Publications

Cancer cells utilize filopodia to explore, adhere to and invade their surrounding microenvironment, yet the protein networks that organize these protrusions remain incompletely defined. To uncover the molecular machinery underlying MYO10-positive filopodia, we targeted the fast biotin ligase TurboID to the motor protein MYO10. Proximity biotinylation in two cell types revealed hundreds of potential MYO10 interactors. A targeted microscopy and siRNA screen identified MINK1, SCRIB, CSNK1A1 and CCT8 as new regulators of MYO10-mediated filopodia formation. Focusing on one of the interactors shared between cell lines, CCT8, known as a subunit of the chaperonin TCP1 ring complex (TRiC), we found that CCT8 associates with the MYO10 motor domain and regulates MYO10 filopodia independently of the TRiC. Depleting CCT8 affected filopodia dynamics and impaired cell spreading, migration and invasion in breast cancer cells. These findings establish CCT8 as a TRiC-independent regulator of MYO10 filopodia across diverse cancer cell types.

Preprints have transformed how biologists share results, yet how well a preprint reads still depends on formatting, a task that has shifted from publishers to authors. Cell biology has also become increasingly quantitative, with conclusions resting on multi-step image analysis whose figures, values, and statistics are revised repeatedly across a project. Keeping a well-formatted manuscript in step with the latest analysis is tedious and error-prone. We introduce Rxiv-Maker, an open-source framework that lets authors write in simple Markdown and produce a formatted article, as a PDF or an editable Word document, with consistent styling, automatic figure and citation numbering, and reliable cross-referencing. For quantitative work, it can optionally regenerate figures and recompute reported values directly from the data, so the text stays consistent with the analysis. Built on familiar tools such as Git and Visual Studio Code, it gives a transparent, collaborative path from analysis to a submission-ready preprint. Researchers have already used Rxiv-Maker for cell-biology studies spanning bacterial cell-cycle analysis, filopodia proteomics, and zebrafish live imaging, showing it supports reproducible reporting of genuine biological findings.

Extravasation, the exit of circulating cancer cells from blood vessels, is a critical yet poorly understood step in metastatic dissemination. Here we show that pancreatic ductal adenocarcinoma (PDAC) cells can breach endothelial barriers through two mechanistically distinct modes of extravasation. MIA PaCa-2 cells breach endothelial junctions via filopodia-like protrusions, enabling access to and spread across the basal extracellular matrix (ECM). By contrast, AsPC-1 cells remain rounded atop the endothelium and cross the barrier by triggering rapid retraction of neighbouring endothelial cells. These distinct extravasation modes were also observed in zebrafish larvae. In the mouse lung, AsPC-1 cells arrest, survive, induce endothelial detachment from the basal lamina, and extravasate through this retraction mechanism before metastatic outgrowth. Mechanistically, AsPC-1-secreted factors are sufficient to destabilise endothelial monolayers, and AsPC-1 cells also induce endothelial apoptosis; however, blocking apoptosis does not prevent barrier disruption. By contrast, treatment with saracatinib, a Src-family kinase inhibitor, protects endothelial barriers, limits early vascular disruption in the lung, and delays metastatic outgrowth. Together, these findings reveal that PDAC cells can extravasate via mechanistically distinct routes, suggesting that effective anti-metastatic strategies may need to target multiple modes of endothelial barrier breach rather than a single pathway.

NucleiSky is a microscopy image registration framework that uses the spatial arrangement of nuclei or other landmarks as constellations to support cross-scale, multimodal registration.

Microscopy is evolving from a descriptive to a predictive tool in biology. Trained on time-lapse images, deep learning can forecast whether a cell will divide, die, or differentiate from how it looks and moves, in some systems, hours or even generations before the usual molecular markers appear, and without added fluorescent labels. Two questions frame this review and stay largely open: which image features actually carry the predictive signal, and whether a model foresees a genuinely future outcome or instead reads a state the cell has already entered. We organise the field by the visual signal that carries fate: changes in cell and nuclear shape over time, changes in brightness and texture, the dynamics of differentiation and competition, and patterns of movement. For each, we ask what must be measured, and over what area and time window, to predict fate. We then set out the main families of models, from networks that read a single image to those that read sequences of images over time and those that compress images into compact numerical summaries, outlining their strengths and limitations. Our central argument is that prediction has outrun validation: most reported performance is checked retrospectively against endpoint markers instead of on genuinely future cells. We close on the shared datasets, benchmarks, and interpretability work this gap demands, and on underexplored fates such as migration and senescence.

Virtual super-resolution deep learning methods provide a powerful solution to overcome the physical and temporal constraints of microscopy imaging. Yet, assessing and choosing an ideal methodological strategy complicates their use in life sciences and creates a lack of trust in these methods. Here we propose an objective comparison of nine popular single-image super-resolution (SISR) models in a collection of publicly available microscopy datasets, including cell components like microtubules, endoplasmic reticulum, and actin, using confocal microscopy, SEM, SIM, SMLM and STED microscopy modalities for fixed and live-cell microscopy data. The proposed models will be assessed quantitatively with a collection of metrics in microscopy and computer vision, and qualitatively by experts in the field. The proposed models will be made accessible through open, user-friendly, containerised notebooks. This systematic assessment of SISR approaches will provide a more comprehensive understanding of these methods' performance and contribute to standardising SISR methods in microscopy.

SHANK3 is a multidomain scaffolding protein critical for neuronal function, which has been linked to neurodevelopmental disorders such as autism spectrum disorder. More recently, SHANK3 has been shown to play a role in cell survival and actin dynamics outside the nervous system. Here, we show that SHANK3 is widely expressed in endothelial cells across different tissues, where its role is not well understood. SHANK3 localised to endothelial cell-cell junctions in cultured endothelial cells, and its depletion compromised endothelial barrier function. SHANK silencing altered cell mechanics including elongated cell morphology, reduced cell-matrix traction forces and alteration of cell migration rate. It further triggered dynamic heterogeneity in endothelial monolayers, with regions of coordinated long-range migration interspersed with areas exhibiting only local velocity fluctuations, consistent with a transition toward more fluid-like tissue behaviour. This change in collective dynamics was accompanied by increased spheroid spreading and fusion, suggestive of altered tissue viscosity, and coincided with disrupted cell-cell junction morphology and mechanical forces in SHANK3-depleted cells. In vivo, SHANK3 depletion impaired endothelial cell migration, resulting in delayed sprouting of intersegmental vessels and disruption of the vascular network in zebrafish embryos. Furthermore, inducible endothelial-specific deletion of SHANK3 in postnatal mice impaired angiogenic sprouting and reduced vascular complexity in the developing retina. Overall, we demonstrate that SHANK3 plays a role in endothelial cell motility and tissue mechanics, with implications for vascular processes during development.

Accurate and reproducible orientation of zebrafish larvae is essential for high-resolution live imaging but remains difficult with standard agarose mounting. Although previous methods have used 3D-printed tools, often fabricated via stereolithography (SLA), here we present an orientation tool made via fused deposition modeling (FDM) and enhanced with a thin resin coating to improve surface smoothness and performance. This design creates consistent, larval-shaped wells that orient larvae dorsoventrally. The molds, designed to fit standard glass-bottom imaging dishes, are inexpensive to produce on common desktop 3D printers and can be easily customized to fit different imaging setups using the open-source design files. This approach enables reliable, long-term live imaging of multiple larvae and extends the use of low-cost additive manufacturing in zebrafish imaging workflows.

Life sciences research depends heavily on open-source academic software, yet many tools remain underused due to installation and distribution barriers. LabConstrictor introduces a GitHub-based packaging workflow that turns Jupyter notebooks into installable desktop applications with version checks and an app-like interface, improving reproducibility and software reuse.

Cell–cell communication in tissues is influenced by contact geometry and molecular signalling. Here, we demonstrate that epithelial intercellular filopodia can penetrate neighbouring cells to form micrometre-long, double-membrane "impact sites" and, in some instances, initiate trans-endocytosis. Using super-resolution live imaging and three-dimensional electron microscopy in cell models, xenografts, and patient-derived tissues, we observe widespread intercellular filopodia and extensive interdigitation at the cell-cell interface. Filopodia impact sites in the recipient cell recruit PACSIN2 into dynamic "PACSIN2 fingers" and are specifically enriched for caveolin-1 and dynamin-2, while clathrin and CLIC pathway markers are not enriched. Most contacts are transient and resolved by retraction, but some undergo scission, with recipient cells internalising filopodial tips across both homotypic and heterotypic interactions, including cancer–endothelium contacts. Correlative light and FIB–SEM analysis reveals that internalised tips are often double-membraned, closely associated with endoplasmic reticulum tubules, and can traffic to lysosomes. These findings define a protrusion-driven, curvature-dependent uptake pathway at cell–cell interfaces and identify filopodia as exchange organelles in epithelial collectives.

Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning (ML) are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap encompasses key aspects of how ML is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of ML for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.

The rapid, transient, and unpredictable nature of interactions between circulating cells and the endothelium challenges the investigation of these events under flow conditions. Here, we developed an imaging and image-analysis framework called FlowVision, which integrates fast, bright-field live-cell imaging with deep-learning-based image analysis to quantitatively track cell landing and arrest on an endothelial monolayer under physiological flow conditions. Using FlowVision, we find that pancreatic ductal adenocarcinoma (PDAC) cells exhibit variable adhesion strength and flow sensitivity. Remarkably, some PDAC cells demonstrate comparable endothelial engagement to leukocytes, preferentially arresting at endothelial junctions, providing them access to the underlying basal extracellular matrix. PDAC cells attach and form clusters in areas with high expression of the endothelial CD44 receptor. Targeting CD44 using siRNA, function-blocking antibodies, or degrading its ligand, hyaluronic acid (HA), strongly reduces PDAC cell attachment. Overall, our label-free live-imaging approach demonstrates that cancer and immune cells share both common and unique features in endothelial adhesion under flow, and allows identification of CD44 and HA as key mediators of PDAC cell arrest.

Researchers often face fragmented parameter input workflows across notebooks and terminal tools, which harms reproducibility. EZInput provides a declarative, write-once run-anywhere interface layer that auto-generates validated user interfaces for Jupyter, Colab, and terminal environments while persisting configurations via YAML.

Constitutive integrin endocytosis and recycling control cell movement and morphology. In contrast, the role of newly synthesized integrins delivered via the biosynthetic pathway has been largely overlooked. We used the retention using selective hooks system to monitor the localization of new integrins exiting the endoplasmic reticulum in space and time. We discovered that new integrin delivery to the plasma membrane is polarized and enhances cell protrusion and focal adhesion growth in an extracellular matrix-ligand–dependent manner. Motor-clutch modeling explained the increased adhesion as higher integrin availability driving recruitment of additional receptors. Unexpectedly, live-cell imaging revealed a small subset of fast-emerging integrin vesicles rapidly transported to the cell surface to facilitate localized spreading. This unconventional secretion depended on cell adhesion and correlated with increased surface levels of immature, high-mannose glycosylated integrin, indicating bypass of the canonical Golgi-dependent secretory pathway. Thus, spatial plasma membrane-targeting of new integrins rapidly alters adhesion receptor availability, providing cells with added plasticity to respond to their environment.

Phototoxicity in live-cell fluorescence microscopy can compromise experimental outcomes, yet quantitative methods to assess its impact remain limited. Here, we present PhotoFiTT (Phototoxicity Fitness Time Trial), an integrated framework combining a standardised experimental protocol with advanced image analysis to quantify light-induced cellular stress in label-free settings. PhotoFiTT leverages machine learning and cell cycle dynamics to analyse mitotic timing, cell size changes, and overall cellular activity in response to controlled light exposure. Using adherent mammalian cells, we demonstrate PhotoFiTT’s ability to detect wavelength- and dose-dependent effects, showcasing that near-UV light induces significant mitotic delays at doses as low as 0.6 J/cm 2, while longer wavelengths require higher doses for comparable deleterious effects. PhotoFiTT enables researchers to establish quantitative benchmarks for acceptable levels of photodamage, facilitating the optimisation of imaging protocols that balance image quality with sample health.

