Flow cytometric detection of the association between cell surface receptors and the cytoskeleton

Nebe, Barbara; Bohn, Wolfgang; Pommerenke, Hagen; Rychly, Joachim

doi:10.1002/(sici)1097-0320(19970501)28:1<66::aid-cyto8>3.0.co;2-f

Cited by 21 publications

(17 citation statements)

References 24 publications

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“…Cross-linking by formaldehyde or through a secondary antibody increased NP40 and TX100 detergent resistance dramatically (data not shown for 3T3-MDR1 cells, but presented below for LS174T cells, see Fig. 3A) (22)(23)(24)(47)(48)(49)(50)(51)(52), suggesting that the anchorage of Pgp is very sensitive to its cross-linking or that cross-linking to already anchored molecules is very efficient. Due to the rather extreme level of Pgp expression in mdr1-transfected NIH 3T3 cells (ϳ5 ϫ 10 5 Pgps/cell; data not shown), partitioning of the protein into the different membrane compartments may not reflect the physiologic behavior of the transporter.…”

Section: Measurements Of Detergent Resistance On High and Low Pgp Expmentioning

confidence: 88%

“…With this technique, we were able to distinguish between the resistances of mAb-labeled Pgp to NP40 and TX100 extractions and show the sensitivity of our method to the nature of associations. Flow cytometric analysis of NP40 extraction was used previously to measure the extent of cytoskeletal associations of cell surface molecules in lymphocytes, and it produced data substantiated by biochemical, microscopic, and electron microscopic results (22)(23)(24)(47)(48)(49). TX100 is the detergent most widely and reliably applied to discriminate biochemically among proteins residing in different lipid domains, i.e., in raft and non-raft milieus (21).…”

Section: Discussion In Low-expressor Cells Kinetic Fcdr Resolvesmentioning

confidence: 99%

“…Biochemical separation focuses on rafts released from cells; all the cytoskeleton-associated membrane constituents ending up in the pellet does not resolve possible population heterogeneities and provides qualitative rather than quantitative information. We have presented a flow cytometric method for the analysis of detergent resistant rafts in an accompanying paper (Gombos et al), and this method uses an approach similar to that used previously for the assessment of cytoskeletal anchorage of different membrane proteins (22)(23)(24). The method is based on the idea that treatment of live cells with nonionic detergents must leave membrane constituents attached to the cytoskeleton, which is amenable to flow cytometric analysis.…”

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confidence: 99%

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

et al. 2004

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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-

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Section: Measurements Of Detergent Resistance On High and Low Pgp Expmentioning

confidence: 88%

Section: Discussion In Low-expressor Cells Kinetic Fcdr Resolvesmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2004

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“…A comparative analysis of the arrangement of actin, tubulin, and vimentin ®lament systems in tsp53 transfected versus non-transfected cells revealed no di erences, indicating that ectopic expression of tsp53 did not alter the normal arrangement of these ®lament systems. To determine binding of tsp53 to the cytoskeleton the cells were lysed with a non-ionic detergent, applying conditions which were shown to produce properly preserved cytoskeletons without remnants of membrane (Bohn et al, 1993;Nebe et al, 1997;Okabe and Hirokawa, 1988). The strong cytoplasmic¯uorescence signal of tsp53 in extracted cells ( Figure 1D) suggested that the bulk of this protein was not lost during cell lysis but was retained on the cytoskeleton.…”

Section: Tsp53 Is Bound To the Cytoskeleton In If (Vimentin) Positivementioning

confidence: 99%

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (Vimentin) scaffold

Klotzsche

Hohenberg

et al. 1998

Oncogene

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The temperature-sensitive mutant tsp53 val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (398C), but is rapidly transported into the cell nucleus at the permissive temperature (308C). tsp53 thus may serve as a model for analysing cellular parameters in¯uencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells di ering in their expression of the intermediate ®lament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate ®lament de®cient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate ®lament system.

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“…These studies applied a relatively high (0.5%) concentration of a medium-strength nonionic detergent (Nonidet-40, NP40) and identified detergentresistant subpopulations of membrane immunoglobulin (IgM) (16), various T-cell surface proteins (17), major histocompatibility class (MHC II) molecules (18), or integrin family proteins (19), all of which were interpreted as a resistance due to constitutive cytoskeletal anchoring. Very recently, another flow cytometric approach has been reported (20), in which detergent solubility was analyzed in cells 30 min after extraction with 1% cold Triton X-100 and formaldehyde fixation.…”

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confidence: 99%

Cholesterol sensitivity of detergent resistance: A rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins

et al. 2004

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Background: Lipid rafts are cholesterol-and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with timeand cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. Methods: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. Results: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37°C efficiently resolved detergent solubility or resistance of

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Flow cytometric detection of the association between cell surface receptors and the cytoskeleton

Cited by 21 publications

References 24 publications

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

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

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (Vimentin) scaffold

Cholesterol sensitivity of detergent resistance: A rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins

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