Kesheng Liu et al. (2002) West Coast Worm Meeting "Functional characterization of a structural motif in UNC-11 that has been implicated in phosphoinositide binding."

Ravit Golan, Kondury Prasad, Kesheng Liu, Aixa Alfonso, Suping Jin, Eileen M Lafer, Andrea M Holgado

[West Coast Worm Meeting, 2002]

In C. elegans, the unc-11 gene encodes a family of proteins with structural and functional homology to the mammalian monomeric clathrin adaptor protein AP180 (Nonet et al., 1999). In the nematode these proteins have a role in the retrieval and sorting of the integral synaptic vesicle protein synaptobrevin from the plasma membrane and to synaptic vesicles (Nonet et al., 1999).

A Holgado et al. (1999) International C. elegans Meeting "A functional analysis of the role of UNC-11, a nematode AP180 homolog, in synaptic vesicles endocytosis."

A Alfonso, A Holgado

[International C. elegans Meeting, 1999]

Endocytosis of synaptic vesicle (SV) membranes via clathrin coated pits involves adaptor/assembly proteins such as AP-2 and AP180. unc-11 encodes an AP180 homolog and has at least two functions: 1) specifying the size of SVs during recycling and 2) sorting of the SV protein synaptobrevin (Nonet et al. , 1999). In vitro , UNC-11 binds to clathrin and promotes assembly of clathrin coats (Golan, Prasad, Lafer and Alfonso, unpublished). Thus loss of unc-11 function results in both exo and endocytosis defects. In our study we were interested in dissecting the contribution of unc-11 function to endocytosis of SV membranes. We used vital fluorescent probes to learn more about the role of this clathrin adaptor/assembly protein in neuronal SV recycling. An optical FM4-64 assay, originally developed by Betz and Bewick (1992), facilitates the direct visualization of endocytosis by the activity-dependent SV uptake of the lipophilic fluorescent dye. Further stimulation of the synapse results in the activity-dependent destaining of the synaptic terminals, which can be attributable to exocytosis of the dye loaded SVs. To study the role of UNC-11 in SV recycling, we induced vesicular membrane internalization in presence of FM4-64, so only loaded SVs are detectable. Finally, we quantify the signal at the presynaptic terminals after staining and following a depolarization-dependent destaining. We have measured dye uptake in mutants of different unc-11 alleles, mutants defective in endocytosis ( dyn-1 ), exocytosis ( unc-18, snb-1 ), as well as the unc-11 double mutant combinations. Our data from unc-11 mutants suggest that UNC-11 is not essential for endocytosis of synaptic membranes. In unc-11(e47) , a null allele, we still see FM4-64 uptake, but there is a significant decrease in this uptake (18% of that of wild type). The decrease could be due to a direct effect in endocytosis, or an indirect defect in endocytosis as a result of a defect in exocytosis. Since we are using experimental conditions in which the ready releasable pool of SVs have been exhausted, exocytosis is not a limitation in the SV loading. And in absence of unc-11 function we still see a decrease in dye uptake, therefore we conclude that UNC-11 must play an important role in endocytosis.