Raft and cytoskeleton associations of an ABC transporter: P‐glycoprotein

Abstract

Background:A novel flow cytometric assay has been described in an accompanying report (Gombos et al., Methods: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. Results: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft mark-

Hyaluronan Synthesis Induces Microvillus-like Cell Surface Protrusions

“…Microvillus-type cell protrusions represent a distinct cellular compartment that harbors important regulatory functions (43). Among others, multidrug resistance transporter proteins (51,52) and ErbB2 receptors (53) reside in microvilli. Hyaluronan synthesis increases ErbB2 activation (9), and ErbB2 stimulates hyaluronan synthesis via a phosphoinositide 3-kinase-dependent positive feedback loop, resulting in enhanced expression and activity of multidrug resistance protein 1 (54).…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…Still, recent studies, examining the ratio of active state conformations of ABCB1 by the conformation sensitive UIC2 antibody, indicate that this protein may be more active in cholesterol-rich, caveolin-positive regions and thus may be regulated by factors altering raft assembly (Bacso et al, 2004). Treatment of cells by β-methyl cyclodextrin (β-MCD) for cholesterol depletion or the application of cholesterol-loaded β-MCD for cholesterol enrichment was reported to modify the transport functions of ABCB1 in several experiments, but in other reports, no significant effects were observed (Luker, Pica, Kumar, Covey, & Piwnica-Worms, 2000;Telbisz et al, 2007).…”

The effects of detergents on the polymerization properties of actin

“…This raises the question, how the detergents influencing actin polymerization in vitro act on DRMs in intact cells. This approach is appealing, since recently, a flow cytometric method was developed for DRM isolation, called FCDR assay (29)(30)(31)(32)(33)(34). In these works, raft-associated proteins labeled by fluorescently tagged antibodies remained cell associated, after detergent solubilization of the whole cells.…”

“…Lipid rafts involved in cell signaling or membrane and protein trafficking are plasma membrane lipid heterogeneities containing elevated amounts of sphingolipids, cholesterol, GPI-anchored and other particular ''raft'' proteins (23)(24)(25)(26)(27)(28). The methodologically defined detergent-resistant membranes (DRMs), which considered to show comparable chemical properties to rafts, are identified applying the same TX100, Brij and CHAPS detergents and used in recently developed cytometric DRM measurements [flow cytometric detergent resistance (FCDR) assay] (29)(30)(31)(32)(33)(34).…”