Cytoskeletal interactions of synapsin I in non-neuronal cells

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.