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BackgroundUpper limb motor paresis is a major symptom of stroke, which limits activities of daily living and compromises the quality of life. Kinematic analysis offers an in-depth and objective means to evaluate poststroke upper limb paresis, with anticipation for its effective application in clinical settings. ObjectiveThis study aims to compare the movement strategies of patients with hemiparesis due to stroke and healthy individuals in forward reach and hand-to-mouth reach, using a simple methodology designed to quantify the contribution of various movement components to the reaching action. MethodsA 3D motion analysis was conducted, using a simplified marker set (placed at the mandible, the seventh cervical vertebra, acromion, lateral epicondyle of the humerus, metacarpophalangeal [MP] joint of the index finger, and greater trochanter of the femur). For the forward reach task, we measured the distance the index finger’s MP joint traveled from its starting position to the forward target location on the anterior-posterior axis. For the hand-to-mouth reach task, the shortening of the vertical distance between the index finger MP joint and the position of the chin at the start of the measurement was measured. For both measurements, the contributions of relevant upper limb and trunk movements were calculated. ResultsA total of 20 healthy individuals and 10 patients with stroke participated in this study. In the forward reach task, the contribution of shoulder or elbow flexion was significantly smaller in participants with stroke than in healthy participants (mean 52.5%, SD 24.5% vs mean 85.2%, SD 4.5%; P

In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate the inheritance of eukaryotic Sec61/oligosaccharyltransferase/translocon-associated protein translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of Candidatus Prometheoarchaeum syntrophicum strain MK-D1 oligosaccharyltransferase 1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet-to-be-translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

The purposes of the present study were: (1) to investigate the relationship between hospital-associated functional decline (HAFD) and non-lying time and (2) to clarify the optimal cut-off value for non-lying time associated with HAFD in older patients undergoing transcatheter aortic valve implantation (TAVI). From January 2021 to December 2022, patients admitted to a university hospital who underwent trans-femoral TAVI were consecutively recruited. We measured short physical performance battery (SPPB) pre and post-TAVI, and non-lying time from post-operative days 3-5. HAFD was defined as at least 1 point decrease in SPPB during pre and post-TAVI. Among 75 patients (47 female, mean age of 84.5 years) enrolled, 14 patients were classified as having HAFD. Non-lying time was significantly shorter in the HAFD group than in the non-HAFD group (371 min vs. 539 min, P < 0.001). Receiver-operating characteristic analysis determined an optimal cut-off value of 477 min for differentiating the patients more likely to experience HAFD (sensitivity, 75%; specificity, 92%; area under the curve, 0.798). The non-lying time could be one of the associated factors of HAFD in older patients with TAVI. Non-lying time of about 480 min (8 h) during hospitalization may be an initial target for preventing HAFD., (© 2023. The Author(s).)

Objective: To investigate the prevalence of post-stroke depression in a rehabilitation ward and elucidate its effect on functional improvement and outcomes., Design: Retrospective cohort study., Setting: A convalescent rehabilitation ward at a University Hospital., Participants: A total of 114 patients with stroke (mean [SD] age, 67.2 [13.5] years; men, 76) assessed at 2 weeks after admission using the Mini-International Neuropsychiatric Interview were enrolled., Main Outcome Measure: Functional independence measure (FIM) efficiency during hospitalization in the ward., Results: Eleven patients (9.6%) had depression based on the Mini-International Neuropsychiatric Interview. Total FIM efficiency and FIM efficiency in the subtotal of motor items were significantly higher in the non-depression group than in the depression group (median [interquartile range]: 0.69 [0.39-0.95] vs 0.41 [0.24-0.63], P =.027; and 0.56 [0.38-0.80] vs 0.42 [0.18-0.49], P =.023, respectively). Patients in the non-depression group had higher FIM scores at discharge (median [interquartile range]: 116.0 [104.5-123.0] vs 104.0 [82.5-112.0], P =.013, respectively), and were more likely to be discharged home (80.6% vs 36.4%, P =.003). Furthermore, patients in the depression group also stayed significantly longer in the ward (71.0 [36.1] vs 106.1 [43.3], P =.010)., Conclusions: Patients with post-stroke depression showed poorer efficiency of functional recovery than those without depression. A future multicenter study with a larger sample size is needed to verify these findings., (© 2023 The Authors.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest.

