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As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.TeaserCell stretch cycles generate PM evaginations of ≈100 nm which are sensed by IRSp53, triggering a local event of actin polymerization that flattens and recovers PM shape.

Summary The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring., Graphical Abstract, Highlights • The myristoyl group of c-Src interacts with the SH3 domain • The Unique Lipid Binding Region modulates myristoyl-related interactions • Mutations in the Unique and SH3 domains affect membrane anchoring • A fuzzy intramolecular complex is retained in the lipid-anchored form of USH3 Src, Structural Biology; Protein Structure Aspects; Biophysics

Cell membranes interact with a myriad of curvature-active proteins that control membrane morphology and are responsible for mechanosensation and mechanotransduction. Some of these proteins, such as those containing BAR domains, are curved and elongated, and hence may adopt different states of orientational order, from isotropic to maximize entropy to nematic as a result of crowding or to adapt to the curvature of the underlying membrane. Here, extending the work of [Nascimento et. al, Phys. Rev. E, 2017, 96, 022704], we develop a mean-field density functional theory to predict the orientational order and evaluate the free-energy of ensembles of elongated and curved objects on curved membranes. This theory depends on the microscopic properties of the particles and explains how a density-dependent isotropic-to-nematic transition is modified by anisotropic curvature. We also examine the coexistence of isotropic and nematic phases. This theory lays the ground to understand the interplay between membrane reshaping by BAR proteins and molecular order, examined in [Le Roux et. al, Submitted, 2020]., 12 pages, 8 figures

Reproducció del document publicat a: https://doi.org/10.1038/s41467-021-26591-3, In many physiological situations, BAR proteins reshape membranes with pre-existing curvature (templates), contributing to essential cellular processes. However, the mechanism and the biological implications of this reshaping process remain unclear. Here we show, both experimentally and through modelling, that BAR proteins reshape low curvature membrane templates through a mechanochemical phase transition. This phenomenon depends on initial template shape and involves the co-existence and progressive transition between distinct local states in terms of molecular organization (protein arrangement and density) and membrane shape (template size and spherical versus cylindrical curvature). Further, we demonstrate in cells that this phenomenon enables a mechanotransduction mode, in which cellular stretch leads to the mechanical formation of membrane templates, which are then reshaped into tubules by BAR proteins. Our results demonstrate the interplay between membrane mechanics and BAR protein molecular organization, integrating curvature sensing and generation in a comprehensive framework with implications for cell mechanical responses., This work was supported by the Spanish Ministry of Science and Innovation (PGC2018-099645-B-I00 to X.T., PID2019-110949GB-I00 to M.A., BFU2016-79916-P and PID2019-110298GB-I00 to P.R.-C.), the Spanish Ministry of Economy and Competitiveness/FEDER (BES-2016-078220 to C.T., the European Commission (H2020-FETPROACT-01-2016-731957), the European Research Council (Adv-883739 to X.T., CoG-681434 to M.A.), the Generalitat de Catalunya (2017-SGR-1602 to X.T. and P.R.-C., 2017-SGR-1278 to M.A.), the prize “ICREA Academia” for excellence in research to P.R.-C. and to M.A., Fundació la Marató de TV3 (201936-30-31 and 201903-30-31-32), and “la Caixa” Foundation (Agreement LCF/PR/HR20/52400004). IBEC and CIMNE are recipients of a Severo Ochoa Award of Excellence from the MINECO. We would like to thank all the members of P. Roca-Cusachs, X. Trepat, and M. Arroyo laboratories for technical assistance and discussions. We thank M. Pons, X. Menino, M.G. Parajo, M.-A. Rodriguez, N. Castro, R. Sunyer, I. Granero, V. González, the Unitat de Criomicroscòpia Electrònica (Centres Científics i Tecnològics de la Universitat de Barcelona, CCiTUB), and the MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS “NANBIOSIS” from CIBER-BBN at IBEC, for their excellent technical assistance.

The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring.

Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface.