Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that the sphingomyelin (SM)-sequestered cholesterol, but not the accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol-independent. Whereas, the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein which activates actin nucleation, is recruited to IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. Importance: IAV infects the cells by harnessing cellular endocytic machineries. Better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies, and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol-independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results would provide new insights into IAV infection and pathway/cargo-specific involvement of cholesterol pool(s).

The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

Chikungunya virus (CHIKV) is a representative alphavirus causing debilitating arthritogenic disease in humans. Alphavirus particles assemble into two icosahedral protein layers: the glycoprotein spike shell embedded in a lipid envelope and the inner nucleocapsid (NC) core. In contrast to matrix-driven assembly of some enveloped viruses, the assembly/budding process of two-layered icosahedral particles remains poorly understood. Here we used cryogenic electron tomography (cryoET) to capture snapshots of the CHIKV assembly process in infected human cells. Subvolume classification of the snapshots revealed 12 intermediate structures, representing different stages of assembly/budding at the plasma membrane. Further subtomogram average structures ranging from subnanometer to nanometer resolutions show that immature, non-icosahedral NCs function as rough scaffolds to trigger icosahedral assembly of the glycoprotein spike lattice, which in turn progressively transforms the underlying NCs into icosahedral cores during budding. Here we resolve a long-standing mechanistic question about the role of spikes and NCs in assembly of two-layered icosahedral shells. Further, data of CHIKV-infected cells treated with budding-inhibiting antibodies shows that spacing spikes apart to prevent their lateral interactions prevents the plasma membrane bending around NC cores, thus blocking virus budding. These findings provide the molecular details of icosahedral enveloped virus formation and antibodies against assembly/budding.

Curvature thylakoid 1 proteins modulate prolamellar body morphology and promote organized thylakoid biogenesis in Arabidopsis thaliana

The term “de-etiolation” refers to the light-dependent differentiation of etioplasts to chloroplasts in angiosperms. The underlying process involves reorganization of prolamellar bodies (PLBs) and prothylakoids into thylakoids, with concurrent changes in protein, lipid, and pigment composition, which together lead to the assembly of active photosynthetic complexes. Despite the highly conserved structure of PLBs among land plants, the processes that mediate PLB maintenance and their disassembly during de-etiolation are poorly understood. Among chloroplast thylakoid membrane–localized proteins, to date, only Curvature thylakoid 1 (CURT1) proteins were shown to exhibit intrinsic membrane-bending capacity. Here, we show that CURT1 proteins, which play a critical role in grana margin architecture and thylakoid plasticity, also participate in de-etiolation and modulate PLB geometry and density. Lack of CURT1 proteins severely perturbs PLB organization and vesicle fusion, leading to reduced accumulation of the light-dependent enzyme protochlorophyllide oxidoreductase (LPOR) and a delay in the onset of photosynthesis. In contrast, overexpression of CURT1A induces excessive bending of PLB membranes, which upon illumination show retarded disassembly and concomitant overaccumulation of LPOR, though without affecting greening or the establishment of photosynthesis. We conclude that CURT1 proteins contribute to the maintenance of the paracrystalline PLB morphology and are necessary for efficient and organized thylakoid membrane maturation during de-etiolation.

Morphological control of receptor-mediated endocytosis

Receptor-mediated endocytosis is the primary process for nanoparticle uptake in cells and one of the main entry mechanisms for viral infection. The cell membrane adheres to the particle (nanoparticle or virus) and then wraps it to form a vesicle delivered to the cytosol. Previous findings identified a minimum radius for a spherical particle below which endocytosis cannot occur. This is due to the insufficient driving force, from receptor-ligand affinity, to overcome the energy barrier created by membrane bending. In this paper, we extend this result to the case of clathrin-mediated endocytosis, which is the most common pathway for virus entry. Moreover, we investigate the effect of ligand inhibitors on the particle surface, motivated by viral an- tibodies, peptides or phage capsids nanoparticles. We determine the necessary conditions for endocytosis by considering the additional energy barrier due to the membrane bending to wrap such inhibiting protrusions. We find that the density and size of inhibitors determine the size range of internalized particles, and endo- cytosis is completely blocked above critical thresholds. The assembly of a clathrin coat with a spontaneous curvature increases the energy barrier and sets a maximum particle size (in agreement with experimental observations on smooth particles). Our investigation suggests that morphological considerations can inform the optimal design of neutralizing viral antibodies and new strategies for targeted nanomedicine.

Colony-like Protocell Superstructures

We report the formation, growth, and dynamics of model protocell superstructures on solid surfaces, resembling single cell colonies. These structures, consisting of several layers of lipidic compartments enveloped in a dome-shaped outer lipid bilayer, emerged as a result of spontaneous shape transformation of lipid agglomerates deposited on thin film aluminum surfaces. Collective protocell structures were observed to be mechanically more stable compared to isolated spherical compartments. We show that the model colonies encapsulate DNA and accommodate non-enzymatic, strand displacement DNA reactions. The membrane envelope is able to disassemble and expose individual daughter protocells, which can migrate and attach via nano-tethers to distant surface locations, while maintaining their encapsulated contents. Some colonies feature ‘exo-compartments’, which spontaneously extend out of the enveloping bilayer, internalize DNA, and merge again with the superstructure. A continuum elastohydrodynamic theory that we developed reveals that the subcompartment formation must be governed by attractive van der Waals (vdW) interactions between the membrane and surface. The balance between membrane bending and vdW interactions yields a critical length scale of 273 nm, above which the membrane invaginations can form subcompartments. The findings support our hypotheses that in extension of the ‘lipid world hypothesis’, protocells may have existed in the form of colonies, potentially benefiting from the increased mechanical stability provided by a superstructure.

A Hydraulic Soft Microhand with an Application of Live Insect Manipulation

We have developed micro scale hydraulic/pneumatic soft grippers and demonstrated the handling of an insect. These grippers are built on Polydimethylsiloxane (PDMS) with the soft material casting technique to form three finger-like columns, which are placed on a circular membrane. The fingers have a length of 1.5mm/2mm and a diameter of 300µm each; the distance between two fingers is 600µm of center-to-center distance. Membrane is built as a 150µm soft skin on the top of a cylindrical void. Applying a pressure difference between the interior of the void and the exterior can bend the membrane. Bending the membrane causes the motion of opening/closing of the gripper, and as a result, the three fingers can grip an object or release it. The PDMS was characterized and the experimental results were used later in Abaqus software to simulate the gripping motion. The produced force and range of deformation of the grippers were investigated by simulation and experiment. The results of the simulation agrees with the experiments. The maximum 543 µN force was measured for the microfluidic compatible microgrippers.Using this microhand gripper, the an ant was manipulated successfully without any damage. Results showed fabricated devices have great potential as a micro/bio manipulator.