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Frances M Brodsky - One of the best experts on this subject based on the ideXlab platform.
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actin scaffolding by Clathrin Heavy Chain is required for skeletal muscle sarcomere organization
Journal of Cell Biology, 2014Co-Authors: Stephane Vassilopoulos, Jeanne Lainé, Christel Gentil, Pierreolivier Buclez, Agathe Franck, Arnaud Ferry, Guillaume Precigout, Robyn Roth, John E Heuser, Frances M BrodskyAbstract:The ubiquitous Clathrin Heavy Chain (CHC), the main component of Clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM–sarcomere attachment sites also known as costameres. We show that Clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.
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conformation switching of Clathrin light Chain regulates Clathrin lattice assembly
Developmental Cell, 2010Co-Authors: Jeremy D Wilbur, Peter K Hwang, Michael Lane, Benjamin D Sellers, Matthew P Jacobson, Robert J Fletterick, Frances M BrodskyAbstract:Summary Clathrin-coated vesicle formation is responsible for membrane traffic to and from the endocytic pathway during receptor-mediated endocytosis and organelle biogenesis, influencing how cells relate to their environment. Generating these vesicles involves self-assembly of Clathrin molecules into a latticed coat on membranes that recruits receptors and organizes protein machinery necessary for budding. Here we define a molecular mechanism regulating Clathrin lattice formation by obtaining structural information from co-crystals of Clathrin subunits. Low resolution X-ray diffraction data (7.9–9.0 A) was analyzed using a combination of molecular replacement with an energy-minimized model and noncrystallographic symmetry averaging. Resulting topological information revealed two conformations of the regulatory Clathrin light Chain bound to Clathrin Heavy Chain. Based on protein domain positions, mutagenesis, and biochemical assays, we identify an electrostatic interaction between the Clathrin subunits that allows the observed conformational variation in Clathrin light Chains to alter the conformation of the Clathrin Heavy Chain and thereby regulates assembly.
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the Clathrin Heavy Chain isoform chc22 functions in a novel endosomal sorting step
Journal of Cell Biology, 2010Co-Authors: Christopher Esk, Chihying Chen, Ludger Johannes, Frances M BrodskyAbstract:Clathrin Heavy Chain 22 (CHC22) is an isoform of the well-characterized CHC17 Clathrin Heavy Chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome–TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.
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a role for the chc22 Clathrin Heavy Chain isoform in human glucose metabolism
Science, 2009Co-Authors: Stephane Vassilopoulos, Chihying Chen, Sachiko Hoshino, Birgit Funke, Alex M Plocik, Woodring E Wright, Raju Kucherlapati, Frances M BrodskyAbstract:Intracellular trafficking of the glucose transporter GLUT4 from storage compartments to the plasma membrane is triggered in muscle and fat during the body's response to insulin. Clathrin is involved in intracellular trafficking, and in humans, the Clathrin Heavy-Chain isoform CHC22 is highly expressed in skeletal muscle. We found a role for CHC22 in the formation of insulin-responsive GLUT4 compartments in human muscle and adipocytes. CHC22 also associated with expanded GLUT4 compartments in muscle from type 2 diabetic patients. Tissue-specific introduction of CHC22 in mice, which have only a pseudogene for this protein, caused aberrant localization of GLUT4 transport pathway components in their muscle, as well as features of diabetes. Thus, CHC22-dependent membrane trafficking constitutes a species-restricted pathway in human muscle and fat with potential implications for type 2 diabetes.
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t cell receptor engagement leads to phosphorylation of Clathrin Heavy Chain during receptor internalization
Journal of Experimental Medicine, 2004Co-Authors: Victoria L Crotzer, Allan Mabardy, Arthur Weiss, Frances M BrodskyAbstract:T cell receptor (TCR) internalization by Clathrin-coated vesicles after encounter with antigen has been implicated in the regulation of T cell responses. We demonstrate that TCR internalization after receptor engagement and TCR signaling involves inducible phosphorylation of Clathrin Heavy Chain (CHC) in both CD4+ and CD8+ human T cells. Studies with mutant Jurkat T cells implicate the Src family kinase Lck as the responsible enzyme and its activity in this process is influenced by the functional integrity of the downstream signaling molecule ZAP-70. CHC phosphorylation positively correlates with ligand-induced TCR internalization in both CD4+ and CD8+ T cells, and CHC phosphorylation as a result of basal Lck activity is also implicated in constitutive TCR endocytosis by CD4+ T cells. Remarkably, irreversible CHC phosphorylation in the presence of pervanadate reduced both constitutive and ligand-induced TCR internalization in CD4+ T cells, and immunofluorescence studies revealed that this inhibition affected the early stages of TCR endocytosis from the plasma membrane. Thus, we propose that CHC phosphorylation and dephosphorylation are involved in TCR internalization and that this is a regulatory mechanism linking TCR signaling to endocytosis.
