Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging shows that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Cellular cryo-electron tomography reveals dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and find that S protein recognizes integrin αvß3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin α v ß 3 . Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

In situ cryo-electron tomography reveals local cellular machineries for axon branch development.

Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows the formation of new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to the lack of direct in-depth observations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches accompanied by filopodia-like protrusions. Microtubules and ER comigrated into preformed branches to support outgrowth together with accumulating compact, ∼500-nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events supporting the hypothesis of local protein synthesis selectively taking place at axon branches, allowing them to serve as unique control hubs for axon development and downstream neural network formation.

Cytoskeleton and Membrane Organization at Axon Branches.

Axon branching is a critical process ensuring a high degree of interconnectivity for neural network formation. As branching occurs at sites distant from the soma, it is necessary that axons have a local system to dynamically control and regulate axonal growth. This machinery depends on the orchestration of cellular functions such as cytoskeleton, subcellular transport, energy production, protein- and membrane synthesis that are adapted for branch formation. Compared to the axon shaft, branching sites show a distinct and dynamic arrangement of cytoskeleton components, endoplasmic reticulum and mitochondria. This review discusses the regulation of axon branching in the context of cytoskeleton and membrane remodeling.

Direct induction of microtubule branching by microtubule nucleation factor SSNA1.

Microtubules are central elements of the eukaryotic cytoskeleton that often function as part of branched networks. Current models for branching include nucleation of new microtubules from severed microtubule seeds or from γ-tubulin recruited to the side of a pre-existing microtubule. Here, we found that microtubules can be directly remodelled into branched structures by the microtubule-remodelling factor SSNA1 (also known as NA14 or DIP13). The branching activity of SSNA1 relies on its ability to self-assemble into fibrils in a head-to-tail fashion. SSNA1 fibrils guide protofilaments of a microtubule to split apart to form daughter microtubules. We further found that SSNA1 localizes at axon branching sites and has a key role in neuronal development. SSNA1 mutants that abolish microtubule branching in vitro also fail to promote axon development and branching when overexpressed in neurons. We have, therefore, discovered a mechanism for microtubule branching and implicated its role in neuronal development.

Structural insights into the cooperative remodeling of membranes by amphiphysin/BIN1.

Amphiphysin2/BIN1 is a crescent-shaped N-BAR protein playing a key role in forming deeply invaginated tubes in muscle T-tubules. Amphiphysin2/BIN1 structurally stabilizes tubular formations in contrast to other N-BAR proteins involved in dynamic membrane scission processes; however, the molecular mechanism of the stabilizing effect is poorly understood. Using cryo-EM, we investigated the assembly of the amphiphysin/BIN1 on a membrane tube. We found that the N-BAR domains self-assemble on the membrane surface in a highly cooperative manner. Our biochemical assays and 3D reconstructions indicate that the N-terminal amphipathic helix H0 plays an important role in the initiation of the tube assembly and further in organizing BAR-mediated polymerization by locking adjacent N-BAR domains. Mutants that lack H0 or the tip portion, which is also involved in interactions of the neighboring BAR unit, lead to a disruption of the polymer organization, even though tubulation can still be observed. The regulatory region of amphiphysin/BIN1 including an SH3 domain does not have any apparent involvement in the polymer lattice. Our study indicates that the H0 helix and the BAR tip are necessary for efficient and organized self-assembly of amphiphysin/N-BAR.

Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca(2+) entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca(2+) import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.