm, UNC-116/KHC and KLC-2 constitute a conventional kinesin-1 complex and this complex physically interacts with UNC-16 (Sakamoto et al, in preparation). However, transport of synaptic vesicles also requires the UNC-104 kinesin; synaptic vesicles (SV's) are retained in the cell bodies of
unc-104 mutants (Hall and Hedgecock, 1991). By genetic double mutant analysis, we have made an intriguing observation that
unc-16 mutations, but not
unc-116 , partially suppresses the SV retention defect of
unc-104 mutants. This observation implies that UNC-16 has functions that are independent of UNC-116 (Byrd and Jin, [
wm2003ab577]). We report here our recent progress on understanding this kinesin-independent function of UNC-16. Using a set of GFP markers generated by M. Rolls (2002), we first addressed where in the secretory pathway UNC-16 and two kinesins, UNC-116 and UNC-104 function. We found that the ER markers were not affected in
unc-16 and kinesin mutant animals. 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 SV's 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. In
unc-16 mutant adult animals, the fluorescent puncta from SNB-1::GFP are larger than normal. By quantitative confocal imaging analysis, we found that the size of SNB-1::GFP puncta was increased, on average 1.25 fold compared to wild type. However, by ultrastructural analysis we detected an overall reduction of morphologically normal synaptic vesicles at synapses in
unc-16 mutants. We interpret these observations to mean that SNB-1::GFP may be retained in some kind of 'intermediate' vesicular compartment; and such compartments likely contribute to the generation of morphologically mature synaptic vesicles. We reason that a key to this interpretation is to determine precisely the vesicular compartment containing UNC-16. Towards this goal, we have begun a series of colocalization studies between UNC-16 and various markers to membrane components of the secretory pathway (MansII/Golgi, SNB-1/synaptic vesicles and Rab-5/Endosome). Results from these cell biological and genetic analyses will be presented at the meeting.