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The AENTH tetramer interfaces a Surface representation of the A2E2 tetramer, with ANTH subunits in cyan and ENTH subunits in gray. The different interfaces are colored: ANTH-ENTH interface 1 in red, ANTH-ENTH interface 2 in green and ANTH-ANTH interface in blue. b Cartoon representation of the tetramer area within the black rectangle in a showing the different interfacial residues as sticks (same color code as in a).
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During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagina...
Citations
... During clathrin-mediated endocytosis, these proteins form a phosphatidylinositol 4,5-bisphosphate (PIP 2 )-dependent complex, essential for membrane remodelling and invagination. It has been shown that their membrane binding domains, ANTH and ENTH, oligomerize in-vitro into different assemblies through lipid interfaces forming the AENTH complex Lizarrondo et al., 2021). Here, DLS has been a powerful tool to assess the aggregation of the individual ANTH and ENTH domains upon mixing with PIP 2 (Supplementary Figure S4A) and during the AENTH complex formation. ...
... Importantly, the macromolecular complex species identified as 12mers and 16mers by MP cannot be resolved by DLS (see the orange and red lines between 6 and 10 nm on the right panel of Supplementary Figure S4C). These assemblies were later on visualized on cryo-EM micrographs from where the structure for 12mers and 16-mers could be resolved (Supplementary Figure S4E, and Lizarrondo et al., 2021). ...
Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.
... The recruitment of clathrin to the plasma membrane requires several proteins that form the pioneer module, a complex with several characterized components, such as F-BAR domain only protein 1 and 2 complex (FCHO1/2), adaptor protein 2 (AP-2), and epidermal growth factor receptor substrate 15 (EPS15) (Shih et al., 1995;Ma et al., 2016;Kaksonen and Roux, 2018). In addition, several molecules such as actin-related protein 2/3 complex (Arp2/3), protein-rich protein Las17 (Las17), type I myosins (Myo3/5), Sla2 and epsin 1 (Ent1) (Galletta et al., 2008;Cheng et al., 2012;Feliciano and Di Pietro, 2012;Kaksonen and Roux, 2018;Lizarrondo et al., 2021), contribute to polymerization of the actin cytoskeleton. Finally, the progressive invagination of the membrane is associated with a constriction process, which culminates in membrane scission (Kaksonen and Roux, 2018). ...
Trypanosoma brucei is one of only a few unicellular pathogens that thrives extracellularly in the vertebrate host. Consequently, the cell surface plays a critical role in both immune recognition and immune evasion. The variant surface glycoprotein (VSG) coats the entire surface of the parasite and acts as a flexible shield to protect invariant proteins against immune recognition. Antigenic variation of the VSG coat is the major virulence mechanism of trypanosomes. In addition, incessant motility of the parasite contributes to its immune evasion, as the resulting fluid flow on the cell surface drags immunocomplexes toward the flagellar pocket, where they are internalized. The flagellar pocket is the sole site of endo- and exocytosis in this organism. After internalization, VSG is rapidly recycled back to the surface, whereas host antibodies are thought to be transported to the lysosome for degradation. For this essential step to work, effective machineries for both sorting and recycling of VSGs must have evolved in trypanosomes. Our understanding of the mechanisms behind VSG recycling and VSG secretion, is by far not complete. This review provides an overview of the trypanosome secretory and endosomal pathways. Longstanding questions are pinpointed that, with the advent of novel technologies, might be answered in the near future.
Membrane proteins are responsible for a large variety of tasks in organisms and of particular interesting as drug targets. At the same time, they are notoriously difficult to work with and require a thorough characterization before proceeding with structural studies. Here, we present a biophysical pipeline to characterize membrane proteins focusing on the optimization of stability, aggregation behavior, and homogeneity. The pipeline shown here is built on three biophysical techniques: differential scanning fluorimetry using native protein fluorescence (nano differential scanning fluorimetry), dynamic light scattering, and mass photometry. For each of these techniques, we provide detailed protocols for performing experiments and data analysis.
In this chapter, we discuss the various sample delivery systems that are available at most biological SAXS beamlines. The focus is laid on the EMBL Biosaxs beamline P12 at the Petra 3 storage ring on the DESY site in Hamburg, Germany.
The minimal requirements necessary to prepare samples are described specifically for macromolecular samples in solution and the background is given on how the physical properties of the scattering process itself determine the sample requirements. We provide a number of exemplary applications as well as guidelines for selecting and performing the right data collection strategy depending on the scientific question at hand. This chapter is aimed at novices to the SAXS technique with biochemical background as well as more experienced users setting out to employ more advanced SAXS set-ups.
