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Abstract One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

Macropinocytosis is a cellular process that enables cells to engulf extracellular material, such as nutrients, growth factors, and even whole cells. It is involved in several physiological functions as well as pathological conditions. In cancer cells, macropinocytosis plays a crucial role in promoting tumor growth and survival under nutrient-limited conditions. In particular KRAS mutations have been identified as main drivers of macropinocytosis in pancreatic, breast, and non-small cell lung cancers. We performed a high-content screening to identify inhibitors of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC)-derived cells, aiming to prevent nutrient scavenging of PDAC tumors. The screening campaign was conducted in a well-known pancreatic KRAS-mutated cell line (MIAPaCa-2) cultured under nutrient deprivation and using FITC-dextran to precisely quantify macropinocytosis. We assembled a collection of 3584 small molecules, including drugs approved by the Food and Drug Administration (FDA), drug-like molecules against molecular targets, kinase-targeted compounds, and molecules designed to hamper protein-protein interactions. We identified 28 molecules that inhibited macropinocytosis, with potency ranging from 0.4 to 29.9 μM (EC50). A few of them interfered with other endocytic pathways, while 11 compounds did not and were therefore considered specific “bona fide” macropinocytosis inhibitors and further characterized. Four compounds (Ivermectin, Tyrphostin A9, LY2090314, and Pyrvinium Pamoate) selectively hampered nutrient scavenging in KRAS-mutated cancer cells. Their ability to impair albumin-dependent proliferation was replicated both in different 2D cell culture systems and 3D organotypic models.These findings provide a new set of compounds specifically targeting macropinocytosis, which could have therapeutic applications in cancer and infectious diseases.

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, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale 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 I-BAR proteins, 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.

Summary: Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.

The cell cortex that supports the plasma membrane contains spectrin, a protein that interacts with the acto-myosin cytoskeleton. Here, the authors analyze spectrin behavior during cellular mechanoresponse and membrane trafficking, and observe spectrin regulation by myosin-driven contractility.

The nucleus is a mechanically stiff organelle of the cell and the DNA damage response protein ATR can localize to the nuclear envelope upon mechanical stress. Here, the authors show that ATR may contribute to the integrity of the nuclear envelope and may play a role in cell migration.

The I-BAR protein IRSp53 senses membrane curvature but its physiological role is unclear. Here, the authors show that during early lumen morphogenesis, IRSp53 controls the shape of the apical plasma membrane and polarized trafficking and ensures the correct epithelial tubular architecture and if deleted, affects renal tubules morphogenesis in various organisms.

It is unclear if genetic alterations in endocytic proteins play a causal role in high incidence human cancers. Here, the authors report the oncogenic role of Epsin3 (EPN3) in breast cancer, and show EPN3 to drive tumorigenesis through induction of a partial epithelial mesenchymal transition state and a TGFβ-dependent regulatory loop that promotes cellular plasticity and invasive behaviour.

This work investigates the mechano-biological features of cells cultured in monolayers in response to different osmotic conditions. In-vitro experiments have been performed to quantify the long-term effects of prolonged osmotic stresses on the morphology and proliferation capacity of glioblastoma cells. The experimental results highlight that both hypotonic and hypertonic conditions affect the proliferative rate of glioblastoma cells on different cell cycle phases. Moreover, glioblastoma cells in hypertonic conditions display a flattened and elongated shape. The latter effect is explained using a nonlinear elastic model for the single cell. Due to a crossover between the free energy contributions related to the cytosol and the cytoskeletal fibers, a critical osmotic stress determines a morphological transition from a uniformly compressed to an elongated shape.

Summary: Antimicrobial peptides (AMPs) are the body’s natural innate immune defense against a spectrum of pathogens and can also modulate cell proliferation, chemotaxis, angiogenesis, wound healing, and immune cell activity. Harnessing these diverse functions for prophylactic use is contingent upon understanding the regulatory mechanisms governing their unconventional secretion from cells. Analysis of the secretion of S100A7 (Psoriasin), an abundant AMP stored in differentiated keratinocytes of the skin, has revealed an unexpected biphasic secretory response to bacterial exposure. The core components regulating S100A7 secretion are NFκB/p38MAPK, caspase-1, and interleukin (IL)-1α. The initial activation of this core machinery is mediated by Toll-like receptor signaling, whereas the chronic response is mediated by Caspase-8 downregulation. Interestingly, there is a concomitant downregulation of Caspase-8 in inflammatory skin diseases wherein S100A7 is constitutively released. These results highlight the potential of targeting these components to control the release of AMPs from the skin in both homeostatic and disease conditions. : The global explosion of antibiotic-resistant microorganisms has spurred interest in alternative strategies to combat these “superbugs.” Antimicrobial peptides (AMPs) have emerged as a promising solution. Bhatt et al. show downregulation of epidermal caspase-8 can mediate sustained release of AMPs from the skin and provide an effective defense against infection. keywords: caspase, antimicrobial peptides, skin, TLR, IL-1, NFkB, psoriasis, antibiotic resistance

Several endocytic pathways operate simultaneously at the cell surface, including the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. Here the authors show that small GTPases and BAR domain proteins regulate branched actin to make clathrin and dynamin-independent endocytic vesicles.

Circular dorsal ruffles (CDRs) are apical actin enriched structures involved in the interpretation of growth factor gradients during cell migration. Here, the authors find that a RAB35/PI3K axis is necessary and sufficient for the formation and stabilization of polarized CDRs and persistent directional migration.

