ABSTRACT
Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. Quantitative live cell imaging reveals that CCDC32, a poorly characterized endocytic accessory protein, regulates CCP stabilization and is required for efficient CCP invagination. CCDC32 interacts with the α-appendage domain (AD) of AP2 via its coiled-coil domain to exert this function. Furthermore, we showed that the clinically observed nonsense mutations in CCDC32, which result in the development of cardio-facio-neuro-developmental syndrome (CFNDS), inhibit CME by abolishing CCDC32-AP2 interactions. Overall, our data demonstrates the function and molecular mechanism of a novel endocytic accessory protein, CCDC32, in CME regulation. Significance Statement: Clathrin-mediated endocytosis (CME) happens via the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). In this study, we used a combination of quantitative live cell imaging, ultrastructure electron microscopy and biochemical experiments to show that CCDC32, a poorly studied and functional ambiguous protein, acts as an important endocytic accessory protein that regulates CCP stabilization and invagination. Specifically, CCDC32 exerts this function via its interactions with AP2, and the coiled-coil domain of CCDC32 and the α-appendage domain (AD) of AP2 are essential in mediating CCDC32-AP2 interactions. Importantly, we demonstrate that clinically observed loss-of-function mutations in CCDC32 lose AP2 interaction capacity and inhibit CME, resulting in the development of cardio-facio-neuro-developmental syndrome (CFNDS).
ABSTRACT
Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.
Subject(s)
Exosomes , Exosomes/metabolism , Endosomes/metabolism , Phosphatidylinositols/metabolism , Multivesicular Bodies/metabolismABSTRACT
Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
Subject(s)
Actins , Septins , MicrotubulesABSTRACT
The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the regulation of endothelial junctions as dependent on the feedback between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Simulations reveal that active cell tension can stabilize cadherin bonds, but excessive RhoA signaling can drive bond dissociation and junction failure. While actin polymerization aids gap closure, high levels of Rac1 can induce junction weakening. Combining the modeling framework with experiments, our model predicts the influence of pharmacological treatments on the junction state and identifies that a critical balance between RhoA and Rac1 expression is required to maintain junction stability. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.
Subject(s)
Actins , Endothelial Cells , Endothelial Cells/metabolism , Actins/metabolism , Feedback , Signal Transduction , Actin Cytoskeleton/metabolismABSTRACT
Clathrin-mediated endocytosis (CME) requires energy input from actin polymerization in mechanically challenging conditions. The roles of actin in CME are poorly understood due to inadequate knowledge of actin organization at clathrin-coated structures (CCSs). Using platinum replica electron microscopy of mammalian cells, we show that Arp2/3 complex-dependent branched actin networks, which often emerge from microtubule tips, assemble along the CCS perimeter, lack interaction with the apical clathrin lattice, and have barbed ends oriented toward the CCS. This structure is hardly compatible with the widely held "apical pulling" model describing actin functions in CME. Arp2/3 complex inhibition or epsin knockout produce large flat non-dynamic CCSs, which split into invaginating subdomains upon recovery from Arp2/3 inhibition. Moreover, epsin localization to CCSs depends on Arp2/3 activity. We propose an "edge pushing" model for CME, wherein branched actin polymerization promotes severing and invagination of flat CCSs in an epsin-dependent manner by pushing at the CCS boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices.
Subject(s)
Actins , Platinum , Actin-Related Protein 2-3 Complex , Animals , Clathrin , Clathrin-Coated Vesicles , Endocytosis , Mammals , PolymerizationABSTRACT
The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.
Subject(s)
Exosomes , Melanoma , Animals , B7-H1 Antigen , CD8-Positive T-Lymphocytes , Cell Line, Tumor , Exosomes/metabolism , Humans , Immunotherapy , Mice , Phosphorylation , Programmed Cell Death 1 Receptor , Tumor MicroenvironmentABSTRACT
The Age of Information (AoI) measures the freshness of information and is a critic performance metric for time-sensitive applications. In this paper, we consider a radio frequency energy-harvesting cognitive radio network, where the secondary user harvests energy from the primary users' transmissions and opportunistically accesses the primary users' licensed spectrum to deliver the status-update data pack. We aim to minimize the AoI subject to the energy causality and spectrum constraints by optimizing the sensing and update decisions. We formulate the AoI minimization problem as a partially observable Markov decision process and solve it via dynamic programming. Simulation results verify that our proposed policy is significantly superior to the myopic policy under different parameter settings.