Richard G. W. Anderson - One of the best experts on this subject based on the ideXlab platform.
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a requirement for ankyrin binding to Clathrin during coated pit budding
Journal of Biological Chemistry, 1999Co-Authors: Peter Michaely, Adeela Kamal, Richard G. W. AndersonAbstract:Abstract Recent studies suggest that the mobility of Clathrin-coated pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during coated pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrinR binds to the N-terminal domain of Clathrin Heavy Chain with high affinity. Addition of peptides containing the D4 region inhibited Clathrin-coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). AnkyrinR peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of Clathrin-coated pits during endocytosis.
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Clathrin Heavy Chain is required for pinocytosis the presence of large vacuoles and development in dictyostelium
Journal of Cell Biology, 1992Co-Authors: Theresa J Ohalloran, Richard G. W. AndersonAbstract:To investigate the intracellular role of the Clathrin Heavy Chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in Clathrin Heavy Chain expression. Immunoblots stained with an anti-Clathrin Heavy Chain antiserum revealed that mutant cells contained undetectable amounts of Clathrin Heavy Chain protein. Similarly, Northern blots showed an absence of Clathrin Heavy Chain mRNA. Clathrin Heavy Chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of Clathrin Heavy Chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.
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Characterization of the Clathrin Heavy Chain from Dictyostelium discoideum
DNA and Cell Biology, 1992Co-Authors: Theresa J. O'halloran, Richard G. W. AndersonAbstract:ABSTRACT We report the cloning and analysis of a Clathrin Heavy-Chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the Clathrin Heavy Chain ...
Sandra K. Lemmon - One of the best experts on this subject based on the ideXlab platform.
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Novel functions of Clathrin light Chains: Clathrin Heavy Chain trimerization is defective in light Chain-deficient yeast.
Journal of Cell Science, 1997Co-Authors: Kristen M. Huang, Lisa Gullberg, Karen K. Nelson, Christopher J. Stefan, Kendall J. Blumer, Sandra K. LemmonAbstract:Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The Clathrin triskelion contains three Heavy Chains, which provide the structural backbone of the Clathrin lattice and three light Chains, which are thought to regulate the formation or disassembly of Clathrin coats. To better understand the function of the Clathrin light Chain, we characterized yeast strains carrying a disruption of the Clathrin light Chain gene (CLC1). Light Chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the Clathrin Heavy Chain gene (CHC1). In clc1-delta cells, the steady state level of the Clathrin Heavy Chain was reduced to 20%-25% of wild-type levels and most of the Heavy Chain was not trimerized. If CHC1 was overexpressed in clc1-delta cells, Heavy Chain trimers were detected and several clc1-delta phenotypes were partially rescued. These results indicate that the light Chain is important for Heavy Chain trimerization and the Heavy Chain still has some function in the absence of the light Chain. In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the Heavy Chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-delta scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-delta scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the Heavy Chain, the light Chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the light Chain can function independently of the Clathrin Heavy Chain in yeast.
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sequence of the Clathrin Heavy Chain from saccharomyces cerevisiae and requirement of the cooh terminus for Clathrin function
Journal of Cell Biology, 1991Co-Authors: Sandra K. Lemmon, Alexandra Pellicenapalle, Kathleen Conley, Carol L FreundAbstract:The sequence of the Clathrin Heavy Chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat Clathrin Heavy Chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for Clathrin light Chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for Clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in Clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal Clathrin-coated vesicle structure and function.
Theresa J Ohalloran - One of the best experts on this subject based on the ideXlab platform.
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Clathrin Heavy Chain is required for spore cell but not stalk cell differentiation in dictyostelium discoideum
Development, 1997Co-Authors: Maria L Niswonger, Theresa J OhalloranAbstract:Previous studies of a Clathrin-minus Dictyostelium cell line revealed important roles for Clathrin Heavy Chain (Clathrin) in endocytosis, secretion of lysosomal hydrolases and osmoregulation. In this paper, we examine the contribution of Clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, Clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although Clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of Clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using Clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of Clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, Clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for Clathrin during Dictyostelium development. First, Clathrin increases the efficiency of early development. Second, Clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, Clathrin is essential for differentiation of mature spore cells.
