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 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.

Light Chain C-Terminal Region Reinforces the Stability of Clathrin Heavy Chain Trimers

Traffic ◽  
2007 ◽  
Vol 8 (8) ◽  
pp. 1101-1110 ◽  
Author(s):  
Joel A. Ybe ◽  
Samantha Perez-Miller ◽  
Qian Niu ◽  
David A. Coates ◽  
Michael W. Drazer ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Terminal Region ◽  
Clathrin Heavy Chain ◽  
The Stability

A Chimeric Streptokinase with Unexpected Fibrinolytic Selectivity

1996 ◽  
Vol 76 (03) ◽  
pp. 429-438 ◽  
Author(s):  
Joel Goldstein ◽  
Gary R Matsueda ◽  
Shyh-Yu Shaw
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Limited Proteolysis ◽  
Clot Lysis ◽  
Wild Type ◽  
Fab Fragment ◽  
Plasma Clot ◽  
N Terminus ◽  
Lag Period ◽  
Similar Kinetic

SummaryChimeric 59D8-SK was designed to confer fibrin-selectivity to streptokinase by fusion of the Fab fragment of anti-fibrin antibody 59D8 to the N-terminus of streptokinase (SK: Ile1-Lys414). It was expressed in a mouse hybridoma cell line and purified by affinity chromatography on a 59D8-antigen column. Chimeric 59D8-SK is a disulfide-linked heterodimer composed of an antibody light chain (Mr 27,000) and a N-glycosylated chimeric heavy chain (Mr 90,000). The fibrin targeting by 59D8 increased plasma clot lysis by 2-fold, but connecting 59D8 to SK has provided 59D8-SK several unique properties: (i) 59D8-SK activated human Glu-plasminogen with a significant lag period that coincided with limited proteolysis of 59D8-SK similar to that observed for wild-type SK. In a kinetic study, both gave very similar kinetic parameters for the activation of Glu-plasminogen even though 59D8-SK was N-glycosylated in its SK portion; (ii) 59D8-SK was relatively inactive in human plasma, compared to SK, but it became activated in the presence of clots; (iii) 59D8-SK lysed clots slowly but completely whereas SK lysed clots rapidly but incompletely. Even though the mechanism behind these new properties is not fully understood, they are characteristics of a second-generation plasminogen activator.

scholarly journals Clathrin heavy chain, light chain interactions.

1983 ◽  
Vol 2 (8) ◽  
pp. 1393-1400 ◽  
Author(s):  
F.K. Winkler ◽  
K.K. Stanley
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Clathrin Heavy Chain

Abstract 437: Lipoprotein(a) Internalization in HepG2 Cells is Regulated by Proprotein Convertase Subtilisin Kexin Type 9 Through the Low-Density Lipoprotein Receptor

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Rocco Romagnuolo ◽  
Nabil G Seidah ◽  
Marlys L Koschinsky
Keyword(s):  
Heavy Chain ◽  
Hepg2 Cells ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Proprotein Convertase ◽  
Human Hepatoma Cells ◽  
Lipoprotein A ◽  
Clathrin Heavy Chain

Elevated levels of lipoprotein(a) (Lp(a)) have been identified as an independent and causal risk factor for coronary heart disease. Lp(a) consists of an LDL-like moiety covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). The mechanism by which Lp(a) is catabolized is currently unknown, but may form the basis for the development of drug therapy to reduce high levels of plasma Lp(a). Although the role of the LDL receptor (LDLR) in Lp(a) catabolism is controversial, recent evidence has shown that Lp(a) levels are significantly reduced with an antibody against proprotein convertase subtilisin kexin type 9 (PCSK9) in patients with hypercholesterolemia receiving statin therapy. Therefore, we explored the role of PCSK9 in Lp(a)/apo(a) internalization by hepatic cells. Lp(a) or apo(a) internalization is significantly reduced in HepG2 (human hepatoma) cells either by overexpressing PCSK9 or by treatment with purified PCSK9. The ability of Lp(a) and apo(a) to be internalized was significantly reduced in the presence of the lysine analogue, ε-ACA, indicating lysine-dependent interactions with cellular receptors. Mutation of the strong lysine binding site in a recombinant apo(a) variant resulted in a reduced ability to be internalized. While LDL can bind to PCSK9 and inhibit its ability to degrade the LDLR, we found that Lp(a) lacked these properties. Interestingly, overexpressing the LDLR on HepG2 cells significantly increased the ability of Lp(a) to be internalized, an effect that was partially reduced by the addition of PCSK9. This indicates a potential key role for the LDLR in regulating Lp(a) catabolism. Furthermore, knockdown of clathrin heavy chain resulted in a significant decrease in apo(a) internalization and apo(a) internalization was not further reduced by pre-treatment of PCSK9 in the context of clathrin heavy chain knockdown. Treatment of HepG2 cells with a lysosomal inhibitor, but not a proteosomal inhibitor, resulted in accumulation of Lp(a) in HepG2 cells indicating that Lp(a) is potentially targeted for degradation through lysosomes. Taken together, these results indicate that Lp(a)/apo(a) uptake can be regulated in HepG2 cells by PCSK9 and the LDLR through clathrin-mediated endocytosis and lysosomal degradation.

Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast

1997 ◽  
Vol 110 (7) ◽  
pp. 899-910 ◽  
Author(s):  
K.M. Huang ◽  
L. Gullberg ◽  
K.K. Nelson ◽  
C.J. Stefan ◽  
K. Blumer ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
State Level ◽  
Light Chains ◽  
Chain Gene ◽  
Light Chain Gene ◽  
Heavy Chains ◽  
Clathrin Heavy Chain ◽  
Multicopy Suppressors ◽  
Delta Cells

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.

The Regulatory Role of Myosin Light Chain Kinase as an Actin-Binding Protein1

1994 ◽  
Vol 116 (6) ◽  
pp. 1377-1382 ◽  
Author(s):  
Li-Hong Ye ◽  
Kohichi Hayakawa ◽  
Yuan Lin ◽  
Tsuyoshi Okagaki ◽  
Koichiro Fujita ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Regulatory Role ◽  
Actin Binding

scholarly journals Kinetics of Factor VIII Light-Chain Cleavage by Thrombin and Factor Xa. A Regulatory Role of the Factor VIII Heavy-Chain Region Lys713-Arg740

1996 ◽  
Vol 240 (2) ◽  
pp. 365-372 ◽  
Author(s):  
Marie-Jose S. H. Donath ◽  
Peter J. Lenting ◽  
Jan A. Mourik ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Xa ◽  
Regulatory Role ◽  
Chain Cleavage ◽  
Kinetics Of

scholarly journals The occurrence of disulphide bonds in purified clathrin light chains

1989 ◽  
Vol 257 (3) ◽  
pp. 775-781 ◽  
Author(s):  
P Parham ◽  
F M Brodsky ◽  
K Drickamer
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Reduced Form ◽  
Light Chains ◽  
Cysteine Residues ◽  
Free Light Chains ◽  
Clathrin Heavy Chain ◽  
Disulphide Bonds ◽  
Antigenic Properties ◽  
Reduced Forms

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.

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