In clathrin-mediated endocytosis, a principal membrane trafficking route of all eukaryotic cells, forces are applied to invaginate the plasma membrane and form endocytic vesicles. These forces are provided by specific endocytic proteins and the polymerizing actin cytoskeleton. One of the best-studied endocytic systems is endocytosis in yeast, known for its simplicity, experimental amenability, and overall similarity to human endocytosis. Importantly, the yeast endocytic protein machinery generates and transmits tremendous force to bend the plasma membrane, making this system beneficial for mechanistic studies of cellular force-driven membrane reshaping. This review summarizes important protein players, molecular functions, applied forces, and open questions and perspectives of this robust, actin-powered membrane-remodeling protein machine.
Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.
Shell‐stabilized gas microbubbles (MB) and nanobubbles (NB) are frequently used for biomedical ultrasound imaging and therapeutic applications. While it is widely recognized that monodisperse bubbles can be more effective in these applications, the efficient formulation of uniform bubbles at high concentrations is difficult to achieve. Here, it is demonstrated that a standard mini‐extruder setup, commonly used to make vesicles or liposomes, can be used to quickly and efficiently generate monodisperse NBs with high yield. In this highly reproducible technique, the NBs obtained have an average diameter of 0.16 ± 0.05 µm and concentration of 6.2 ± 1.8 × 1010 NBs mL−1 compared to 0.32 ± 0.1 µm and 3.2 ± 0.7 × 1011 mL−1 for NBs made using mechanical agitation. Parameters affecting the extrusion and NB generation process including the temperature, concentration of the lipid solution, and the number of passages through the extruder are also examined. Moreover, it is demonstrated that extruded NBs show a strong acoustic response in vitro and a strong and persistent US signal enhancement under nonlinear contrast enhanced ultrasound imaging in mice. The extrusion process is a new, efficient, and scalable technique that can be used to easily produce high yield smaller monodispersed nanobubbles. Shell‐stabilized, gas nanobubbles are used in a variety of biomedical and industrial applications, but are difficult to make. Here a high yield, scalable method for direct production of monodisperse lipid‐shelled C3F8 nanobubbles for biomedical applications via a simple extrusion process is presented. Compared to self‐assembly via mechanical agitation, the process is efficient, and requires no postprocessing for size isolation.
Subcellular localization of proteins acting on the endomembrane system is primarily regulated via membrane trafficking. To obtain and maintain the correct protein composition of the plasma membrane and membrane‐bound organelles, the loading of selected cargos into transport vesicles is critically regulated at donor compartments by adaptor proteins binding to the donor membrane, the cargo molecules, and the coat‐protein complexes, including the clathrin coat. The ANTH/ENTH/VHS domain‐containing protein superfamily generally comprises a structurally related ENTH, ANTH, or VHS domain in the N‐terminal region and a variable C‐terminal region, which is thought to act as an adaptor during transport vesicle formation. This protein family is involved in various plant processes, including pollen tube growth, abiotic stress response, and development. In this review, we provide an overview of the recent findings on ANTH/ENTH/VHS domain‐containing proteins in plants. AP180 N‐terminal homology (ANTH) domain‐containing PICALM proteins mediate endocytic and exocytic transport of plasma‐membrane proteins in plant cells.
Giant lipid vesicles form unusual multispherical or “multi-balloon” shapes consisting of several spheres that are connected by membrane necks. Such multispherical shapes have been recently observed when the two sides of the membranes were exposed to different sugar solutions. This sugar asymmetry induced a spontaneous curvature, the sign of which could be reversed by swapping the interior with the exterior solution. Here, previous studies of multispherical shapes are reviewed and extended to develop a comprehensive theory for these shapes. Each multisphere consists of large and small spheres, characterized by two radii, the large-sphere radius, Rl, and the small-sphere radius, Rs. For positive spontaneous curvature, the multisphere can be built up from variable numbers Nl and Ns of large and small spheres. In addition, multispheres consisting of N* = Nl + Ns equally sized spheres are also possible and provide examples for constant-mean-curvature surfaces. For negative spontaneous curvature, all multispheres consist of one large sphere that encloses a variable number Ns of small spheres. These general features of multispheres arise from two basic properties of curvature elasticity: the local shape equation for spherical membrane segments and the stability conditions for closed membrane necks. In addition, the (Nl + Ns)-multispheres can form several (Nl + Ns)-patterns that differ in the way, in which the spheres are mutually connected. These patterns may involve multispherical junctions consisting of individual spheres that are connected to more than two neighboring spheres. The geometry of the multispheres is governed by two polynomial equations which imply that (Nl + Ns)-multispheres can only be formed within a certain restricted range of vesicle volumes. Each (Nl + Ns)-pattern can be characterized by a certain stability regime that depends both on the stability of the closed necks and on the multispherical geometry. Interesting and challenging topics for future studies include the response of multispheres to locally applied external forces, membrane fusion between spheres to create multispherical shapes of higher-genus topology, and the enlarged morphological complexity of multispheres arising from lipid phase separation and intramembrane domains.
Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.
Teaser
Simulating the binding of 100 Pleckstrin homology domains to cell membranes reveals patterns in their lipid interactions.
