Phosphatidylinositol-4,5-bisphosphate-rich plasma membrane patches organize active zones of endocytosis and ruffling in cultured adipocytes
Huang, Shaohui ; Lifshitz, Lawrence M. ; Patki, Varsha ; Tuft, Richard A. ; Fogarty, Kevin E. ; Czech, Michael P.
Citations
Student Authors
Faculty Advisor
Academic Program
UMass Chan Affiliations
Document Type
Publication Date
Keywords
Actins
Adipocytes
Animals
Cell Membrane
Cell Membrane Structures
Cells, Cultured
Clathrin
Clathrin-Coated Vesicles
Cytoskeleton
Endocytosis
Fluorescent Dyes
Isoenzymes
Mice
Microscopy, Fluorescence
Models, Biological
Myosins
Phosphatidylinositol 4,5-Diphosphate
Phospholipase C delta
Protein Structure, Tertiary
Recombinant Fusion Proteins
Transferrin
Type C Phospholipases
Life Sciences
Medicine and Health Sciences
Subject Area
Embargo Expiration Date
Link to Full Text
Abstract
A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cdelta1 (PLCdelta1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCdelta1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.
Source
Mol Cell Biol. 2004 Oct;24(20):9102-23. Link to article on publisher's site