ABSTRACT
This study aims to identify the factors that influence Chinese professional caregivers' bereavement experiences after patient deaths. Through a content analysis, the study reanalyzed the qualitative data initially collected to understand the lived experiences of professional bereavement in Mainland China. Specifically, the study assessed semi-structured interview transcripts conducted with 24 Chinese physicians and nurses and generated 15 open codes, reflecting the influencing factors. These were further categorized into four themes: dying and death conditions, professional caregivers' characteristics, professional caregivers' involvement, and the bereaved family. The results revealed that professional bereavement experiences and the unveiled factors relate to both the personal and professional lives of the interviewees. Overall, the health care system and cultural backgrounds should be listed as influencing factors for professional bereavement experiences in addition to the aforementioned four.
Subject(s)
Bereavement , Caregivers , China , Grief , Humans , Qualitative ResearchABSTRACT
Human fibroblasts can switch between lamellipodia-dependent and -independent migration mechanisms on two-dimensional surfaces and in three-dimensional (3D) matrices. RhoA GTPase activity governs the switch from low-pressure lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway, which acts through nonmuscle myosin II (NMII) to disrupt lamellipodia downstream from Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit lamellipodia formation via two distinct pathways in high-pressure cells.
Subject(s)
Osmotic Pressure/physiology , Pseudopodia/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actin-Related Protein 2-3 Complex/physiology , Actomyosin/metabolism , Cell Culture Techniques , Cell Movement/physiology , Cytoskeletal Proteins/metabolism , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Humans , Myosin Type II/metabolism , Myosin Type II/physiology , Signal TransductionABSTRACT
Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.
Subject(s)
Cell Movement/physiology , Cell Surface Extensions/metabolism , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , CRISPR-Cas Systems , Cell Adhesion , Cell Line , Cytoskeleton/metabolism , Extracellular Matrix/metabolism , Gene Knockout Techniques , Humans , Kinetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/ultrastructure , Microfilament Proteins/genetics , Microfilament Proteins/ultrastructure , Myosins/metabolism , Pseudopodia/metabolism , Receptor, EphB2ABSTRACT
Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.
Subject(s)
Actins/metabolism , Actin Cytoskeleton/metabolism , Actins/genetics , Actins/ultrastructure , Cell Movement/genetics , Cells, Cultured , Collagen/chemistry , Gene Expression Regulation , Humans , Intestinal Pseudo-Obstruction/genetics , Muscle Contraction/genetics , Muscle, Smooth/cytology , MutationABSTRACT
SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a significant decrease in the rate of leading edge actin extension in WAVE2 KO cells, and an increase in WAVE1 KO cells. The faster rates of actin extension in WAVE1 KO cells are offset by faster retrograde flow, and therefore do not translate into faster lamellipodium protrusion. Thus, WAVE1 restricts the rate of actin extension at the leading edge, and appears to couple actin networks to the membrane to drive protrusion. Overall, these results suggest that WAVE1 and WAVE2 have redundant roles in promoting Arp2/3-dependent actin nucleation and lamellipodia formation, but distinct roles in controlling actin network extension and harnessing network growth to cell protrusion.
Subject(s)
Actins/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cell Surface Extensions/metabolism , Humans , Microfilament Proteins/metabolism , Pseudopodia/metabolism , Wiskott-Aldrich Syndrome Protein Family/geneticsABSTRACT
Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.
