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

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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 markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and GM1‐gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. Conclusions By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS‐174‐T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.
Funding Information
  • OTKA (T032563, 034393, T046945)
  • Hungarian Academy of Sciences
  • Hungarian Ministry of Health (OMFB 02692/2000, ETT 138/2001, ETT 031/2001, ETT 614/2003)