scholarly journals Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization

2011 ◽  
Vol 195 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Matteo Bonazzi ◽  
Lavanya Vasudevan ◽  
Adeline Mallet ◽  
Martin Sachse ◽  
Anna Sartori ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Actin Binding ◽  
Coated Pits ◽  
Surface Receptors ◽  
Clathrin Heavy Chain ◽  
Adhesion Sites ◽  
Infection Models ◽  
Large Particles

Bacterial pathogens recruit clathrin upon interaction with host surface receptors during infection. Here, using three different infection models, we observed that host–pathogen interactions induce tyrosine phosphorylation of clathrin heavy chain. This modification was critical for recruitment of actin at bacteria–host adhesion sites during bacterial internalization or pedestal formation. At the bacterial interface, clathrin assembled to form coated pits of conventional size. Because such structures cannot internalize large particles such as bacteria, we propose that during infection, clathrin-coated pits serve as platforms to initiate actin rearrangements at bacteria–host adhesion sites. We then showed that the clathrin–actin interdependency is initiated by Dab2 and depends on the presence of clathrin light chain and its actin-binding partner Hip1R, and that the fully assembled machinery can recruit Myosin VI. Together, our study highlights a physiological role for clathrin heavy chain phosphorylation and reinforces the increasingly recognized function of clathrin in actin cytoskeletal organization in mammalian cells.

scholarly journals Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

2020 ◽  
Author(s):  
Ridim D Mote ◽  
Jyoti Yadav ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Shivprasad Patil ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Young’S Modulus ◽  
Heavy Chain ◽  
Young's Modulus ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Actin Binding ◽  
Clathrin Heavy Chain

AbstractMouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Young’s modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young’s modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Internalization of cholera toxin by different endocytic mechanisms

2001 ◽  
Vol 114 (20) ◽  
pp. 3737-3747 ◽  
Author(s):  
Maria L. Torgersen ◽  
Grethe Skretting ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Cholera Toxin ◽  
Heavy Chain ◽  
Large Fraction ◽  
Cell Types ◽  
Coated Pits ◽  
Bhk Cells ◽  
Clathrin Heavy Chain ◽  
Antisense Mrna ◽  
Inducible Synthesis ◽  
Different Cell Types

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-β-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

scholarly journals Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

2011 ◽  
Vol 22 (19) ◽  
pp. 3699-3714 ◽  
Author(s):  
Douglas R. Boettner ◽  
Helena Friesen ◽  
Brenda Andrews ◽  
Sandra K. Lemmon
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Deletion Mutation ◽  
Actin Binding ◽  
Synthetic Genetic Array ◽  
Array Analysis ◽  
Clathrin Heavy Chain ◽  
N Terminus ◽  
Mechanistic Basis

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin–binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.

scholarly journals Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

1992 ◽  
Vol 118 (6) ◽  
pp. 1371-1377 ◽  
Author(s):  
T J O'Halloran ◽  
R G Anderson
Keyword(s):  
Heavy Chain ◽  
Antisense Rna ◽  
Fluid Phase ◽  
Solid Particles ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Clathrin Heavy Chain ◽  
Development Cycle ◽  
Mutant Cells

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.

scholarly journals Clathrin Heavy Chain 1 Plays Essential Roles During Oocyte Meiotic Spindle Formation and Early Embryonic Development in Sheep

2021 ◽  
Vol 9 ◽  
Author(s):  
Zhe Han ◽  
Xin Hao ◽  
Cheng-Jie Zhou ◽  
Jun Wang ◽  
Xin Wen ◽  
...  
Keyword(s):  
Embryo Development ◽  
Heavy Chain ◽  
Spindle Assembly ◽  
Early Embryo ◽  
Meiotic Spindle ◽  
Coated Pits ◽  
Spindle Formation ◽  
Early Embryo Development ◽  
Clathrin Heavy Chain ◽  
First Polar Body

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.

scholarly journals The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1119-1134 ◽  
Author(s):  
C Bazinet ◽  
A L Katzen ◽  
M Morgan ◽  
A P Mahowald ◽  
S K Lemmon
Keyword(s):  
X Chromosome ◽  
Heavy Chain ◽  
P Element ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Wild Type ◽  
Degenerate Primers ◽  
Coated Pits ◽  
Lethal Mutations ◽  
Clathrin Heavy Chain

Abstract The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

Developmental and morphological regulation of clathrin-mediated endocytosis inTrypanosoma brucei

2001 ◽  
Vol 114 (14) ◽  
pp. 2605-2615 ◽  
Author(s):  
Gareth W. Morgan ◽  
Clare L. Allen ◽  
Tim R. Jeffries ◽  
Michael Hollinshead ◽  
Mark C. Field
Keyword(s):  
Heavy Chain ◽  
Developmental Regulation ◽  
Bloodstream Form ◽  
Host Immune System ◽  
Coat Proteins ◽  
Flagellar Pocket ◽  
Coated Pits ◽  
Ultrastructural Studies ◽  
Clathrin Heavy Chain ◽  
Collecting Tubules

Essentially all macromolecular communication between Trypanosoma brucei and its host is confined to vesicular trafficking events occurring at or around the flagellar pocket. The vertebrate stage bloodstream form trypomastigote exhibits an extremely high rate of endocytosis required for nutrient uptake and probably also evasion of the host immune system. However, the rate of endocytosis is very low in the procyclic vector parasite, indicating that endocytosis is subject to a marked level of developmental regulation. Previous ultrastructural studies and crude biochemical fractionations have indicated the presence of coated pits and vesicles that are analogous to clathrin coats in the bloodstream form, but not in the procyclic. However, a definitive description of the components of this coat and its molecular function in T. brucei has remained elusive. We describe the molecular cloning and initial characterisation of components of the T. brucei endocytic coats: clathrin heavy chain (TbCLH) and a β-adaptin (TbAPβ1). TbCLH is markedly upregulated in the bloodstream form compared with the procyclic, whereas TbAPβ1 is subject to more limited developmental regulation. We generated antisera against both proteins and show that the clathrin coat is tightly associated with the flagellar pocket in both major life stages. However, in bloodstream parasites TbCLH is also extensively distributed throughout the posterior end of the cell on numerous large vesicular and tubular structures. By cryoimmuno EM, clathrin is localised to collecting tubules at the flagellar pocket and is also associated with the trans-Golgi network. These EM data confirm that the electron dense coats reported on trypanosome vesicles and tubules contain clathrin. The TbAPβ1 exhibits an atypical distribution relative to previously characterised adaptins, associating not only with the trans-Golgi but also with other tubular-vesicular elements. Localisation of TbAPβ1 is also subject to developmental regulation. These data describe major endocytic coat proteins in T. brucei for the first time, and indicate stage-specific expression of the clathrin heavy chain. Modulation of clathrin expression is likely to be an important factor in the developmental regulation of endocytosis and recycling in the African trypanosome.

scholarly journals Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Gilles Moulay ◽  
Jeanne Lainé ◽  
Mégane Lemaître ◽  
Masayuki Nakamori ◽  
Ichizo Nishino ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Genetic Control ◽  
Heavy Chain ◽  
Muscle Development ◽  
Tissue Homeostasis ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Coated Pits ◽  
Clathrin Heavy Chain

Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.

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