ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) is a phosphoinositide involved in a variety of cellular functions, including signal transduction, organelle trafficking, and actin dynamics. Although the role of PtdIns[4,5]P2 in endocytosis is well established, the precise trafficking steps relying on normal PtdIns[4,5]P2 balance in the endosomal pathway have not yet been elucidated. Here we show that decrease in intracellular PtdIns[4,5]P2 levels achieved by the overexpression of the 5-phosphatase domain of synaptojanin 1 or by siRNA knock-down of PIP5Ks expression lead to severe defects in the internalization of transferrin as well as in the recycling of internalized transferrin back to the cell surface in COS-7 cells. These defects suggest that PtdIns[4,5]P2 participates in multiple trafficking and/or sorting events during endocytosis. Coexpression of the PtdIns[4,5]P2 synthesizing enzyme, PIP5KI gamma, was able to rescue these endocytic defects. Furthermore, decreased levels of PtdIns[4,5]P2 caused delays in rapid and slow membrane recycling pathways as well as a severe backup of endocytosed membrane. Taken together, our results demonstrate that PtdIns[4,5]P2 availability regulates multiple steps in the endocytic cycle in non-neuronal cells.
Subject(s)
Phosphatidylinositol 4,5-Diphosphate/metabolism , Transferrin/metabolism , Animals , Biological Transport , COS Cells , Chlorocebus aethiops , Chromatography, Thin Layer , Endocytosis , Kinetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin-regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD-95. Its overexpression increased the number and length of dendritic filopodia/spines via an N-WASP-independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD-95-induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long-term potentiation, SPIN90/WISH associated with PSD-95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD-95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD-95-dependent synaptic remodeling.