A biological tissue is an ensemble of soft cells in close physical contact. Events such as cell-shape changes and, more rarely, cell-divisions and apoptosis continuously occur in a tissue, whose collective behavior is set by the cumulative occurrence of such events. In this complex environment, quantifying the single-cell dynamics is key to extract quantitative information to be used to capture the fundamental ingredients of this collective tissue dynamics for validating the predictions of models and numerical simulations. However, tracking the motion of each cell in a dense assembly, even in controlled in vitro settings, is a demanding task, because of a combination of different factors, such as poor image quality, cell shape variability and cell deformability. Here we show that Differential Dynamic Microscopy (DDM), an approach that provides a characterization of the sample structure and dynamics at various spatial frequencies (wave-vectors), can be used successfully to extract quantitative information about a confluent monolayer of Madin-Darby Canine Kidney (MDCK) epithelial cells. In particular, combining structural and dynamical information obtained at different wave-vectors, we show that DDM can provide the single-cell mean squared displacement and the cell division rate at various stages during the temporal evolution of the monolayer. In contrast with tracking algorithms, which require expert supervision and a considerate choice of the analysis parameters, DDM analysis can be run in an automated fashion and yields an unbiased quantification of the dynamic processes under scrutiny, thus providing a powerful means to probe the single-cell dynamics within dense cell collectives.

Basic scientific research generates knowledge that has intrinsic value which is independent of future applications. Basic research may also lead to practical benefits, such as a new drug or diagnostic method. Building on our previous study of basic biomedical and biological researchers at Harvard, we present findings from a new survey of similar scientists from three countries. The goal of this study was to design policies to enhance both the public health potential and the work satisfaction and test scientists’ attitudes towards these factors. The present survey asked about the scientists’ motivations, goals and perspectives along with their attitudes concerning policies designed to increase both the practical (i.e. public health) benefits of basic research as well as their own personal satisfaction. Close to 900 basic investigators responded to the survey; results corroborate the main findings from the previous survey of Harvard scientists. In addition, we find that most bioscientists disfavor present policies that require a discussion of the public health potential of their proposals in grants but generally favor softer policies aimed at increasing the quality of work and the potential practical benefits of basic research. In particular, bioscientists are generally supportive of those policies entailing the organization of more meetings between scientists and the general public, the organization of more academic discussion about the role of scientists in the society, and the implementation of a “basic bibliography” for each new approved drug.

The originally published version of this Article contained an error in the name of the author Salvatore Corallino, which was incorrectly given as Corallino Salvatore. This has now been corrected in both the PDF and HTML versions of the Article.

Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia.

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape.

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.

BACKGROUND: In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that knockout mice for Eps8, a regulator of actin dynamics, display reduced body weight, partial resistance to age- or diet-induced obesity, and overall improved metabolic status. Alteration in the liver gene expression profile, in behavior and metabolism point to a calorie restriction-like phenotype in Eps8 knockout mice. Additionally, and consistent with a calorie restricted metabolism, Eps8 knockout mice show increased lifespan. The metabolic alterations in Eps8 knockout mice correlated with a significant reduction in intestinal fat absorption presumably caused by a 25% reduction in intestinal microvilli length. CONCLUSIONS/SIGNIFICANCE: Our findings implicate actin dynamics as a novel variable in the determination of longevity. Additionally, our observations suggest that subtle differences in energy balance can, over time, significantly affect bodyweight and metabolic status in mice.

The TOCA family of F-BAR-containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell-cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP-dependent actin-dynamics.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (DeltaGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of DeltaGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that DeltaGBD-mDia2-induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.

Background: Exchanges between scientists and nonscientists are critical to realizing the social value of basic research. These exchanges rest in part on the willingness and ability of scientists to engage effectively in science communication activities. In this paper, we discuss the perception and willingness of basic scientists in the biological and biomedical fields to engage in science outreach. Methods: The analysis is based on qualitative data collected as part of a survey on the social value of basic research and is framed by the theory of planned behavior. This is a well-established theory of human behavior that relies on the premise that a person’s intention to engage in a behavior is the single best predictor of whether that person will in fact engage in that behavior. Results: Our data show that, while bioscientists maintain a positive attitude towards science communication, their intentions are influenced by some negative feelings with regard to how nonscientists react to science communication efforts. Interactions with institutional actors, governmental bodies and the public are particularly problematic. On the other hand, interactions with clinicians and patients are framed in positive terms. Finally, some study participants raised concerns as to their ability to communicate science effectively, the availability of time and resources, and the lack of proper rewards, particularly in terms of career advancement, for those who engage in science efforts. Conclusions: Our findings suggest that bioscientists' intentions to engage in science communication efforts must be better studied to develop empirically-informed interventions to increase scientists’ participation in science outreach efforts.

Basic scientific research generates knowledge that has intrinsic value which is independent of future applications. Basic research may also lead to practical benefits, such as a new drug or diagnostic method. Building on our previous study of basic biomedical and biological researchers at Harvard, we present findings from a new survey of similar scientists from three countries. This survey asked about the scientists’ motivations, goals and perspectives along with their attitudes concerning policies designed to increase both the practical (i.e. public health) benefits of basic research as well as their own personal satisfaction. Close to 900 basic investigators responded to the survey; results corroborate the main findings from the previous survey of Harvard scientists. In addition, we find that most bioscientists disfavor present policies that require a discussion of the public health potential of their proposals in grants but generally favor softer policies aimed at increasing the quality of work and the potential practical benefits of basic research. In particular, bioscientists are generally supportive of those policies entailing the organization of more meetings between scientists and the general public, the organization of more academic discussion about the role of scientists in the society, and the implementation of a “basic bibliography” for each new approved drug.