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Clathrin Heavy Chain functions in sorting and secretion of lysosomal enzymes in dictyostelium discoideum
Journal of Cell Biology, 1994Co-Authors: Tracy Ruscetti, James A Cardelli, Maria L Niswonger, Theresa J OhalloranAbstract:The Clathrin Heavy Chain is a major component of Clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the Clathrin Heavy Chain gene (chcA) in Dictyostelium discoideum to generate a stable Clathrin Heavy Chain-deficient cell line. Measurement of pinocytosis in the Clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of Clathrin Heavy Chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that Clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in Clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in Clathrin-minus and wild-type cells. Like wild-type cells, Clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the Clathrin Heavy Chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.
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Clathrin Heavy Chain is required for pinocytosis the presence of large vacuoles and development in dictyostelium
Journal of Cell Biology, 1992Co-Authors: Theresa J Ohalloran, Richard G. W. AndersonAbstract:To investigate the intracellular role of the Clathrin Heavy Chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in Clathrin Heavy Chain expression. Immunoblots stained with an anti-Clathrin Heavy Chain antiserum revealed that mutant cells contained undetectable amounts of Clathrin Heavy Chain protein. Similarly, Northern blots showed an absence of Clathrin Heavy Chain mRNA. Clathrin Heavy Chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of Clathrin Heavy Chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.
Cheng-guang Liang - One of the best experts on this subject based on the ideXlab platform.
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Clathrin Heavy Chain 1 Plays Essential Roles During Oocyte Meiotic Spindle Formation and Early Embryonic Development in Sheep.
Frontiers in cell and developmental biology, 2021Co-Authors: Zhe Han, Xin Hao, Cheng-jie Zhou, Jun Wang, Xin Wen, Xing-yue Wang, De-jian Zhang, Cheng-guang LiangAbstract:As a major protein of the polyhedral coat of coated pits and vesicles, Clathrin molecules have been shown to play a stabilization role for kinetochore fibers of the mitotic spindle by acting as inter-microtubule bridges. Clathrin Heavy Chain 1 (CLTC), the basic subunit of the Clathrin coat, plays vital roles in both spindle assembly and chromosome congression during somatic-cell mitosis. However, its function in oocyte meiotic maturation and early embryo development in mammals, especially in domesticated animals, has not been fully investigated. In this study, the expression profiles and functional roles of CLTC in sheep oocytes were investigated. Our results showed that the expression of CLTC was maintained at a high level from the germinal vesicle (GV) stage to metaphase II stage and that CLTC was distributed diffusely in the cytoplasm of cells at interphase, from the GV stage to the blastocyst stage. After GV breakdown (GVBD), CLTC co-localized with beta-tubulin during metaphase. Oocyte treatments with taxol, nocodazole, or cold did not affect CLTC expression levels but led to disorders of its distribution. Functional impairment of CLTC by specific morpholino injections in GV-stage oocytes led to disruptions in spindle assembly and chromosomal alignment, accompanied by impaired first polar body (PB1) emissions. In addition, knockdown of CLTC before parthenogenetic activation disrupted spindle formation and impaired early embryo development. Taken together, the results demonstrate that CLTC plays a vital role in sheep oocyte maturation via the regulation of spindle dynamics and an essential role during early embryo development.
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cytoskeleton associated protein 5 and Clathrin Heavy Chain binding regulates spindle assembly in mouse oocytes
Oncotarget, 2017Co-Authors: Cheng-jie Zhou, Zhe Han, Donghui Wang, Xiangwei Kong, Zhizhong Yun, Cheng-guang LiangAbstract:Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to Clathrin Heavy Chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation.
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Clathrin Heavy Chain 1 is required for spindle assembly and chromosome congression in mouse oocytes
Microscopy and microanalysis : the official journal of Microscopy Society of America Microbeam Analysis Society Microscopical Society of Canada, 2013Co-Authors: Jie Zhao, Li Liu, Lu Wang, Hong-xia Zhou, Heide Schatten, Cheng-guang LiangAbstract:Clathrin Heavy Chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.