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Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development. Funding: Q.T.-R. acknowledges a grant funded by ‘The Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE)’ (PRE2018-084393). M.I. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-63759) and the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P). S.W. acknowledges support from the Government of Spain (MINECO’s Plan Nacional (PGC2018-098532-A-I00), Severo Ochoa (CEX2019- 000910-S) and Generalitat de Catalunya (CERCA, AGAUR). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and MINECO’s Plan Nacional (BFU2017-86296-P)

The sensation of mechanical force underlies many of our daily activities. As the sense of touch determines the quality of life, the subconscious sense of proprioception and visceral mechanosensation is indispensible for survival. Many internal organs change shape, either as an active part of their physiology or passively due to body movements. Importantly, these shape changes need to be sensed and balanced properly to prevent organ failure and dysfunction. Consequently, a failure to properly sense volume changes of internal organs has a huge clinical relevance, manifested by a plethora of congenital and age-related diseases. Here we review novel data on mammalian stretch reception as well as classical studies from insect and nematode proprioceptors with the aim to highlight the missing link between organ-level deformation and mechanosensing on the molecular level.

Fever is an evolutionary conserved response able to stimulate and boost both innate and adaptive immunity. Temperature variations in general modify the homeostatic setpoint such as blood flow in warm blooded animals and can change gene-expression patterns on the single cell level [1] . Neutrophils and dendritic cells (DCs) patrol tissues under various temperature levels and their effective encounter and response to ‘danger signals’ is key for optimal immune defence [2] . Still, the thermo-regulation of immune cell dynamics remains largely elusive. Here we show that the local temperature controls immune cell dynamics, such as shape changes, cell migration speed and persistence in 2D/2.5D/3D biomimetic micro-environments.

SUMMARY Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity., Graphical Abstract, In Brief Gómez-García et al. show that the Pou5f1 gene folds into nucleosome clutches, with larger clutches in differentiated cells than in stem cells. These clutch changes are accompanied by enhanced hierarchical looping in differentiated cells. H2B dynamics is cell-type specific, correlates with clutch patterns, and is regulated by linker histone H1.

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment. Funding: V.V. acknowledges support from the ICFOstepstone PhD Programme funded by the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement 665884. F.P. and Q.T.-R. acknowledge grants funded by Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE) (BES2017-080523-SO, PRE2018-084393). M.A.V. acknowledges support from the Spanish Ministry of Science, Education and Universities through grant RTI2018-099718-B-100 and an institutional “Maria de Maeztu Programme” for Units of Excellence in R&D (CEX2018-000792-M) and FEDER funds.S.W. and M.K. acknowledge support from the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa program for Centres of Excellence in R&D (CEX2019-000910-S), from Fundació Privada Cellex, Fundación Mig-Puig, and from Generalitat de Catalunya through the CERCA program and LaserLab (654148). M.K. acknowledges support through Spanish Ministry of Economy and Competitiveness (RYC-2015-17935, EQC2018-005048-P, AEI-010500-2018-228, and PGC2018-097882-A-I00),Generalitat de Catalunya (2017 SGR 1012), the ERC (715243), and the HFSPO (CDA00023/2018). S.W. acknowledges support through the Spanish Ministry of Economy and Competitiveness via MINECO’s Plan Nacional (PGC2018-098532-A-I00). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, and the MINECO’s Plan Nacional (BFU2017-86296-P)

In vitro reconstitutions reveal the essential components of a mammalian, cytoplasmic mRNA transport system and their functions., Through the asymmetric distribution of messenger RNAs (mRNAs), cells spatially regulate gene expression to create cytoplasmic domains with specialized functions. In neurons, mRNA localization is required for essential processes such as cell polarization, migration, and synaptic plasticity underlying long-term memory formation. The essential components driving cytoplasmic mRNA transport in neurons and mammalian cells are not known. We report the first reconstitution of a mammalian mRNA transport system revealing that the tumor suppressor adenomatous polyposis coli (APC) forms stable complexes with the axonally localized β-actin and β2B-tubulin mRNAs, which are linked to a kinesin-2 via the cargo adaptor KAP3. APC activates kinesin-2, and both proteins are sufficient to drive specific transport of defined mRNA packages. Guanine-rich sequences located in 3′UTRs of axonal mRNAs increase transport efficiency and balance the access of different mRNAs to the transport system. Our findings reveal a minimal set of proteins sufficient to transport mammalian mRNAs.