Subject(s)
Actins/metabolism , Adenomatous Polyposis Coli Protein/metabolism , Adenomatous Polyposis Coli/metabolism , Microtubules/metabolism , Actin Cytoskeleton/metabolism , Animals , Cell Movement/physiology , Cells, Cultured , Growth Cones/metabolism , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Neurons/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Cell Communication , Drosophila melanogaster/metabolism , Dynamins/metabolism , Endocytosis , Actin-Related Protein 2-3 Complex/metabolism , Actins/genetics , Amino Acid Sequence , Animals , Drosophila melanogaster/genetics , Dynamins/genetics , Female , Guanosine Triphosphate/metabolism , Male , Myoblasts/cytology , Myoblasts/metabolism , Protein Binding , Sequence HomologyABSTRACT
Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.
Subject(s)
Actins/genetics , Cytoskeleton/ultrastructure , Mitochondria/ultrastructure , Mitochondrial Dynamics/genetics , Myosin Type II/genetics , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Constriction , Cytoskeleton/metabolism , Ionomycin/pharmacology , Mitochondria/genetics , Mitochondrial Dynamics/drug effects , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Myosin Type II/chemistry , Myosin Type II/metabolismABSTRACT
Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Binding Sites , Cattle , Cell Membrane/metabolism , Humans , Lipid Bilayers , Molecular Dynamics SimulationABSTRACT
PICK1 is a modular scaffold implicated in synaptic receptor trafficking. It features a PDZ domain, a BAR domain, and an acidic C-terminal tail (ACT). Analysis by small- angle x-ray scattering suggests a structural model that places the receptor-binding site of the PDZ domain and membrane-binding surfaces of the BAR and PDZ domains adjacent to each other on the concave side of the banana-shaped PICK1 dimer. In the model, the ACT of one subunit of the dimer interacts with the PDZ and BAR domains of the other subunit, possibly accounting for autoinhibition. Consistently, full-length PICK1 shows diffuse cytoplasmic localization, but it clusters on vesicle-like structures that colocalize with the trans-Golgi network marker TGN38 upon deletion of either the ACT or PDZ domain. This localization is driven by the BAR domain. Live-cell imaging further reveals that PICK1-associated vesicles undergo fast, nondirectional motility in an F-actin-dependent manner, but deleting the ACT dramatically reduces vesicle speed. Thus the ACT links PICK1-associated vesicles to a motility factor, likely myosin, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Transport Vesicles/physiology , Amino Acid Sequence , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Transport Vesicles/metabolismABSTRACT
The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.
Subject(s)
Nerve Tissue Proteins/antagonists & inhibitors , cdc42 GTP-Binding Protein/chemistry , Amino Acid Motifs , Binding Sites , Calorimetry , Crystallography, X-Ray , Humans , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Structure, Tertiary , Pseudopodia/metabolism , cdc42 GTP-Binding Protein/metabolism , src Homology DomainsABSTRACT
CARMIL is an approximately 1,370-amino-acid cytoskeletal scaffold that has crucial roles in cell motility and tissue development through interactions with cytoskeletal effectors and regulation of capping protein at the leading edge. However, the mechanism of CARMIL leading edge localization is unknown. Here we show that CARMIL interacts directly with the plasma membrane through its amino-terminal region. The crystal structure of CARMIL1-668 reveals that this region harbours a non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat domain. Lipid binding is mediated by the PH domain, but is further enhanced by a central helical domain. Small-angle X-ray scattering reveals that the helical domain mediates antiparallel dimerization, properly positioning the PH domains for simultaneous membrane interaction. In cells, deletion of the PH domain impairs leading edge localization. The results support a direct membrane-binding mechanism for CARMIL localization at the leading edge, where it regulates cytoskeletal effectors and motility.
Subject(s)
Carrier Proteins/chemistry , Cell Membrane/chemistry , Cytoskeleton/chemistry , Lipids/chemistry , Amino Acid Sequence , Animals , Binding Sites , Brain Chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement , Crystallography, X-Ray , Cytoskeleton/metabolism , Kinetics , Lipids/isolation & purification , Mice , Microfilament Proteins , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis.