Regulation of actin dynamics by PI(4,5)P-2 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)P-2 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)P-2-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)P-2 and actin-binding proteins control the actin cytoskeleton in cells.Peer reviewe

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

Funding Information: Acknowledgments: The computations were supported by the University of Chicago Research Funding Information: The computations were supported by the University of Chicago Research Computing Center (RCC). We thank E. Coudrier and C. Simon for insightful discussions. We also thank F. Di Federico for handling plasmids, F. Tabarin-Cayrac for cell sorting, and A.-S. Mace for ImageJ programming assistance. F.-C.T., C.L.C., and P.B. are members of the CNRS consortium AQV. F.-C.T. and P.B. are members of the Labex Cell(n)Scale (ANR-11-LABX0038) and Paris Sciences et Lettres (ANR-10-IDEX-0001-02). We acknowledge the Cell and Tissue Imaging Core facility (PICT IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). This work was supported by Human Frontier Science Program (HFSP) grant RGP0005/2016 (to F.-C.T., J.M.H., G.A.V., P.L., and P.B.), Institut Curie and the Centre National de la Recherche Scientifique (CNRS) (to F.-C.T., J.M.H., and P.B.), Marie Curie actions H2020-MSCA-IF-2014 (to F.-C.T.), EMBO Long-Term fellowship ALTF 1527-2014 (to F.-C.T.), Pasteur Foundation Fellowship (to J.M.H.), Agence Nationale pour la Recherche ANR-20-CE13-0032 (to J.M.H. and P.B.) and ANR-20-CE11-0010-01 (to F.-C.T), Université Paris Sciences et Lettres-QLife Institute ANR-17-CONV-0005 Q-LIFE (to P.B.), FY 2015 Researcher Exchange Program between the Japan Society for the Promotion of Science and Academy of Finland (to Y.S.), the Takeda Science Foundation (to Y.S.), the Wesco Scientific Promotion Foundation (to Y.S.), Agence Nationale pour la Recherche ANR-18-CE13-0026-01 and ANR-21-CE13-0010-03 (to C.L.C.), Cancer Society Finland 4705949 (to P.L.), and U.S. National Institutes of Health (NIH) Institute of General Medical Sciences (NIGMS) grant R01-GM063796 (to G.A.V. and Z.J.) Publisher Copyright: Copyright © 2022 The Authors, some rights reserved.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.Peer reviewe

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.Peer reviewe

Real-World Incidence of Febrile Neutropenia among Patients Treated with Single-Agent Amrubicin: Necessity of the Primary Prophylactic Administration of Granulocyte Colony-Stimulating Factor

Background: Single-agent amrubicin chemotherapy is a key regimen, especially for small cell lung cancer (SCLC); however, it can cause severe myelosuppression. Purpose: The purpose of this study was to determine the real-world incidence of febrile neutropenia (FN) among patients treated with single-agent amrubicin chemotherapy for thoracic malignancies. Patients and methods: The medical records of consecutive patients with thoracic malignancies, including SCLC and non-small cell lung cancer (NSCLC), who were treated with single-agent amrubicin chemotherapy in cycle 1 between January 2010 and March 2020, were retrospectively analyzed. Results: One hundred and fifty-six patients from four institutions were enrolled. Their characteristics were as follows: median age (range): 68 (32–86); male/female: 126/30; performance status (0/1/2): 9/108/39; SCLC/NSCLC/others: 111/30/15; and prior treatment (0/1/2/3-): 1/96/31/28. One hundred and thirty-four (86%) and 97 (62%) patients experienced grade 3/4 and grade 4 neutropenia, respectively. One hundred and twelve patients (72%) required therapeutic G-CSF treatment, and 47 (30%) developed FN. Prophylactic PEG-G-CSF was not used in cycle 1 in any case. The median overall survival of the patients with FN was significantly shorter than that of the patients without FN (7.2 vs. 10.0 months, p = 0.025). Conclusions: The real-world incidence rate of FN among patients with thoracic malignancies that were treated with single-agent amrubicin chemotherapy was 30%. It is suggested that prophylactic G-CSF should be administered during the practical use of single-agent amrubicin chemotherapy for patients who have already received chemotherapy

Diminished Medial Prefrontal Activity behind Autistic Social Judgments of Incongruent Information

Individuals with autism spectrum disorders (ASD) tend to make inadequate social judgments, particularly when the nonverbal and verbal emotional expressions of other people are incongruent. Although previous behavioral studies have suggested that ASD individuals have difficulty in using nonverbal cues when presented with incongruent verbal-nonverbal information, the neural mechanisms underlying this symptom of ASD remain unclear. In the present functional magnetic resonance imaging study, we compared brain activity in 15 non-medicated adult males with high-functioning ASD to that of 17 age-, parental-background-, socioeconomic-, and intelligence-quotient-matched typically-developed (TD) male participants. Brain activity was measured while each participant made friend or foe judgments of realistic movies in which professional actors spoke with conflicting nonverbal facial expressions and voice prosody. We found that the ASD group made significantly less judgments primarily based on the nonverbal information than the TD group, and they exhibited significantly less brain activity in the right inferior frontal gyrus, bilateral anterior insula, anterior cingulate cortex/ventral medial prefrontal cortex (ACC/vmPFC), and dorsal medial prefrontal cortex (dmPFC) than the TD group. Among these five regions, the ACC/vmPFC and dmPFC were most involved in nonverbal-information-biased judgments in the TD group. Furthermore, the degree of decrease of the brain activity in these two brain regions predicted the severity of autistic communication deficits. The findings indicate that diminished activity in the ACC/vmPFC and dmPFC underlies the impaired abilities of individuals with ASD to use nonverbal content when making judgments regarding other people based on incongruent social information

