PubMed · 11212701
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H Okada. 2000. [HRF20(CD59, MACIF) deficiency].. https://pubmed.ncbi.nlm.nih.gov/11212701/
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It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 degrees C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.
Delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface takes place by transcytosis in hepatocytes and also probably in epithelial Madin-Darby canine cells. The integral protein MAL2 was demonstrated to be essential for basolateral-to-apical transcytosis in hepatoma HepG2 cells. Reduction of endogenous MAL2 levels impedes cargo delivery to the apical membrane, but, paradoxically, cargo does not accumulate in the subapical compartment where MAL2 predominantly resides but in distant endosome elements. To understand how transcytosis can be apparently mediated at a distance, we have analyzed the dynamics of machinery and cargo by live-cell imaging of MAL2 and transcytosing CD59, a GPI-anchored protein, in HepG2 cells. MAL2 was revealed as being a highly dynamic protein. Soon after basolateral endocytosis of CD59, a fraction of MAL2 redistributed into peripheral vesicular clusters that concentrated CD59 and that were accessible to transferrin (Tf) receptor, a basolateral recycling protein. Following Tf receptor segregation, the clusters fused in a MAL2(+)globular structure and moved toward the apical surface for CD59 delivery. All these processes were impaired in cells with reduced MAL2 content. Other GPI-anchored proteins examined behave similarly. As MAL2 is expressed by many types of epithelia, the sorting events described herein are probably of quite general utility.
Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins in the affected blood cell membranes. Analysis of blood cells by flow cytometry is useful to identify the affected blood cells with PNH-specific phenotypes. Because PNH-affected red blood cells (RBC) have shortened life-spans in the circulation, ratios of PNH-affected populations analyzed by flow cytometry in whole RBC are lower than those in PNH-affected erythropoiesis. We developed a reticulocyte-gated 2-color flow cytometry of RBC and revealed that the percentages of PNH-affected, CD59--populations in reticulocytes were higher than in whole RBC in patients with PNH. A serial analysis of a patient with PNH who underwent peripheral blood stem cell transplantation confirmed the usefulness of this method to evaluate PNH-affected RBC populations with high sensitivity; ie, the presence of CD59- reticulocytes in the circulation could be a sensitive marker for PNH-affected erythropoiesis.