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Understanding of the range and mechanisms of clathrin functions has developed exponentially since clathrin's discovery in 1975. Here, newly established molecular mechanisms that regulate clathrin activity and connect clathrin pathways to differentiation, disease and physiological processes such as glucose metabolism are reviewed. Diversity and commonalities of clathrin pathways across the tree of life reveal species-specific differences enabling functional plasticity in both membrane traffic and cytokinesis. New structural information on clathrin coat formation and cargo interactions emphasizes the interplay between clathrin, adaptor proteins, lipids and cargo, and how this interplay regulates quality control of clathrin function and is compromised in infection and neurological disease. Roles for balancing clathrin-mediated cargo transport are defined in stem cell development and additional disease states.

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

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.

Deformation of the plasma membrane into clathrin‐coated vesicles is a critical step in clathrin‐mediated endocytosis and requires the orchestrated assembly of clathrin and endocytic adaptors into a membrane‐associated protein coat. The individual role of these membrane‐bending and curvature‐stabilizing factors is subject to current debate. As such, it is unclear whether the clathrin coat itself is stiff enough to impose curvature and if so, whether this could be effectively transferred to the membrane by the linking adaptor proteins. We have recently demonstrated that clathrin alone is sufficient to form membrane buds in vitro. Here, we use atomic force microscopy to assess the contributions of clathrin and its membrane adaptor protein 2 (AP2) to clathrin coat stiffness, which determines the mechanics of vesicle formation. We found that clathrin coats are less than 10‐fold stiffer than the membrane they enclose, suggesting a delicate balance between the forces harnessed from clathrin coat formation and those required for membrane bending. We observed that clathrin adaptor protein AP2 increased the stiffness of coats formed from native clathrin, but did not affect less‐flexible coats formed from clathrin lacking the light chain subunits. We thus propose that clathrin light chains are important for clathrin coat flexibility and that AP2 facilitates efficient cargo sequestration during coated vesicle formation by modulating clathrin coat stiffness.

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