Objective: To compare the functional outcomes of patients with lower limb amputations receiving haemodialysis and those not receiving haemodialysis., Design: A retrospective cohort study., Patients: Patients with lower limb amputation who were admitted to a convalescent rehabilitation ward between January 2018 and December 2021., Methods: The primary outcome was the effectiveness of the Functional Independence Measure (FIM) during hospitalisation in the ward. Secondary outcomes included the total and subtotal (motor/cognitive) FIM scores at discharge, gain in the total and subtotal (motor/cognitive) FIM scores, K-level at discharge, length of hospital stay in the ward, rehabilitation time, and discharge destination. Outcomes were compared between the non-haemodialysis and haemodialysis groups., Results: A total of 28 patients (mean [standard deviation] age, 67.0 [11.9] years; men, 20) were enrolled in this study. Among them, 11 patients underwent haemodialysis. The FIM effectiveness was significantly higher in the non-haemodialysis group than in the haemodialysis group (median [interquartile range], 0.78 [0.72 - 0.81] vs 0.65 [0.28 - 0.75], p = 0.038). The amount of rehabilitation and all secondary outcomes were not significantly different between the groups ( p > 0.05)., Conclusion: Patients with lower limb amputation who were receiving haemodialysis had poorer FIM effectiveness than those not receiving haemodialysis., Competing Interests: The authors have no conflicts of interest to declare., (© Published by Medical Journals Sweden, on behalf of the Foundation for Rehabilitation Information.)

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 PIP 2 . 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.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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)P 2 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., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022.)

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., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The International Classification of Functioning, Disability, and Health (ICF) Generic-30 (Rehabilitation) Set is a tool used to assess the functioning of a clinical population in rehabilitation. The ICF Generic-30 consists of nine ICF categories from the component "body functions" and 21 from the component "activities and participation". This study aimed to develop a rating reference guide for the nine body function categories of the ICF Generic-30 Set using a predefined, structured process and to examine the interrater reliability of the ratings using the rating reference guide., Methods: The development of the first version of the rating reference guide involved the following steps: (1) a trial of rating patients by several raters; (2) cognitive interviews with each rater to analyze the thought process involved in each rating; (3) the drafting of the rating reference guide by a multidisciplinary panel; and (4) a review by ICF specialists to confirm consistency with the ICF. Subsequently, we conducted a first field test to gain insight into the use of the guide in practice. The reference guide was modified based on the raters' feedback in the field test, and an inter-rater reliability test was conducted thereafter. Interrater agreement was evaluated using weighted kappa statistics with linear weights., Results: The first version of the rating reference guide was successfully developed and tested. The weighted kappa coefficient in the field testing ranged from 0.25 to 0.92. The interrater reliability testing of the rating reference guide modified based on the field test results yielded an improved weighted kappa coefficient ranging from 0.53 to 0.78. Relative improvements in the weighted kappa coefficients were observed in seven out of the nine categories. Consequently, seven out of nine categories were found to have a weighted kappa coefficient of 0.61 or higher., Conclusions: In this study, we developed and modified a rating reference guide for the body function categories of the ICF Generic-30 Set. The interrater reliability test using the final version of the rating reference guide showed moderate to substantial interrater agreement, which encouraged the use of the ICF in rehabilitation practice.

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.

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.