Through the asymmetric distribution of mRNAs cells spatially regulate gene expression to create cyto-plasmic domains with specialized functions. In mammalian neurons, mRNA localization is required for essential processes as cell polarization, migration and synaptic plasticity underlying long-term memory formation. The essential components driving cytoplasmic mRNA transport in neurons and mammalian cells are not known. Here, we report the first reconstitution of a mammalian mRNA transport system revealing that the tumour suppressor adenomatous polyposis coli (APC) forms stable complexes with the axonally localised β-actin and β2B-tubulin mRNAs which are linked to a heterotrimeric kinesin-2 via the cargo adaptor KAP3. APC activates kinesin-2 and both proteins are sufficient to drive specific transport of defined mRNA packages. Guanine-rich sequences located in 3’UTRs of axonal mRNAs increase transport efficiency and balance the access of different mRNAs to the transport system. Our findings reveal for the first time a minimal set of proteins capable of driving kinesin-based, mammalian mRNA transport.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families(1). Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Cell migration has important functions in immune response, development and cancer proliferation. Cell movement essentially requires the establishment of an axis of polarity. To maintain migration in one direction - called persistent cell migration - the polarity axis needs to be stabilized in the direction of movement.Biochemical feedback loops between signaling molecules have been recognized to play an important role for stabilizing cell polarity by reinforcing actin polymerization at the leading edge. Here we describe a reverse feedback mechanism coupling the retrograde flowing actin cytoskeleton in migrating cells back to cell polarity and persistent migration supporting an essential mechano-chemical coupling of signaling cascades and the flowing cytoskeletal meshwork in migrating cells (Maiuri et al., 2015). We used genetic, physical and chemical tools to modulate cytoskeletal flows in migrating dendritic cells and found a strong increase in the stability of cellular polarization and migration persistence upon increasing retrograde flow speed. We provide experimental evidence that the spatial distribution of different actin binding proteins (Myosin-GFP, Lifeact-GFP and Utrophin-GFP) with varying actin-binding strength gets significantly shifted towards the cell rear for increasing retrograde flow speeds upon sufficient coupling to the flowing actin network. Finally we show that a minimal theoretical model build on the mechano-chemical coupling between polarity markers and cytoskeletal flows can predict the stability of cell polarity in dependence on retrograde flow speeds and can capture the broad range of cell migration phenotypes from Brownian, to persistent and intermittent random walks.Maiuri, P.∗, Rupprecht, J.-F.∗, Wieser, S.∗, Ruprecht, V., Benichou, O., Carpi, N., Coppey, M., De Beco, S., Gov, N., Heisenberg, C.-P., et al. (2015). Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence. Cell 161, 374-386. ∗contributed equally.

Nonadherent polarized cells have been observed to have a pearlike, elongated shape. Using a minimal model that describes the cell cortex as a thin layer of contractile active gel, we show that the anisotropy of active stresses, controlled by cortical viscosity and filament ordering, can account for this morphology. The predicted shapes can be determined from the flow pattern only; they prove to be independent of the mechanism at the origin of the cortical flow, and are only weakly sensitive to the cytoplasmic rheology. In the case of actin flows resulting from a contractile instability, we propose a phase diagram of three-dimensional cell shapes that encompasses nonpolarized spherical, elongated, as well as oblate shapes, all of which have been observed in experiment.

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides—in particular a CAMP with Lysine–Leucine–Lysine repeats (termed KLK)—affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents., Research highlights ► Cationic antimicrobial peptide KLK affects eukaryotic host cells ► KLK induces accumulation of plasma mebrane proteins/lipids ► Dependent on sialic acid residues ► Application of KLK leads to a recycling inhibition of sialyted molecules

