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PubMed2006

A role for clathrin in reassembly of the Golgi apparatus.

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.

1-Butanol↗
PubMed2000

Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like).

Anaplastic lymphoma kinase (ALK)-positive lymphomas are characterized by expression of a hybrid protein, comprising the cytoplasmic portion of the ALK tyrosine kinase fused to a partner protein. This hybrid kinase is often encoded by the nucleophosmin (NPM) NPM-ALK fusion gene resulting from the (2;5)(p23;q35) chromosomal translocation. However, the ALK gene at 2p23 may also be involved in 2 variant translocations, namely t(1;2)(q25;p23) and t(2;3)(p23;q21), which create the TPM3-ALK and TFG-ALK fusion genes, respectively. We report here 2 lymphomas with an unusual finely granular cytoplasmic ALK staining pattern, clearly different from the pattern observed in ALK-positive lymphomas carrying NPM-ALK or its variants. A cloned complementary DNA sequence from 1 of these 2 lymphomas contained the ALK gene fused to the second clathrin heavy chain gene (also referred to as clathrin heavy polypeptide-like gene) (CLTCL). The distinctive granular cytoplasmic staining pattern for ALK was likely to be due to binding of the fusion protein to clathrin-coated vesicles. The CLTCL gene is constitutively expressed in lymphoid cells and therefore presumably contributes an active promoter for the CLTCL-ALK gene. The fusion protein had a molecular weight (250 kd) that differs from all known ALK products, and it was autophosphorylated in an in vitro kinase assay, confirming that it is constitutively active and hence capable of contributing to malignant transformation. These 2 cases, therefore, represent a hitherto undescribed mechanism of ALK activation in lymphoma and further illustrate the diversity of fusion partners for the ALK gene.

Amino Acid Sequence↗
PubMed1988

Characterization of yeast clathrin and anticlathrin heavy-chain monoclonal antibodies.

Clathrin-coated vesicles (CVs) were isolated from Saccharomyces cerevisiae by using procedures developed by Mueller and Branton [17]. Triskelions were purified from this material by extraction of CVs to release clathrin and by subsequent fractionation on Sepharose CL-4B. Triskelions were composed of approximately 180,000 Mr heavy chains and a single light-chain type of approximately 38,000 Mr and were able to undergo self-assembly into polyhedral cages. Trypsin digestion of such reassembled cages showed a peptide pattern very similar to that obtained for mammalian clathrin with two fragments of 125,000 and 110,000 Mr, which represent the major portion of the heavy-chain arm, and a polypeptide of approximately 43,000 Mr, which is the presumptive terminal domain. Eight monoclonal antibodies reacting with yeast clathrin heavy chains were produced. All eight bind to the major portion of the heavy-chain arm, and none bind to the terminal domain fragment. Peptide digestion experiments also indicated that at least three major regions on the arm are recognized by these antibodies. These will be useful in further structural and functional studies of clathrin from yeast.

Antibodies, Monoclonal↗
PubMed2000

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation.

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.

Animals↗
PubMed1985

A test of clathrin function in protein secretion and cell growth.

Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible. The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion. Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11. Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA. Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains. Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate. Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles.

Biological Transport↗
PubMed2005

Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells.

Vesicular stomatitis virus (VSV) is well established to enter cells by pH-dependent endocytosis, but mechanistic aspects of its internalization have remained unclear. Here, we examined the functional role of clathrin in VSV entry by expression of a dominant-negative mutant of Eps15 (GFP-Eps15Delta95/295), a protein essential for clathrin-mediated endocytosis. Whereas expression of GFP alone had no effect on VSV infection, expression of GFP-Eps15Delta95/295 severely limited infection. As independent ways to examine clathrin function, we also examined cells that had been treated with chlorpromazine and utilized small interfering RNA (siRNA) techniques. Inhibition of clathrin-mediated endocytosis by chlorpromazine treatment, as well as clathrin knock-down using siRNA duplexes directed against the clathrin heavy chain, also prevented VSV infection. In combination with previous morphological approaches, these experiments establish clathrin as an essential component needed for endocytosis of VSV.

Adaptor Proteins, Signal Transducing↗
PubMed2006

EGF-induced activation of the EGF receptor does not trigger mobilization of caveolae.

Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-beta-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.

Caveolae↗
PubMed1997

A novel structural model for regulation of clathrin function.

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.

Amino Acid Sequence↗
PubMed2002

Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization.

A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization.

Antigen Presentation↗
PubMed2001

Hrs recruits clathrin to early endosomes.

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

Amino Acid Sequence↗
PubMed2000

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Animals↗
PubMed2003

Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis.

PURPOSE: The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis. METHODS: PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K+ depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000TM reagent) with specific antisense oligonucleotides designed against the rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated. RESULTS: Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs. CONCLUSIONS: Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in rabbit conjunctival epithelial cells.

Animals↗
PubMed1985

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.

Animals↗
PubMed1996

SCD5, a suppressor of clathrin deficiency, encodes a novel protein with a late secretory function in yeast.

Clathrin and its associated proteins constitute a major class of coat proteins involved in vesicle budding during membrane transport. An interesting characteristic of the yeast clathrin heavy chain gene (CHC1) is that in some strains a CHC1 deletion is lethal, while in others it is not. Recently, our laboratory developed a screen that identified five multicopy suppressors that can rescue lethal strains of clathrin heavy chain-deficient yeast (Chc - scd1-i) to viability. One of these suppressors, SCD5, encodes a novel protein of 872 amino acids containing two regions of repeated motifs of unknown function. Deletion of SCD5 has shown that it is essential for cell growth at 30 degrees C. scd5-delta strains carrying low copy plasmids encoding C-terminal truncations of Scd5p are temperature sensitive for growth at 37 degrees C. At the nonpermissive temperature, cells expressing a 338-amino acid deletion (Scd5P-delta 338) accumulate an internal pool of fully glycosylated invertase and mature alpha-factor, while processing and sorting of the vacuolar hydrolase carboxypeptidase Y is normal. The truncation mutant also accumulates 80- to 100-nm vesicles similar to many late sec mutants. Moreover, at 34 degrees C, overexpression of Scd5p suppresses the temperature sensitivity of a sec2 mutant, which is blocked at a post-Golgi step of the secretory pathway. Biochemical analyses indicate that approximately 50% of Scd5p sediments with a 100,000 x g membrane fraction and is associated as a peripheral membrane protein. Overall, these results indicate that Scd5p is involved in vesicular transport at a late stage of the secretory pathway. Furthermore, this suggests that the lethality of clathrin-deficient yeast can be rescued by modulation of vesicular transport at this late secretory step.

Amino Acid Sequence↗
PubMed2000

NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking.

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes-organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.

Adaptor Protein Complex 2↗
PubMed2006

The high-affinity immunoglobulin-E receptor (FcepsilonRI) is endocytosed by an AP-2/clathrin-independent, dynamin-dependent mechanism.

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FepsiloncRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcepsilonRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.

Animals↗
PubMed1999

Human monocyte activation by cleaved form of alpha-1-antitrypsin involvement of the phagocytic pathway.

Production of alpha-1-antitrypsin (AAT) by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases, of four- to eightfold, in numbers of macrophages and levels of AAT and its cleavage fragments have been found in various inflammatory loci. We have found that the C-terminal peptide (C-36) of AAT, produced by specific proteinase cleavage when added in its fibrillar form at concentrations >/=5 microM to monocytes in culture for 24 h, significantly increases low density lipoprotein (LDL) binding and uptake, up-regulates levels of LDL receptors and also induces proinflammatory cytokine (interleukin-1, interleukin-6 and tumour necrosis factor alpha) production and glutathione reductase activity. Because it is known that various cells selectively internalize surface receptors and their ligands through receptor-mediated endocytosis via clathrin-coated pits, we tested whether antibodies raised against the clathrin heavy chain would block the effects of the fibrillar form of C-36 on human monocytes in culture. Addition of excess anti-(clathrin HC) with 10 microM fibrillar C-36 diminished the stimulatory effects of the latter on LDL binding, uptake and LDL receptor levels. In contrast, however, in the presence of anti-(clathrin HC), the potentially cytotoxic effects of fibrils, such as induction of cytokines, free radicals and cytosolic activity of cathepsin D, were much greater than those observed when cells were treated with fibrils alone. These results suggest that endocytosis is the pathway by which C-36 fibrils upregulate LDL receptors, and may be the natural mechanism for fibril clearance. We infer that human monocytes clear C-36 fibrils by a clathrin-dependent pathway, presumably endocytotic, and that loss of this pathway amplifies the cytotoxic effects of the fibrils by increasing their availability to other specific or nonspecific sites through which they exert their cytotoxic effects.

Base Sequence↗
PubMed2004

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration.

The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

Animals↗