Interaction of Proteins with Biomembranes

Many proteins interact with cell and subcellular membranes. The plasma and intracellular membranes are characterized by their different lipid compositions that enable membrane-binding proteins to localize to distinct subcellular compartments. These lipid–protein interactions also regulate protein conformation and protein–protein interactions, which precisely regulate the activation of molecular complexes at the respective membranes. Furthermore, membrane-bound proteins can control lipid lateral diffusion, membrane tension/fluidity, and lipid phase separation. These membrane properties can induce the intracellular signaling that plays crucial roles in various cellular processes such as cell migration, morphogenesis, membrane trafficking, and signal transduction. However, due to the complexity of the abundant protein–protein interactions within a cell, the exact molecular mechanisms underlying protein interactions with lipids contain a lot of unclarity. This Membranes Special Issue, entitled “Interaction of Proteins with Biomembrane”, discusses the recent progress in lipid–protein interactions from various perspectives, including cell biology, biochemistry, and biophysics. These studies will elucidate the mechanisms by which membranes regulate protein localizations/functions, and thus provide new insights into the fundamental principles of lipid–protein interactions. A summary of the research articles is presented here. Motegi et al. [1] studied the formation of phosphatidylinositol (PI)-induced microdomains on supported lipid bilayers using atomic force microscopy (AFM) and single-particle tracking. The authors found that the PI-induced microdomains had less fluidity than the surrounding regions where lipids freely diffused, and thus functioning as diffusion barriers. These PI-induced microdomains acted as a scaffold to promote the initial clustering of FBP17, one of the membrane-remodeling Bin/Amphiphysin/Rvs (BAR) domain proteins. The surrounding fluid region promoted FBP17 assembly through lipid lateral diffusion. This study suggests the possible role of lipid microdomains in the self-assembly of membrane-binding proteins according to their lipid composition and physical properties. To et al. [2] studied the non-structural (NS) protein, NS4A, a membrane protein critical for virulence, and thus flavivirus membrane morphogenesis. The authors found that a peptide containing an N-terminal cytoplasmic tail and one-third of the first transmembrane domain of Zika virus (ZIKV) NS4A formed homotrimers. The authors propose that the disruption of this oligomerization is essential for in vitro screening assays for the antiviral discovery. By using a single quantum dot tracking approach, Kovtun et al. [3] studied the lateral diffusion, nanodomain formation, and their implications in signal transduction of the D2 subtype dopamine receptor (D2DR), a class A G-protein-coupled receptor (GPCR), which has naturally occurring genetic variants in schizophrenia. The authors found a significant decrease in the diffusion dynamics of the Val96Ala D2L schizophrenia variant. By measuring the relative frequency of D2L–D2L interactions, the authors found a significant fraction of D2L receptors and their variants transiently colocalized. The authors also compared nanoclusters of D2DR to those of phosphatidylinositol 4,5-bisphosphate in the plasma membrane. Aalst et al. [4] studied the cholesterol binding of the CC motif chemokine receptor 3 (CCR3), a class A GPCR, which is mainly responsible for the cellular trafficking in eosinophils. CCR3 plays vital roles in inflammatory conditions such as asthma, arthritis and the cancer metastasis. The authors analyzed lipid–protein contacts to identify the potential cholesterol-binding sites in the transmembrane region of CCR3 by using in silico coarse-grained molecular dynamics (MD) using PyLipID. Several cholesterol-binding sites of CCR3 contain a cholesterol recognition/interaction amino acid consensus (CRAC) motif and its inverted CARC motif. Generally, the CARC motif in the transmembrane region is located in the outer membrane leaflet, and its mirror motif, CRAC, is in the inner membrane leaflet. Based on the sequence alignment, these cholesterol-binding sites are conserved not only in CCR3 but also in other CC and CXC motif chemokine receptors. Furthermore, the functional residues in and near these sites were implicated in receptor dimerization, ligand binding, and signal transduction. The authors propose that their findings provide insights into the mechanisms underlying cholesterol regulation of the class A GPCR subfamily. Taken together, the papers in this Special Issue will update our current knowledge of the interaction between proteins and biomembranes. The technical approaches presented here, such as AFM, single-particle tracking, and supported lipid bilayers, will help us understand the spatiotemporal dynamics of protein/lipid lateral diffusion, compartmentalization of the cell membrane, and microdomain formations and their physical properties. These studies will provide new insights into the fundamental principles underlying physiological functions of membrane proteins such as GPCRs and membrane-remodeling proteins in cells and tissues

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

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.Peer reviewe

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.Peer reviewe