Glycolipid-enriched membrane domains are assembled into membrane patches by associating with the actin cytoskeleton.
Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.
Rodgers
W
W
Department of Pathology, Yale University School of Medicine, 330 Cedar Street, New Haven, Connecticut 06520-8061, USA.
Zavzavadjian
J
J
D000199:Actins / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017830:Octoxynol / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000199:Actins; D006017:Glycolipids; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008563:Membrane Lipids; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D017830:Octoxynol; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)