Background: Modification of the home environment, together with rehabilitative interventions, is important for maximizing the level of functioning after an individual with disability undergoes rehabilitation in the hospital., Objectives: We developed a simple screening scale - the home environment checklist (HEC) - to identify any mismatch between an individual's abilities and their home environment to help clinicians monitor the appropriateness of the home environment to which individuals with disability will be discharged. We also examined the psychometric properties of the HEC., Methods: The HEC was developed by a multidisciplinary panel of rehabilitation experts using information routinely collected in rehabilitation clinics before discharge. The reliability of the checklist was assessed in 60 individuals undergoing rehabilitation. The inter-rater agreement and internal consistency of the scale were assessed by weighted kappa statistics and Cronbach's alpha, respectively. Rasch analysis was performed with 244 rehabilitation individuals to evaluate the internal construct validity, and the known-groups validity was confirmed by a comparison of the daily activity levels of 30 individuals with disabilities under rehabilitation to the HEC score., Results: The HEC was developed as a simple, 10-item checklist. The weighted kappa statistics ranged from 0.73 to 0.93, indicating excellent inter-rater reliability. Cronbach's alpha was 0.92, indicating high internal consistency. Rasch analysis with a testlet approach on 3 subscales demonstrated a good fit with the Rasch model (χ 2 =13.2, P=0.153), and the demonstrated unidimensionality and absence of differential item functioning supported the internal construct validity of the HEC. HEC scores were significantly different (P<.01) among individuals with disability and 3 levels of restrictions in their activities (no limitation, home-bound, and bed-bound), which demonstrates the known-groups validity of the HEC., Conclusions: The HEC has good reliability and validity, which supports its utility in rehabilitation clinics., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Funahashi R, Mukaino M, Otaka Y, Senju Y, Yoneda C, Ozeki Y, Shimizu Y, Koike T, Saito E. Feasibility of the International Classification of Functioning, Disability and Health Rehabilitation Set for inpatient rehabilitation: Selection and validity of a set of categories for inpatients in a convalescent rehabilitation ward. Jpn J Compr Rehabil Sci 2020; 11: 1-8., Objective: To evaluate the feasibility of the International Classification of Functioning, Disability and Health (ICF) Rehabilitation Set for inpatients in a convalescent (Kaifukuki) rehabilitation ward., Methods: Overall, 295 inpatients in convalescent rehabilitation wards were rated using the ICF Rehabilitation Set, and the proportion of missing values was investigated. Categories with missing values <10% were selected, and internal construct validity of the total score of the selected categories was examined using Rasch analysis., Results: Missing values were detected in 25 items, of which seven had missing values of ≥10%. No ceiling or floor effects were noted. Rasch analysis of 23 categories with missing values <10% showed a good fit to the model after applying testlet solution and item splitting, which supported the internal construct validity of the ICF Rehabilitation Set., Conclusion: This identified the set of categories of the ICF Rehabilitation Set that could be used for evaluating rehabilitation inpatients. These categories had good internal construct validity based on Rasch analysis., (©Kaifukuki Rehabilitation Ward Association 2020.)

Background: The World Health Organization developed the International Classification of Functioning, Disability, and Health (ICF) in 2001 and has been in the process of implementing it in clinics since then. Current international efforts to implement ICF in rehabilitation clinics include the implementation of ICF Core Sets and the development of simple, intuitive descriptions for the ICF Generic-30 Set (also called Rehabilitation Set). The present study was designed to operationalize these ICF tools for clinical practice in Japan. This work included 1) the development of the Japanese version of the simple, intuitive descriptions for the ICF Generic-30 Set, 2) the development of a rating reference guide for Activity and Participation categories, and 3) the examination of the interrater reliability of rating Activity and Participation categories., Methods: The Japanese version of the simple, intuitive descriptions for the ICF Generic-30 Set was developed following the process employed to develop the Chinese and Italian versions. For further operationalization of this ICF Set in practice, a rating reference guide was developed. The development of the rating reference guide involved the following steps: 1) a trial of rating patients by several raters, 2) cognitive interviewing of the raters to analyse the thinking process involved in rating, 3) drafting of the rating reference guide, and 4) review by ICF specialists to confirm consistency with the original ICF concepts. After the rating reference guide was developed, interrater reliability of the rating with the reference guide was determined. Interrater reliability was examined using weighted kappa statistics with linear weight., Results: Through the pre-defined process, the Japanese version of the simple, intuitive descriptions for 30 categories of the ICF Generic-30 Set and the rating reference guides for 21 Activity and Participation categories were successfully developed. The weighted kappa statistics ranged from 0.61 to 0.85, showing substantial to excellent agreement of the ratings between raters., Conclusions: The present study demonstrates that ICF categories can be translated into clinical practice. Collaboration between clinicians and researchers would further enhance the implementation of the ICF in Japan.