Saccharomyces cerevisiae Lpe10p is a homologue of the Mg2+-channel-forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2Δ/lpe10Δ double disruption, indicating that these two proteins cannot substitute for each other. Here, we show that deletion of LPE10 results in a loss of rapid Mg2+ influx into mitochondria, as has been reported for MRS2 deletion. Additionally, we found a considerable loss of the mitochondrial membrane potential (ΔΨ) in the absence of Lpe10p, which was not detected in mrs2Δ cells. Addition of the K+/H+-exchanger nigericin, which artificially increases ΔΨ, led to restoration of Mg2+ influx into mitochondria in lpe10Δ cells, but not in mrs2Δ/lpe10Δ cells. Mutational analysis of Lpe10p and domain swaps between Mrs2p and Lpe10p suggested that the maintenance of ΔΨ and that of Mg2+ influx are functionally separated. Cross-linking and Blue native PAGE experiments indicated interaction of Lpe10p with the Mrs2p-containing channel complex. Using the patch clamp technique, we showed that Lpe10p was not able to mediate high-capacity Mg2+ influx into mitochondrial inner membrane vesicles without the presence of Mrs2p. Instead, coexpression of Lpe10p and Mrs2p yielded a unique, reduced conductance in comparison to that of Mrs2p channels. In summary, the data presented show that the interplay of Lpe10p and Mrs2p is of central significance for the transport of Mg2+ into mitochondria of S. cerevisiae. Structured digital abstract • MINT-7905005: LPE10 (uniprotkb:Q02783) physically interacts (MI:0915) with MRS2 (uniprotkb:Q01926) by anti tag coimmunoprecipitation (MI:0007) • MINT-7905028: LPE10 (uniprotkb:Q02783) and LPE10 (uniprotkb:Q02783) covalently bind (MI:0195) by cross-linking study (MI:0030) • MINT-7905072: LPE10 (uniprotkb:Q02783) and MRS2 (uniprotkb:Q01926) covalently bind (MI:0195) by cross-linking study (MI:0030)

SummaryCell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

Cell migration is key for various biological processes and potentially leads to cancer dissemination if reactivated by tumour cells. Here we used zebrafish embryos from blastula to gastrula stages as a model system to monitor cell transformations under defined culture conditions and within the physiological tissue environment. This experimental framework allowed us to uncover a simple polarization mechanism that drives the transformation of embryonic cells into a highly efficient amoeboid migration mode irrespective of the specific progenitor cell type [1]. The observed amoeboid motility switch strongly depends on mechanical and contractile properties of the cellular cytoskeleton and was triggered by stochastic changes in cortex architecture when cells were exposed to biochemical stimuli or compressive forces from the 3D environment. Supported by theoretical modeling we show that cell transformation is induced by cortical instabilities above a critical threshold level of Myosin II mediated contractility, resulting in polarized cells with exceptionally high retrograde cortical flows and a pronounced increase in cortical actomyosin density towards the cell rear. We further show through a combination of experiments and theory that these cortical flows are essential for stabilization of cell polarity and cell shape and, in addition, provide the intracellular force required for cell locomotion in confined environments. Finally, we provide evidence for the existence of a similar amoeboid migration phenotype within the gastrulating embryo in vivo and show that amoeboid cell transformations can be induced in response to embryo wounding.[1] V. Ruprecht, S. Wieser, A. Callan-Jones, M. Smutny, H. Morita, K. Sako, V. Barone, M. Ritsch-Marte, M. Sixt, R. Voituriez, and C.-P. Heisenberg, “Cortical contractility triggers a stochastic switch to fast amoeboid cell motility”, Cell, vol. 160, no. 4, pp. 673-685, Feb. 2015.

International audience; Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents.

We have developed an assay for quantitative analysis of the interaction between a fluorescently marked protein (prey) and a membrane protein (bait) using microstructured surfaces covered with biotinylated ligands (antibodies) targeted against the bait. The proof-of-concept was demonstrated for the interaction between CD4, a major co-receptor in T-cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signalling. Here we present improvements and a more precise characterization of the method as well as the applicability of the assay for the analysis of protein interactions within lipid rafts in the inner and outer leaflet of the plasma membrane. We stably expressed fluorescently labelled raft and non-raft proteins in the human T24 cell line as prey proteins and determined the degree of interaction with the antibody-targeted bait proteins CD59 (GPI-anchored protein, raft marker) and CD71 (Transferrin-receptor, non-raft marker), respectively. We found strong interaction of CD59 with putative raft markers including various GPI-GFP constructs, the inner-leaflet associated proteins Lck and Flottilin1 and a Pleckstrin-Homology domain fused to GFP. Importantly, we did not find interaction of CD59 with CD71-GFP and other potential non-raft proteins. When CD71 was used as the bait protein we did not find interaction with the putative raft markers. While the detected absence of CD71 from and the presence of CD59 in lipid rafts confirm current knowledge, it is still very unclear if a lipid-raft dependent coupling of proteins and certain especially negatively charged lipids across the plasma-membrane bilayer exists. Thus, our micropatterning assay will be of great interest to address this question.