Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo.

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function.

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance.

Clathrin light chains CLCa and CLCb have non-redundant roles in epithelial lumen formation.

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 with 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, although 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.

Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System.

The early secretory pathway encompasses the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) organelles. The ERGIC is now understood to be a complex cargo sorting hub involved in a variety of cellular and tissue processes, however the traffic pathways to and from the ERGIC are still unclear.Classical methods employed for the analysis of a cargo 's journey along the secretory pathway rely on reversible traffic blocks leading to cargo accumulation in the ER . Although these methods were key to characterize Golgi and post-Golgi traffic routes, their poor specificity to the cargo of interest and limited spatiotemporal resolution make them inadequate for the fine characterization of cargo traffic in the early secretory pathway.In this chapter, we describe a protocol to study the traffic of cargo proteins in the early secretory pathway using the Retention Using Selective Hook (RUSH ) system, a highly specific and sensitive tracking system with a high spatiotemporal resolution. Taking GLUT4 and GLUT1 as examples of unconventionally and conventionally secreted cargo respectively, we describe the steps to clone the cargoes in the RUSH vector and follow and quantify their traffic along the early secretory pathway. This RUSH method can also be used to study the traffic of other cargo proteins in the early secretory pathway.

CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis.

Glucose transporter 4 (GLUT4) is sequestered inside muscle and fat and then released by vesicle traffic to the cell surface in response to postprandial 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 ER-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 downregulation of either p115 or CHC22, but not GM130 or sortilin, abrogates insulin-responsive GLUT4 release. This indicates that 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.

Genetic diversity of CHC22 clathrin impacts its function in 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, altering its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22's role in metabolism and have potential to differentially influence the human insulin response.