Plasma membrane of cultured cells

The immuuoreplica technique is a high-resolution technique for the localization of antigens on the plasma membrane of cultured cells. After immunolabeling, the cells are replicated with carbon and platinum to reveal immunolabeling on the cell surface. 

The immunoreplica technique (14) is used when it is necessary to detect antigenic sites on the plasma membrane of cultured cells. The cells are cultured on coverslips, and are fixed as described above depending on the antibody in question, and immunolabeled in situ as described in Section 3.1.1.2., steps 3-9. After immunolabeling (Section 3.1.1.2., step 9), they are further fixed with 1% osmium tetroxide and are dehydrated in a graded series of ethanol (70, 90, 100%), critically point-dried, and replicated with a layer of carbon and platinum, The replicas are cleaned with sodium hypochlorite and chronic acid before examination with the transmission electron microscope. Large areas of the replicated plasma membrane remain intact for observation. Colloidal gold probes are probably the only probes of sufficient density that can be detected on these surfaces. 

Martin, O.C. and Pagano, R.E., 1987, Transbilayer movement of florescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells../. Biol. Chem., 262 5890-5898. 

Klenk, H.-D., and Choppin, P. W., 1970, Glycosphingolipids of plasma membranes of cultured cells and an enveloped virus (SV5) found in these cells, Proc. Natl. Acad. Sci. U.S. 66 57. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Hansen GH, Belhage B, Schousboe A (1991) Effect of GABA agonist on the expression and distribution of GABAa receptors in the plasma membrane of cultured cerebellar granule cells an immunocytochemical study. Neurosci. Lett., 124, 162-165. 

One of the key differences between RP2 and cofactor C is membrane association mediated via post-translational modification. Dual N-terminal acyl modification by myristoylation and palmitoylation target RP2 to the cytoplasmic face of the plasma membrane in cultured cells (Chappie et al., 2000) and in cells throughout the retina (Grayson et al., 2002). A pathogenic mutation AS6 (Rosenberg et al., 1999 Schwahn et al, 1998) in RP2 prevents the plasma membrane targeting of the protein (Chappie et al, 2000, 2002 Schwahn et al., 2001), illustrating that this post-translational modification is vital for the function of the protein in the retina. RP2 is not partitioned to either the apical or basolateral domains of polarized... 

The total number of polypeptides in the culture medium was 8-20 bands according to electrophoretic analysis (Figure 1). The protein export was inhibited by actinomycin D, cycloheximide, 2-deoxy-D-glucose and also by concanavalin A (Con A). In the presence of Con A (400 pg/ml), the enzymes were accumulated entirely inside the cells but not in the periplasm or the cell wall (Figure 2). At the same time the lectin had no influence upon cell growth and expression of invertase and acid phosphatase associated with the cell envelope. The activity accumulated inside Con A - treated cells was released into the culture medium on a-methylmannoside addition (Figure 3). The process was not affected by cycloheximide but was inhibited by sodium azide. The effect of Con A appears to be conditioned by its interaction with the plasma membrane of intact cells. Besides Con A, we detected peroxidase interaction with the plasmalemma of intact cells and alkaline phosphatase internment under physiological conditions. 

Rosenberg, S. A., and Einstein, Jr., A. B., 1972, Sialic acids on the plasma membrane of cultured human lymphoid cells, J. Cell. Biol. 53 466. 

Purdue, J. F., Kletzien, R., and Miller, K., 1971 z, The isolation and characterization of plasma membrane from cultured cells. I. The chemical composition of membrane isolated from uninfected and oncogenic RNA virus-converted chick embryo fibroblasts, Biochim. Biophys. Acta 249 419-434. 

A final aspect of this topic which should be mentioned is the apparent failure of hexosaminidase which is present in high concentration in fetal calf serum (—2000 units/ml) to cross the plasma membrane of cultured skin and amnio tic cells. Fetal calf serum is usually added to... 

Attempts to study the entry of ES products into cells using markers of fluid phase endocytosis yielded unexpected results. When larvae browse resistant IEC-6 cells in the presence of extracellular fluorescent dextran, dextran enters the cytoplasm of a significant proportion of the cells in the mono-layer (Butcher et al., 2000). The parameters of dextran entry are most compatible with the conclusion that larvae wound the plasma membranes of IEC-6 cells that is, they create transient breaches in the membrane that allow impermeant markers to enter the cell (McNeil and Ito, 1989). Wounding is considered to be a common occurrence in intestinal epithelia (McNeil and Ito, 1989). Injured cells are able to heal their wounds by recruiting vesicles to seal the breach (Steinhardt et al., 1994). In an experimental system, healing allows the injured cell to retain cytoplasmic dextran. In epithelial cell cultures inoculated with T. spiralis larvae, the relationship between glycoprotein delivery and injury of plasma membranes is not clear, i.e. dextran-laden cells do not always stain with Tyv-specific antibodies and... 

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