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Abstract
Differential solubilization of membrane components by cold 1% Triton X-100 extraction is common practice in cell biology and membrane research, used to define components of, or localization within membrane domains called lipid rafts. In this study, extraction of biological membranes was continuously monitored in single cells by confocal microscopy. The distributions of fluorescently-tagged proteins that label raft and non-raft membranes, cytosolic and cytoskeletal proteins were continuously monitored upon addition of the detergent. Membranes containing the non-raft membrane protein VSVG-GFP were immediately extracted from the plasma membrane, whereas raft-membrane proteins were predominantly resistant to the detergent. The morphological characteristics of differential membrane solubilization consisted of the formation of pores that expand and percolate as the detergent-mediated solubilization proceeds. Pore expansion and percolation was much slower and more restricted in non-polarized MDCK cells than in COS-7 cells. Heterologous overexpression in COS-7 cells of the fluorescently-tagged human MAL, a tetra-spanning, lipid-raft-associated protein, significantly slowed and limited membrane pore expansion and percolation. Extensive percolation resulting in large holes in the membrane was observed for the raft-associated, GPI-GFP-labeled membranes in COS-7 cells. Quantitative analysis carried out using pixel intensity variance as an indicator of membrane pore expansion demonstrated that the MAL protein is capable of modifying the plasma membrane, thereby increasing its resistance to detergent-induced pore formation.
| Abbreviations | ||
| detergent-resistant membrane | = | DRM |
| pixel fluorescence intensity variance | = | PFIVar |
| Madin Darby Canine Kidney | = | MDCK |
| fluorescent protein | = | FP |
| Triton X-100 | = | TX-100 |
| Abbreviations | ||
| detergent-resistant membrane | = | DRM |
| pixel fluorescence intensity variance | = | PFIVar |
| Madin Darby Canine Kidney | = | MDCK |
| fluorescent protein | = | FP |
| Triton X-100 | = | TX-100 |