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29
Inactivation of clathrin heavy chain inhibits synaptic
recycling but allows bulk membrane uptake
Jaroslaw Kasprowicz 1 , Sabine Kuenen 1 , Katarzyna Miskiewicz 1 , Patrik Verstreken 1 .
Katholieke Universiteit Leuven 1
Abstract:
Synaptic vesicle cycling depends on clathrin, an abundant protein that polymerizes around
newly forming vesicles and dynamin a protein that mediates fission of newly formed vesicles.
However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test
clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants
combined with chc photoinactivation to circumvent early embryonic lethality associated with
chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads
to substantial membrane internalization visualized by live dye uptake and electron microscopy.
Our data not only indicate that chc is critical for synaptic vesicle recycling but also show that in
the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization.
Furthermore, inactivation of chc in the context of other endocytic mutations like dap 160, synj
as well as shibire ts1 (dynamin mutant) results in membrane uptake. Qualitative similarities
between phenotypes of inactivated chc and recently published data on Dynamin 1 KO mice
stimulated neurons (Hayashi et al. 2009) may suggest that dynamin and clathrin directly
cooperate in vivo. To further scrutinize these interactions in vivo we will employ the FALI
technique to inactivate dynamin in shi null mutant flies. Our work will shed light onto the
regulatory mechanisms of SV formation and the role of clathrin and dynamin in this process.