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The membrane-anchored c-Src tyrosine kinase mediates signaling from a wide range of cell surface receptors controlling cell growth, adhesion, and survival. c-Src deregulation is associated with cancer. Dimerization appears to be a novel layer of regulation through a yet unclear mechanism. Binding of c-Src tyrosine kinase to the plasma membrane is mediated by the myristoylated and strongly positively charged N-terminal SH4 domain. Although activation of c-Src is known to require phosphorylation by a second c-Src molecule, electrostatic repulsion between the charged residues was considered to prevent dimerization. Here we show that a cluster of positively charged lysine residues in c-Src SH4 domain not only does not prevent dimerization but, in fact, enhances it through a lipid-mediated process. Dimerization not only depends on the number of positive charges but also on their position and the nature of the charged residues. Replacement of lysine by arginine increases dimerization in vitro and in vivo and, in HEK293T cells, causes a two-fold increase in tyrosine phosphorylation. Lipid mediated protein-protein interactions induced by clusters of basic residues may represent a general mechanism for modulating cell signaling, consistent with the abundance of positively charged residues in the juxta membrane region of many signaling proteins.

Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

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.

Pontes et al. show that plasma membrane mechanics exerts an upstream control during cell motility. Variations in plasma membrane tension orchestrate the behavior of the cell leading edge, with increase–decrease cycles in tension promoting adhesion row positioning., Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.

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.

The proto-oncogene tyrosine-protein kinase Src is a key ele- ment of signaling cascades involved in the invasive and meta- stasis-forming capacity of cancer cells. While membrane ty- rosine-kinase receptors are known to dimerize, Src is classified as a non-receptor kinase and assumed to remain always mono- meric. Here we demonstrate the formation of stable dimers by the first domains of myristoylated Src previously shown to be sufficient for Src trafficking. Src dimers fused to green fluo- rescent protein (GFP) on supported lipid bilayers were identi- fied using single-molecule photobleaching experiments. Com- petition with a protein containing only native Src domains without GFP confirms that dimerization is a previously over- looked intrinsic property of Src. Dimerization is concomitant to membrane binding by the myristoylated forms of Src and may constitute a new regulation layer for the Src oncogene.

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

SummaryRegulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved., Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.