Uptake of fluorescent nano beads into BY2‐cells involves clathrin‐dependent and clathrin‐independent endocytosis
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To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants.Keywords:
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ABSTRACT During the last decade the term ‘endocytosis’ has become virtually synonymous with the activity of clathrin-coated vesicles. These vesicles, which are derived from cell surface clathrin-coated pits, are transport vehicles responsible for the transfer of plasma membrane receptors and their ligands, between the first two stations of the endocytic pathway: namely, the plasma membrane and early endosomes (Goldstein et al., 1985; van Deurs et al., 1989; Griffiths and Gruenberg, 1991). Despite the irrefutable evidence that clathrin-coated vesicles mediate endocytosis, their contribution to the total endocytic activity of the cell and the composition of the membrane they internalise remains controversial. Here we discuss: (1) the evidence that non-clathrin-mediated endocytic mechanisms operate alongside the clathrin-mediated pathway; (2) the evidence that endocytosis occurs for surface molecules that are not enriched in clathrin-coated pits and; (3) the sorting activities of cell surface clathrincoated pits and the notion that plasma membrane proteins that show particularly slow rates of uptake are actively excluded from the endocytic pathway.
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Key features of clathrin-associated endocytosis are outlined, and current evidence in favor of clathrin-independent endocytosis and of its structural counterpart(s) is reviewed. We next summarize our recent observations on clathrin-independent endocytosis in primary cultures of rat foetal fibroblasts, using two reversible inhibitors of the formation of endocytic clathrin-coated pits, Severe inhibition of clathrin polymerization at the plasma membrane slows down receptor-mediated endocytosis of transferrin by ten-fold, without affecting bulk-flow endocytosis of fluid and membrane. Furthermore, the size of primary endocytic vesicles, identified by ultrastructural cytochemistry; is the same in control and treated cells. Two interpretations are offered. The most provocative one proposes that clathrin plays no role in the formation of primary endocytic vesicles, and is only required to concentrate receptors in endocytic pits, accelerating thereby internalization of ligand-receptor complexes. In the second interpretation, inhibition of clathrin polymerization unmasks an accessory molecular machinery, which is not operating under control conditions. In both cases, another endocytic molecular machinery is required and remains to be identified.
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Amphiphysin
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Clathrin mediated endocytosis is the principle mechanism by which cells internalize cell surface receptors. It can be thought of as a molecular machine which concentrates receptors into patches at the plasma membrane and which bends the membrane into a vesicle that pinches off. Quite how the many structural, enzymatic and cytoskeletal components of the endocytic machine are spatially and temporally organised is not fully understood. In this talk I will describe our efforts to address this problem by analysing single endocytic events using total internal fluorescence microscopy (TIR‐FM).
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A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.
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Recent evidence has proved that in addition to the well-documented clathrin-mediated endocytic route (vesicles of 100-150 nm), at least three distinct non-clathrin-coated endocytic pathways function at the surface of mammalian cells. Endocytosis via these pathways is initiated by caveolae (50-80 nm), macropinosomes (500-2000 nm) and micropinosomes (95-100 nm). The current state of knowledge about these non-clathrin coated endocytic routes is presented and evidence that endocytic routes other than via clathrin-coated vesicles are utilised by viruses is discussed. The recent advances in these areas have provided us with tools to investigate the entry of those viruses which appear to enter cells via endocytosis into non-clathrin-coated vesicles. Data indicate that these four endocytic pathways differ in the absence, presence and/or type of coat on the vesicles, the size of the vesicles, their sensitivity to a variety of inhibitors, and in the ligands endocytosed. A historical perspective of the discovery of these non-clathrin-coated endocytic pathways is provided and recent information is summarised and discussed. The entry of viruses via non-clathrin-coated pits is destined to be an exciting new area of viral-cell entry, as has been indicated recently by the finding that entry of simian virus type 40 into cells occurs via caveolae. Copyright 1997 by John Wiley & Sons, Ltd.
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