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Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by

Spontaneous locomotion is a common feature of most metazoan cells, generally attributed to the fundamental properties of the actomyosin network. This force-producing machinery has been studied down to the most minute molecular details, especially in lamellipodium-driven migration. Nevertheless, how actomyosin networks work inside contraction-driven amoeboid cells still lacks unifying principles. Here, using stable motile blebs as a model amoeboid motile system, we image the dynamics of the actin cortex at the single filament level and reveal the co-existence of three phases of the actin network with distinct rheological properties. Physical modelling shows that these three phases organize spontaneously due to a rigidity percolation transition combined with an active advection of the percolated network. This spontaneous spatial organization of the mechanical properties of the actin network, which we propose to call advected percolation, constitutes a minimal and generic locomotion mechanism. It explains, down to the single actin filament level and up to the scale of the entire cell, how amoeboid cells can propel efficiently through complex 3D environments, a feature shared by immune and cancer cells.

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 application of genetically encoded fluorophores for microscopy has afforded one of the biggest revolutions in the biosciences. Bioluminescence microscopy is an appealing alternative to fluorescence microscopy, because it does not depend on external illumination, and consequently does neither produce spurious background autofluorescence, nor perturb intrinsically photosensitive processes in living cells and animals. The low quantum yield of known luciferases, however, limit the acquisition of high signal-noise images of fast biological dynamics. To increase the versatility of bioluminescence microscopy, we present an improved low-light microscope in combination with deep learning methods to increase the signal to noise ratio in extremely photon-starved samples at millisecond exposures for timelapse and volumetric imaging. We apply our method to image subcellular dynamics in mouse embryonic stem cells, the epithelial morphology during zebrafish development, and DAF-16 FoxO transcription factor shuttling from the cytoplasm to the nucleus under external stress. Finally, we concatenate neural networks for denoising and light-field deconvolution to resolve intracellular calcium dynamics in three dimensions of freely movingCaenorhabditis eleganswith millisecond exposure times. This technology is cost-effective and has the potential to replace standard optical microscopy where external illumination is prohibitive.

Regulation of microtubule (MT) dynamics is key for mitotic spindle assembly and faithful chromosome segregation. Here we show that polyglutamylation, a still understudied post-translational modification of spindle MTs, is essential to define their dynamics within the range required for error-free chromosome segregation. We identify TTLL11 as an enzyme driving MT polyglutamylation in mitosis and show that reducing TTLL11 levels in human cells or zebrafish embryos compromises chromosome segregation fidelity and impairs early embryonic development. Our data reveal a mechanism to ensure genome stability in normal cells that is compromised in cancer cells that systematically downregulate TTLL11. Our data suggest a direct link between MT dynamics regulation, MT polyglutamylation and two salient features of tumour cells, aneuploidy and chromosome instability (CIN). The work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 675737 to I.V., I.Z., and C.J.; the grants from the Spanish Ministry of Economy (MINECO) I + D grant BFU2012-37163 and BFU2015-68726-P to I.V.; C.J. was supported by the French National Research Agency (ANR) award ANR-17-CE13-0021 and Fondation pour la Recherche Medicale (FRM) grant DEQ20170336756. V.R. was supported by the Spanish Ministry of Economy (MINECO) I + D grant PID2020-117011GB-I00. We thank all the members of the Vernos group and the DiviDE ITN for the discussions and the CRG microscopy facility for technical support. We acknowledge the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa” as well as support of the CERCA Programme/Generalitat de Catalunya. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript are in part based upon data obtained from the GTEx Portal on 05/03/2021. The results shown here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes. MK acknowledges financial support from the Spanish Ministry of Economy and Competitiveness through the Plan Nacional (PGC2018-097882-A-I00), FEDER (EQC2018-005048-P), Severo Ochoa program for Centres of Excellence in R&D (CEX2019-000910-S; RYC-2016-21062), from Fundació Privada Cellex, Fundació Mir-Puig, and from Generalitat de Catalunya through the CERCA and Research program (2017 SGR 1012), in addition to funding through ERC (MechanoSystems) and HFSP (CDA00023/2018). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, MINECO's Plan Nacional (BFU2017-86296-P, PID2020-117011GB-I00) and Generalitat de Catalunya (CERCA). 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

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.

Super-resolution fluorescence microscopy has become a powerful tool in cell biology to observe sub-cellular organization and molecular details below the diffraction limit of light. Super-resolution methods are generally classified into three main concepts: stimulated emission depletion (STED), single molecule localization microscopy (SMLM) and structured illumination microscopy (SIM). Here, we highlight the novel concept of modulation-enhanced localization microscopy (meLM) which we designate as the 4th super-resolution method. Recently, a series of modulation-enhanced localization microscopy methods have emerged, namely MINFLUX, SIMPLE, SIMFLUX, ModLoc and ROSE. Although meLM combines key ideas from STED, SIM and SMLM, the main concept of meLM relies on a different idea: isolated emitters are localized by measuring their modulated fluorescence intensities in a precisely shifted structured illumination pattern. To position meLM alongside state-of-the-art super-resolution methods we first highlight the basic principles of existing techniques and show which parts of these principles are utilized by the meLM method. We then present the overall novel super-resolution principle of meLM that can theoretically reach unlimited localization precision whenever illumination patterns are translated by an arbitrarily small distance. L R acknowledges support of a fellowship from 'la Caixa' Foundation (ID 100010434, LCF/BQ/IN18/11660032) and funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 713673. V R acknowledges support from the Spanish Ministry of Economy and Competitiveness through the Program 'Centro de Excelencia Severo Ochoa 2013-2017', the CERCA Programme/Generalitat de Catalunya, MINECO's Plan Nacional (BFU2017-86296-P) and support from the CRG Advanced Light Microscopy Facility. S W acknowledges support from the Spanish Ministry of Economy and Competitiveness through the 'Severo Ochoa' program for Centres of Excellence in R&D (SEV-2015-0522), from Fundació Privada Cellex, and from Generalitat de Catalunya through the CERCA program.

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)

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo.

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 their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as a non-dissipative cellular shape deformation gauge that enables cells to continuously measure shape variations on the time scale of seconds. Inner nuclear membrane unfolding together with the relative spatial intracellular positioning of the nucleus provides physical information on the amplitude and type of cellular shape deformation. This adaptively activates a calcium-dependent mechano-transduction pathway, controlling the level of 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 behaviour to their microenvironment.One Sentence SummaryThe nucleus functions as an active deformation sensor that enables cells to adapt their behavior to the tissue microenvironment.

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.

We present a structured illumination microscopy based point localization estimator (SIMPLE) that achieves a 2-fold increase in single molecule localization precision compared to conventional centroid estimation methods. SIMPLE advances the recently introduced MINFLUX concept by using precisely phase-shifted sinusoidal wave patterns as nanometric rulers for simultaneous particle localization based on photon count variation over a 20 μm field of view. We validate SIMPLE in silico and experimentally on a TIRF-SIM setup using a digital micro-mirror device (DMD) as a spatial light modulator. Funding: “la Caixa” Foundation (ID 100010434, LCF/BQ/IN18/11660032); Marie Skłodowska-Curie Horizon 2020 (713673, 754558, 642157); Spanish Ministry Centro de Excelencia Severo Ochoa (SEV-2015-0522); MINECO’s Plan Nacional (BFU2017-86296-P); Generalitat de Catalunya (CERCA); Fundació Privada Cellex

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

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL 5 KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK―membrane interactions that lead to these alterations require the ability of the peptide to move into an α-helical conformation. We show that KLK induces an increase of the intracellular Ca 2+ concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca 2+ level of T24 cells. We show that, although KLK slightly stimulates Ca 2+ influx into the cell, the bulk increase of Ca 2+ levels is due to KLK-induced depletion of intracellular Ca 2+ stores. Finally, we demonstrate a KLK-induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.

Experimentalists are increasingly confronted with the demand to single out rare interaction events under a vast excess of non-interacting molecules. We recently presented an approach how to virtually dilute fluorescently labeled membrane constituents by combining photobleaching and single molecule microscopy, termed “Thinning Out Clusters while Conserving the Stoichiometry of Labeling” (TOCCSL; Moertelmaier et al., Appl. Phys. Lett., 2005, 87, 263903). Using this approach, single molecule microscopy can be performed even at arbitrarily high surface densities of fluorescent probe molecules. Here, we extended this method for two color microscopy. We provide a detailed statistical description of false positives and false negatives. In particular, we quantified the increase in sensitivity by tracking the colocalized objects over successive images. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin B molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. Each colocalized spot could be further analyzed with respect to its diffusion constant, yielding a clear anticorrelation between occupancy and mobility. We finally demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified.

Applying Scanning Probe Microscopy (SPM) methods to determine structural details of biomolecules (e.g. proteins) is one of the key trends in nanotechnology. Here we present the analysis of a Langmuir-Blodgett film of hydrofobin HFBI. The statistical analysis is based on an accurate procedure of data detrending to study the quasi-crystalline clusters of the HFBI layer. A statistical improvement method of signal to noise ratio was used. It relies on the variation of the crystallographic lattice superimposed on a quasi-periodical data set. The procedure produces optimal values of crystallographic periods and orientations of the symmetry axes. This information is used for selecting coherent data sets within a crystallographic cluster. Superposition of cells from the same cluster and calculation of an average periodical structure improves the signal to noise ratio, which leads from nano to subnano resolution of the protein structure. The number of superimposed cells N defines the resolution gain factor N 1/2 . In the case of hydrofobin, the value of N lies in the range of 25-100. This allows for the determination of fine structural details of HFBI. Average structures obtained from different domains of the protein layer also show a high degree of reproducibility.

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.

Mechanically coupled cells can generate forces driving cell and tissue morphogenesis during development. Visualization and measuring of these forces is of major importance to better understand the complexity of the biomechanic processes that shape cells and tissues. Here, we describe how UV laser ablation can be utilized to quantitatively assess mechanical tension in different tissues of the developing zebrafish and in cultures of primary germ layer progenitor cells ex vivo.

Mechanically coupled cells can generate forces driving cell and tissue morphogenesis during development. Visualization and measuring of these forces is of major importance to better understand the complexity of the biomechanic processes that shape cells and tissues. Here, we describe how UV laser ablation can be utilized to quantitatively assess mechanical tension in different tissues of the developing zebrafish and in cultures of primary germ layer progenitor cells ex vivo.

International audience; In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

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.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” or “membrane rafts”. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analogue Bodipy-GM1 which preferentially partitions into liquid ordered phases. For both markers we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, stable platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures the mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL₅KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK-membrane interactions that lead to these alterations require the ability of the peptide to move into an α-helical conformation. We show that KLK induces an increase of the intracellular Ca²(+) concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca²(+) level of T24 cells. We show that, although KLK slightly stimulates Ca²(+) influx into the cell, the bulk increase of Ca²(+) levels is due to KLK-induced depletion of intracellular Ca²(+) stores. Finally, we demonstrate a KLK-induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.

Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.

Selective uptake of high density lipoprotein (HDL)-associated cholesteryl-esters (CE) by hepatocytes is a crucial process for the removal of cholesterol from the circulation. Together with triglycerides, CE is transported in the hydrophobic core of HDL particles; free cholesterol, phospholipids, and apolipoproteins build up the particles' amphiphilic surface monolayer. The lipid transfer process itself is highly speculative: it is still unclear how lipids are removed from the circulation and transferred from HDL - a main carrier of cholesterol in the blood stream - to cells.In this study we provide a mechanistic understanding of the cargo exchange process between HDL and biomembranes. The interaction between HDL and synthetic lipid membranes was investigated with force spectroscopy and high speed atomic force microscopy; the transfer of single cargo molecules was directly visualized using a combined and simultaneously operating fluorescence and force microscope. Experimental evidence points to the fact that i) cargo transfer requires contact; ii) only amphiphilic cargo is transferred; iii) upon contact the particle incorporates into the hydrophobic core of the bilayer where it can diffuse. In particular, we compared the transfer of the fluorescently labelled lipids DiI, and Bodipy-labelled cholesterol and cholesteryl-ester. Live cell experiments confirmed the data obtained on the synthetic systems and on GPMV's (giant plasma membrane vesicles). Particle incorporation and cargo transfer was abolished at increased membrane cholesterol levels, as a consequence of the reduced membrane elasticity. These observations reveal a mechanism for regulation of lipid uptake based on sensing plasma membrane cholesterol levels. The function of the corresponding receptor SR-B1 is primarily an anchor to hold the particle close to the plasma membrane; once in proximity, elastic properties of the membrane regulate the fusion of the particle.

There is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. Various pictures emerged, which ascribe the plasma membrane a high degree of organization at very short length scales of tens of nanometers. We employed single molecule fluorescence microscopy to study diffusion of CD59, a GPI-anchored protein, in the plasma membrane of living T24 cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. By separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules, rendering direct influences of the membrane skeleton unlikely.In both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. However, a closed analytical theory for these analysis is only available for the simplest models. To address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with Monte Carlo simulations of the process. We demonstrated the working principle for the analysis of free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors.In the recent years increasing evidence was reported for an inherent heterogeneity of cell populations. Our reasoning was that mobility probes nanometer-sized properties of the moving protein and its local environment. Automated and tailored data analysis routines allowed for the analysis of the required large data sets: ∼200.000 trajectories obtained on ∼350 cells were analyzed in total. We found up to five-fold higher variability of the diffusion constant between cells compared to the uncertainty for the determination of the diffusion constant on a single cell.

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3 , a paralog of the neural microexon regulator Srrm4 . Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

Biomimetic materials have long been the (he)art of bioengineering. They usually aim at mimicking in vivo conditions to allow in vitro culture, differentiation and expansion of cells. The past decade has witnessed a considerable amount of progress in soft lithography, bio-inspired micro-fabrication and biochemistry, allowing the design of sophisticated and physiologically relevant micro- and nano-environments. These systems now provide an exquisite toolbox with which we can control a large set of physicochemical environmental parameters that determine cell behavior. Bio-functionalized surfaces have evolved from simple protein-coated solid surfaces or cellular extracts into nano-textured 3D surfaces with controlled rheological and topographical properties. The mechanobiological molecular processes by which cells interact and sense their environment can now be unambiguously understood down to the single-molecule level. This Commentary highlights recent successful examples where bio-functionalized substrates have contributed in raising and answering new questions in the area of extracellular matrix sensing by cells, cell–cell adhesion and cell migration. The use, the availability, the impact and the challenges of such approaches in the field of biology are discussed.

Gathering information on the interplay between different membrane components and the characteristics of protein interactions in live cells requires the design of sensitive methods that allow for the simultaneous study of different probe molecules. Especially the aim to single out rare interaction events under a vast excess of non-interacting molecules remains challenging.The two-color single molecule imaging technique presented here is the advancement of a recently presented approach to virtually dilute fluorescently labeled membrane constituents by photobleaching [1]. Using this technique, single molecule microscopy can be performed at almost arbitrarily high surface densities of fluorescent probe molecules. The method yields information on the fraction of colocalized particles and their position within 40nm accuracy. Supplemental data on the mobility and stoichiometry of the labeled molecule species can also be deduced. The sensitivity of our two-color single molecule imaging technique is significantly increased by tracking colocalized spots over consecutive images. We present a detailed statistical description of false positives and false negatives and quantify the sensitivity of our method.Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. We demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified by tracking colocalized spots.We also present preliminary results on the application to live cells investigating the interaction between Bodipy-GM1 and Ab-Alexa647 labeled CD59.[1] Moertelmair et al., Appl. Phys Lett. 87, 263903, 2005.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, also termed lipid or membrane rafts. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the demonstration that single rafts diffuse as stable platforms in the live cell plasma membrane. Our method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. The special photobleaching protocol TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling1) was used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, we used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidylinositol-anchored monomeric GFP. For both markers we found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of small, mobile, stable rafts hosting these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.1. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schutz, G. J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87, 263903 (2005).