"Molecular dancing with a slippery star - Dissecting the role of PI(4,5)P2
dephosphorylation in clathrin coated pit dynamics"

Derek Toomre, Ph.D.

Department of Cell Biology

Ludwig Institute for Cancer Research

Yale University School of Medicine

SHM-C227/229

333 Cedar Street

P.O. Box 208002

New Haven, Connecticut  06520-8002

Abstract:
Phosphoinositides are thought to play an important role in clathrin-coated pit (CCP) dynamics. In a joint venture between the De Camilli and Toomre labs, we have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatial-temporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner endophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs, suggesting that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP. Rapid PI(4,5)P2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/ FKBP system. PI(4,5)P2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P2 in several steps of clathrin coat dynamics and Arp2/3-dependent actin regulation.