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., Competing Interests: FT, AB, HB, JM, YS, MT, LP, SM, EL, EC No competing interests declared, PL, PB Reviewing editor, eLife, (© 2018, Tsai et al.)

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)P 2 ], 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)P 2 along the lipid bilayer. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely fulfill their specific functions in cytoskeletal dynamics., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

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.

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., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

PACSIN2, a membrane-sculpting BAR domain protein, localizes to caveolae. Here, we found that protein kinase C (PKC) phosphorylates PACSIN2 at serine 313, thereby decreasing its membrane binding and tubulation capacities. Concomitantly, phosphorylation decreased the time span for which caveolae could be tracked at the plasma membrane (the 'tracking duration'). Analyses of the phospho-mimetic S313E mutant suggested that PACSIN2 phosphorylation was sufficient to reduce caveolar-tracking durations. Both hypotonic treatment and isotonic drug-induced PKC activation increased PACSIN2 phosphorylation at serine 313 and shortened caveolar-tracking durations. Caveolar-tracking durations were also reduced upon the expression of other membrane-binding-deficient PACSIN2 mutants or upon RNA interference (RNAi)-mediated PACSIN2 depletion, pointing to a role for PACSIN2 levels in modulating the lifetime of caveolae. Interestingly, the decrease in membrane-bound PACSIN2 was inversely correlated with the recruitment and activity of dynamin 2, a GTPase that mediates membrane scission. Furthermore, expression of EHD2, which stabilizes caveolae and binds to PACSIN2, restored the tracking durations of cells with reduced PACSIN2 levels. These findings suggest that the PACSIN2 phosphorylation decreases its membrane-binding activity, thereby decreasing its stabilizing effect on caveolae and triggering dynamin-mediated removal of caveolae., (© 2015. Published by The Company of Biologists Ltd.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Caveolae are flask-shaped invaginations of the plasma membrane. The BAR domain proteins form crescent-shaped dimers, and their oligomeric filaments are considered to form spirals at the necks of invaginations, such as clathrin-coated pits and caveolae. PACSIN2/Syndapin II is one of the BAR domain-containing proteins, and is localized at the necks of caveolae. PACSIN2 is thought to function in the scission and stabilization of caveolae, through binding to dynamin-2 and EHD2, respectively. These two functions are considered to be switched by PACSIN2 phosphorylation by protein kinase C (PKC) upon hypotonic stress and sheer stress. The phosphorylation decreases the membrane binding affinity of PACSIN2, leading to its removal from caveolae. The removal of the putative oligomeric spiral of PACSIN2 from caveolar membrane invaginations could lead to the deformation of caveolae. Indeed, PACSIN2 removal from caveolae is accompanied by the recruitment of dynamin-2, suggesting that the removal provides space for the function of dynamin-2. Otherwise, the removal of PACSIN2 decreases the stability of caveolae, which could result in the flattening of caveolae. In contrast, an increase in the amount of EHD2 restored caveolar stability. Therefore, PACSIN2 at caveolae stabilizes caveolae, but its removal by phosphorylation could induce both caveolar endocytosis and flattening.

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes., (© 2014 The Authors.)

