UMMS Affiliation

Program in Molecular Medicine; Department of Physiology

Publication Date

10-1-2004

Document Type

Article

Subjects

3T3 Cells; 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

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: Mol Cell Biol. 2004 Oct;24(20):9102-23. Link to article on publisher's site

DOI of Published Version

10.1128/MCB.24.20.9102-9123.2004

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

Molecular and cellular biology

PubMed ID

15456883

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