Meiyan Jin

Protein homeostasis (proteostasis) refers to the balance of the cellular protein environment, tightly regulated by pathways governing protein synthesis, folding, trafficking, and degradation. Growing evidence supports the interconnection of these pathways to ensure the robustness of the proteo-stasis network. A recent study by Park et al. showed that, in macroautophagy/autophagy-deficient cells, the loss of proteasome or nuclear pore components causes synthetic lethality, as cytoplasmic proteins that accumulate under impaired autophagy are transported to the nucleus and degraded by nuclear proteasomes. The authors illustrated the mechanistic basis for why cells with conditions such as Huntington disease, where both autophagy and cytoplasm-to-nuclear shuttling are compromised, are more vulnerable to proteostasis perturbation.Abbreviation: UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

Summary: During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane. Coordination of endocytic protein recruitment in time and space is important for efficient CME. Here, we show that the multivalent scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. By live-cell imaging of genome-edited cells, we observed that endogenously labeled ITSN1 is recruited to CME sites shortly after they begin to assemble. Knocking down ITSN1 impaired endocytic protein recruitment during the stabilization stage of CME site assembly. Artificially locating ITSN1 to the mitochondria surface was sufficient to assemble puncta consisting of CME initiation proteins, including EPS15, FCHO, adaptor proteins, the AP2 complex and epsin1 (EPN1), and the vesicle scission GTPase dynamin2 (DNM2). ITSN1 can form puncta and recruit DNM2 independently of EPS15/FCHO or EPN1. Our work redefines ITSN1’s primary endocytic role as organizing and stabilizing the CME protein interaction networks rather than a previously suggested role in initiation and provides new insights into the multi-step and multi-zone organization of CME site assembly.

Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood. Here, branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches. When endocytic dynamics of unperturbed CME sites are compared, sites with actin assembly show a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. In addition, N-WASP and the Arp2/3 complex are recruited to one side of CME sites, where they are positioned to stimulate asymmetric actin assembly and force production. We propose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetrically, much as a bottle opener pulls off a bottle cap.

Actin filament assembly facilitates vesicle formation in several trafficking pathways including clathrin-mediated endocytosis (CME)^1,2. However, how actin assembly forces are harnessed has not been fully revealed for any vesicle forming process. In this study, three-dimensional (3D) super-resolution microscopy, live-cell imaging of triple-genome-edited, induced pluripotent stem cells (iPSCs), and newly developed machine-learning-based computational analysis tools, were used to comprehensively analyze assembly dynamics and geometry of proteins representing three different CME functional modules. When hundreds of CME events with and without associated Arp2/3-dependent actin network assembly were compared, sites with actin assembly showed a distinct delay between completion of endocytic coat expansion and vesicle scission, consistent with the notion that these were stalled sites requiring actin assembly forces to complete vesicle formation. Importantly, our analysis showed that N-WASP is preferentially recruited to one side of CME sites, where it stimulates actin assembly asymmetrically. These results indicate that in mammalian cells actin assembly is induced at stalled CME sites, where asymmetric forces pull the plasma membrane into the cell much like a bottle opener pulls off a bottle cap.

Significance: Most cell-cell fusogens have tall ectodomains that drive fusion by undergoing conformational changes that pull two membranes together, akin to the mechanism used by viral fusogens during enveloped virus entry. In contrast, fusion-associated small transmembrane (FAST) proteins from reovirus have short, structurally minimal, membrane-disruptive ectodomains. In this work, we show that evolutionarily distant FAST proteins from aquareovirus and orthoreovirus use different adaptor proteins to hijack host actin assembly and push two membranes together during cell-cell fusion. Despite minimal sequence similarity, the cytoplasmic tails from divergent FAST proteins can be functionally exchanged and even replaced with different actin nucleators while maintaining fusogenicity. This feature suggests a conserved biophysical strategy shared by FAST proteins that could be used by other cell-cell fusogens.