Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin–glycan interactions in clathrin-independent endocytosis
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Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin–glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin–glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes. Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin–glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin–glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.Keywords:
Galectin
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.
Clathrin adaptor proteins
Bulk endocytosis
Cell membrane
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Az blocks the association of Hsc70 with clathrin and consequently suppresses the disassembly of CCVs during clathrin-mediated endocytosis.
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ABSTRACT Clathrin-mediated endocytosis is the main entry route for most cell surface receptors and their ligands. It is regulated by clathrin-coated structures that are endowed with the ability to cluster receptors and to locally bend the plasma membrane, resulting in the formation of receptor-containing vesicles that bud into the cytoplasm. This canonical role of clathrin-coated structures has been shown to play a fundamental part in many different aspects of cell physiology. However, it has recently become clear that the ability of clathrin-coated structures to deform membranes can be perturbed. In addition to chemical or genetic alterations, numerous environmental conditions can physically prevent or slow down membrane bending and/or budding at clathrin-coated structures. The resulting ‘frustrated endocytosis’ is emerging as not merely a passive consequence, but one that actually fulfils some very specific and important cellular functions. In this Review, we provide an historical and defining perspective on frustrated endocytosis in the clathrin pathway of mammalian cells, before discussing its causes and highlighting the possible functional consequences in physiology and diseases.
Cell membrane
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Internalization
Cell membrane
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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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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.
Pinocytosis
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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.
Pinocytosis
Wortmannin
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Internalization
Clathrin adaptor proteins
Lipid microdomain
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Clathrin adaptor proteins
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Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers. PMID: 17306539
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