Mutations in the ankyrin repeat domain (ARD) of TRPV4 are responsible for several channelopathies, including Charcot-Marie-Tooth disease type 2C and congenital distal and scapuloperoneal spinal muscular atrophy. However, the molecular pathogenesis mediated by these mutations remains elusive, mainly due to limited understanding of the TRPV4 ARD function. Here we show that phosphoinositide binding to the TRPV4 ARD leads to suppression of the channel activity. Among the phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) most potently binds to the TRPV4 ARD. The crystal structure of the TRPV4 ARD in complex with inositol-1,4,5-trisphosphate, the head-group of PI(4,5)P2, and the molecular-dynamics simulations revealed the PI(4,5)P2-binding amino-acid residues. The TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2. Notably, disease-associated TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity. These findings identify TRPV4 ARD as a lipid-binding domain in which interactions with PI(4,5)P2 normalize the channel activity in TRPV4.

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.

Caveolae are flask-shaped invaginations of the plasma membrane that are associated with tumor formation, pathogen entry and muscular dystrophy, through the regulation of lipids, signal transduction and endocytosis. Caveolae are generated by the fusion of caveolin-1-containing vesicles with the plasma membrane, which then participate in endocytosis via dynamin. Proteins containing membrane-sculpting F-BAR (or EFC) domains organize the membrane in clathrin-mediated endocytosis. Here, we show that the F-BAR protein PACSIN2 sculpts the plasma membrane of the caveola. The PACSIN2 F-BAR domain interacts directly with caveolin-1 by unmasking autoinhibition of PACSIN2. Furthermore, the membrane invaginations induced by the PACSIN2 F-BAR domain contained caveolin-1. Knockdown of PACSIN2 resulted in abnormal morphology of caveolin-1-associated plasma membranes, presumably as a result of decreased recruitment of dynamin-2 to caveolin-1. These results indicate that PACSIN2 mediates membrane sculpting by caveolin-1 in caveola morphology and recruits dynamin-2 for caveola fission.

The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily consists of proteins containing the BAR domain, the extended FCH (EFC)/FCH-BAR (F-BAR) domain, or the IRSp53-MIM homology domain (IMD)/inverse BAR (I-BAR) domain. These domains bind membranes through electrostatic interactions between the negative charges of the membranes and the positive charges on the structural surface of homo-dimeric BAR domain superfamily members. Some BAR superfamily members have membrane-penetrating insertion loops, which also contribute to the membrane binding by the proteins. The membrane-binding surface of each BAR domain superfamily member has its own unique curvature that governs or senses the curvature of the membrane for BAR-domain binding. The wide range of BAR-domain surface curvatures correlates with the various invaginations and protrusions of cells. Therefore, each BAR domain superfamily member may generate and recognize the curvature of the membrane of each subcellular structure, such as clathrin-coated pits or filopodia. The BAR domain superfamily proteins may regulate their own catalytic activity or that of their binding proteins, depending on the membrane curvature of their corresponding subcellular structures., ((c) 2009 Elsevier Ltd. All rights reserved.)

We studied the process of formation of stress fibres and involvement of phosphorylation of myosin-II during spreading of Swiss 3T3 fibroblasts. In cells that were allowed to spread for 1 h on a glass surface, circular bundles of actin and myosin-II filament were present. At 2-3 h after the plating, cells showed a polygonal and polarized shape. The proportion of the cells having circular bundles was decreased, whereas that of the cells with straight bundles of actin filaments was increased. At 4 h after the plating, cells were completely polarized and stress fibres were present at the periphery and the dorsal and ventral surfaces of the cells. Thus, spreading cells possessed different forms of actomyosin bundles corresponding to the cell shape. In circular bundles and stress fibres, myosin regulatory light chains were diphosphorylated. Formation of circular bundles and stress fibres was suppressed after the treatment of the cells with Y-27632, a Rho-kinase inhibitor, or blebbistatin, a myosin-II inhibitor. In digitonin-extracted cells, circular bundles as well as stress fibres contracted following the addition of Mg-ATP. These results suggest that circular bundles are contractile structures containing actin and phosphorylated myosin-II filaments, and the formation of circular bundles is regulated by Rho-kinase.