Biology subjects

Brodsky, F. M.

Publications and source records attributed to Brodsky, F. M..

6 recordsLinked to original sources

bioRxiv2026

Clathrin differentially adapts its trimerisation domain during mammalian evolution to traffic the insulin-responsive GLUT4 glucose transporter.

In humans, the CHC22 isoform of clathrin regulates glucose metabolism by trafficking the GLUT4 glucose transporter for intracellular storage in skeletal muscle and release following insulin signalling. Some vertebrate lineages have lost the gene encoding CHC22 but operate the same insulin-stimulated GLUT4 trafficking pathway. Here, we show that species lacking CHC22 exclusively produce an alternatively-spliced form of the universally expressed CHC17 clathrin isoform (CHC17-SAS) with a truncated C-terminus similar to CHC22, expressed predominantly in skeletal muscle. Through its trimerisation domain, CHC17-SAS binds the CHC22-specific adaptor SNX5 that enables CHC22's distinct intracellular function. The 2.3 [A] crystal structure of the CHC22 trimerisation domain demonstrates conservation of the core trimeric fold from CHC17 but differences in electrostatic surface charge that may account for their differential properties. Using GLUT4 translocation assays in HeLa cell models, we show that CHC17-SAS is a functional surrogate for CHC22. Identification of CHC17-SAS resolves the evolutionary conundrum posed by CHC22 absence in some vertebrate lineages, and reveals a common mechanism for mammalian GLUT4 trafficking.

cell biology↗

bioRxiv2023

Epithelial lumen development requires differential participation of the vertebrate clathrin light chain isoforms with dominance of CLCa in vivo

To identify functional differences between vertebrate clathrin light chains (CLCa or CLCb), phenotypes of mice lacking genes encoding either isoform were characterised. Mice without CLCa displayed 50% neonatal mortality, reduced body weight, reduced fertility, and [~]40% of aged females developed uterine pyometra. Mice lacking CLCb displayed a less severe weight reduction phenotype compared to those lacking CLCa, and had no survival or reproductive system defects. Analysis of female mice lacking CLCa that developed pyometra revealed ectopic expression of epithelial differentiation markers (FOXA2 and K14) and a reduced number of endometrial glands, indicating defects in the lumenal epithelium. Defects in lumen formation and polarity of epithelial cysts derived from uterine or gut cell lines were also observed when either CLCa or CLCb were depleted, with more severe effects from CLCa depletion. In cysts, the CLC isoforms had different distributions relative to each other, while they converge in tissue. Together, these findings suggest differential and cooperative roles for CLC isoforms in epithelial lumen formation, with a dominant function for CLCa.

cell biology↗

bioRxiv2022

CHC22 clathrin membrane recruitment uses SNX5 in bipartite interaction with secretory tether p115

The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, transports the glucose transporter GLUT4 from the endoplasmic-reticulum-to-Golgi intermediate compartment (ERGIC) directly to an intracellular GLUT4 storage compartment (GSC) from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, the molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. The C-terminal trimerization domain of CHC22 binds SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both heavy chain termini, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi bypass route for GLUT4 traffic. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.

cell biology↗

bioRxiv2020

Identifying a GSK3-TRARG1 signaling axis reveals BCL9L as a novel regulator of GLUT4 trafficking

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

cell biology↗

bioRxiv2018

Evolution of the CLTCL1 Gene Encoding CHC22 Clathrin Reveals Selection Influencing CHC22’s Role in Human Glucose Metabolism

CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans. We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene, revealing independent gene loss in at least two vertebrate lineages, after arising from gene duplication. All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin, which mediates endocytosis. For vertebrates retaining CLTCL1, strong evidence for purifying selection supports CHC22 functionality. All human populations maintained two high frequency CLTCL1 allelic variants, encoding either methionine or valine at position 1316. Functional studies indicated that CHC22-V1316, which is more frequent in farming populations than in hunter-gatherers, has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic, attenuating its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22s role in metabolism and have potential to differentially influence the human insulin response.

genetics↗

bioRxiv2018

CHC22 Clathrin Diverts GLUT4 from the ER-to-Golgi Intermediate Compartment for Intracellular Sequestration

Glucose Transporter 4 (GLUT4) is sequestered inside muscle and fat, then released by vesicle traffic to the cell surface in response to post-prandial insulin for blood glucose clearance. Here we map the biogenesis of this GLUT4 traffic pathway in humans, which involves clathrin isoform CHC22. We observe that GLUT4 transits through the early secretory pathway more slowly than the constitutively-secreted GLUT1 transporter and localize CHC22 to the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). CHC22 functions in transport from the ERGIC, as demonstrated by an essential role in forming the replication vacuole of Legionella pneumophila bacteria, which requires ERGIC-derived membrane. CHC22 complexes with ERGIC tether p115, GLUT4 and sortilin and down-regulation of either p115 or CHC22, but not GM130 or sortilin abrogate insulin-responsive GLUT4 release. This indicates CHC22 traffic initiates human GLUT4 sequestration from the ERGIC, and defines a role for CHC22 in addition to retrograde sorting of GLUT4 after endocytic recapture, enhancing pathways for GLUT4 sequestration in humans relative to mice, which lack CHC22.\n\nSummaryBlood glucose clearance relies on insulin-mediated exocytosis of glucose transporter 4 (GLUT4) from sites of intracellular sequestration. We show that in humans, CHC22 clathrin mediates membrane traffic from the ER-to-Golgi Intermediate Compartment, which is needed for GLUT4 sequestration during GLUT4 pathway biogenesis.

cell biology↗