The eukaryotic-like characteristics of small GTPase, roadblock and TRAPPC3 proteins from Asgard archaea.

Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes.

Membrane Proteins: Function, Structure, and Dynamics.

Plasma and intracellular membranes are characterized by different lipid compositions that enable proteins to localize to distinct subcellular compartments [...].

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis.

Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota.

A biophysical perspective of the regulatory mechanisms of ezrin/radixin/moesin proteins.

Many signal transductions resulting from ligand-receptor interactions occur at the cell surface. These signaling pathways play essential roles in cell polarization, membrane morphogenesis, and the modulation of membrane tension at the cell surface. However, due to the large number of membrane-binding proteins, including actin-membrane linkers, and transmembrane proteins present at the cell surface, the molecular mechanisms underlying the regulation at the cell surface are yet unclear. Here, we describe the molecular functions of one of the key players at the cell surface, ezrin/radixin/moesin (ERM) proteins from a biophysical point of view. We focus our discussion on biophysical properties of ERM proteins revealed by using biophysical tools in live cells and in vitro reconstitution systems. We first describe the structural properties of ERM proteins and then discuss the interactions of ERM proteins with PI(4,5)P2 and the actin cytoskeleton. These properties of ERM proteins revealed by using biophysical approaches have led to a better understanding of their physiological functions in cells and tissues. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00928-0.

Interaction of Proteins with Biomembranes.

Many proteins interact with cell and subcellular membranes [...].

Bex1 is essential for ciliogenesis and harbours biomolecular condensate-forming capacity.

BACKGROUND: Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia. RESULTS: Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system. CONCLUSIONS: Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.

Dynamin 2 and BAR domain protein pacsin 2 cooperatively regulate formation and maturation of podosomes.

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.

Liposome Co-sedimentation and Co-flotation Assays to Study Lipid-Protein Interactions.

A large proportion of proteins are expected to interact with cellular membranes to carry out their physiological functions in processes such as membrane transport, morphogenesis, cytoskeletal organization, and signal transduction. The recruitment of proteins at the membrane-cytoplasm interface and their activities are precisely regulated by phosphoinositides, which are negatively charged phospholipids found on the cytoplasmic leaflet of cellular membranes and play critical roles in membrane homeostasis and cellular signaling. Thus, it is important to reveal which proteins interact with phosphoinositides and to elucidate the underlying mechanisms. Here, we present two standard in vitro methods, liposome co-sedimentation and co-flotation assays, to study lipid-protein interactions. Liposomes can mimic various biological membranes in these assays because their lipid compositions and concentrations can be varied. Thus, in addition to mechanisms of lipid-protein interactions, these methods provide information on the possible specificities of proteins toward certain lipids such as specific phosphoinositide species and can hence shed light on the roles of membrane interactions on the functions of membrane-associated proteins.

Fluorescence Assays to Study Membrane Penetration of Proteins.

Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide-protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane-protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane-protein association and uncover molecular details of protein-lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.

Spatiotemporal Analysis of Caveolae Dynamics Using Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence microscopy enables to analyze the localizations and dynamics of cellular events that occur at or near the plasma membrane. Total internal reflection fluorescence microscopy exclusively illuminates molecules in the close vicinity of the glass surface, thereby reducing background fluorescence and enabling observation of the plasma membrane in the glass-attached cells with a high signal-to-noise ratio. Here, we describe the application of total internal reflection fluorescence microscopy to analyze the dynamics of caveolae, which play essential physiological functions, including membrane tension buffering, endocytosis, and signaling at the plasma membrane.

Regulation of actin dynamics by PI(4,5)P2 in cell migration and endocytosis.

The actin cytoskeleton is indispensable for several cellular processes, including migration, morphogenesis, polarized growth, endocytosis, and phagocytosis. The organization and dynamics of the actin cytoskeleton in these processes are regulated by Rho family small GTPases and kinase-phosphatase pathways. Moreover, membrane phospholipids, especially the phosphatidylinositol phosphates have emerged as important regulators of actin dynamics. From these, PI(4,5)P2 is the most abundant at the plasma membrane, and directly regulates the activities and subcellular localizations of numerous actin-binding proteins. Here, we discuss recent studies demonstrating that actin-binding proteins interact with PI(4,5)P2-rich membranes through drastically different affinities and dynamics correlating with their roles in cytoskeletal dynamics. Moreover, by using mesenchymal cell migration and clathrin-mediated endocytosis as examples, we present a model for how interplay between PI(4,5)P2 and actin-binding proteins control the actin cytoskeleton in cells.

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner.

One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using in vitro and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.

Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides.

The actin cytoskeleton powers membrane deformation during many cellular processes, such as migration, morphogenesis, and endocytosis. Membrane phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], regulate the activities of many actin-binding proteins (ABPs), including profilin, cofilin, Dia2, N-WASP, ezrin, and moesin, but the underlying molecular mechanisms have remained elusive. Moreover, because of a lack of available methodology, the dynamics of membrane interactions have not been experimentally determined for any ABP. Here, we applied a combination of biochemical assays, photobleaching/activation approaches, and atomistic molecular dynamics simulations to uncover the molecular principles by which ABPs interact with phosphoinositide-rich membranes. We show that, despite using different domains for lipid binding, these proteins associate with membranes through similar multivalent electrostatic interactions, without specific binding pockets or penetration into the lipid bilayer. Strikingly, our experiments reveal that these proteins display enormous differences in the dynamics of membrane interactions and in the ranges of phosphoinositide densities that they sense. Profilin and cofilin display transient, low-affinity interactions with phosphoinositide-rich membranes, whereas F-actin assembly factors Dia2 and N-WASP reside on phosphoinositide-rich membranes for longer periods to perform their functions. Ezrin and moesin, which link the actin cytoskeleton to the plasma membrane, bind membranes with very high affinity and slow dissociation dynamics. Unlike profilin, cofilin, Dia2, and N-WASP, they do not require high "stimulus-responsive" phosphoinositide density for membrane binding. Moreover, ezrin can limit the lateral diffusion of PI(4,5)P2 along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics.

Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension.

Caveolae are abundant flask-shaped invaginations of plasma membranes that buffer membrane tension through their deformation. Few quantitative studies on the deformation of caveolae have been reported. Each caveola contains approximately 150 caveolin-1 proteins. In this study, we estimated the extent of caveolar deformation by measuring the density of caveolin-1 projected onto a two-dimensional (2D) plane. The caveolin-1 in a flattened caveola is assumed to have approximately one-quarter of the density of the caveolin-1 in a flask-shaped caveola. The proportion of one-quarter-density caveolin-1 increased after increasing the tension of the plasma membrane through hypo-osmotic treatment. The one-quarter-density caveolin-1 was soluble in detergent and formed a continuous population with the caveolin-1 in the caveolae of cells under isotonic culture. The distinct, dispersed lower-density caveolin-1 was soluble in detergent and increased after the application of tension, suggesting that the hypo-osmotic tension induced the dispersion of caveolin-1 from the caveolae, possibly through flattened caveolar intermediates.

ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends.

Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells.

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation.

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

MIM-Induced Membrane Bending Promotes Dendritic Spine Initiation.

Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.