From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReD’s capabilities on various fluorescence microscopy images. Quantitative analyses of different datasets highlight SReD’s utility: estimating the periodicity of spectrin rings in neurons, detecting Human Immunodeficiency Virus type-1 viral assembly, and evaluating microtubule dynamics modulated by End-binding protein 3. Our open-source plugin for ImageJ or FIJI enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.

This preprint reports that LSD1 serine 166 phosphorylation acts as a molecular switch linking chromatin regulation to transcriptional activation and tissue remodeling programs.

Data exploration is an essential step in quantitative cell biology, bridging raw data and scientific insights. Unlike polished, published figures, effective data exploration requires a flexible, hands-on approach that reveals trends, identifies outliers and refines hypotheses. This Opinion offers simple, practical advice for building a structured data exploration workflow, drawing on the authors' personal experience in analyzing bioimage datasets. In addition, the increasing availability of generative artificial intelligence and large language models makes coding and improving data workflows easier than ever before. By embracing these practices, researchers can streamline their workflows, produce more reliable conclusions and foster a collaborative, transparent approach to data analysis in cell biology.

Cancer's notorious heterogeneity poses significant challenges, as each tumor comprises a unique ecosystem. While single‐cell and spatial transcriptomics advancements have transformed our understanding of spatial diversity within tumors, the temporal dimension remains underexplored. Tumors are dynamic entities that continuously evolve and adapt, and relying solely on static snapshots obscures the intricate interplay between cancer cells and their microenvironment. Here, we advocate for integrating temporal dynamics into cancer research, emphasizing a fundamental shift from traditional endpoint experiments to data‐driven, continuous approaches. This integration involves, for instance, the development of advanced live imaging techniques, innovative temporal omics methodologies, and novel computational tools.

Developing user-friendly image analysis software is essential for advancing biological and life science research. However, the interdisciplinary gap between software developers and life scientists presents challenges to software adoption. In this Essay, we provide practical recommendations to guide bioimage analysts and developers in creating accessible and usable software for biological research. These recommendations are presented in three phases, covering software design, user involvement in early development stages and the importance of software dissemination. Additionally, two software development case studies are presented to highlight the practical application of these principles, showing how thoughtful development, user-centric design and thorough documentation can bridge the gap between software developers and biologists, fostering wider adoption of the software and enabling further scientific discovery.

Talin-1 (TLN1) is best known to activate integrin receptors and transmit mechanical stimuli to the actin cytoskeleton at focal adhesions. However, the localization of TLN1 is not restricted to focal adhesions. By utilizing both subcellular fractionations and confocal microscopy analyses, we show that TLN1 localizes to the nucleus in several human cell lines, where it is tightly associated with the chromatin. Importantly, small interfering RNA (siRNA)-mediated depletion of endogenous TLN1 triggers extensive changes in the gene expression profile of human breast epithelial cells. To determine the functional impact of nuclear TLN1, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our findings revealed that the accumulation of nuclear TLN1 alters the expression of a subset of genes and impairs the formation of cell-cell clusters. This study introduces an additional perspective on the canonical view of TLN1 subcellular localization and function.

The expanding scale and complexity of microscopy image datasets require accelerated analytical workflows. NanoPyx meets this need through an adaptive framework enhanced for high-speed analysis. At the core of NanoPyx, the Liquid Engine dynamically generates optimized central processing unit and graphics processing unit code variations, learning and predicting the fastest based on input data and hardware. This data-driven optimization achieves considerably faster processing, becoming broadly relevant to reactive microscopy and computing fields requiring efficiency.

From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReD's capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReD's utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.

In life sciences, tracking objects from movies enables researchers to quantify the behavior of single particles, organelles, bacteria, cells, and even whole animals. While numerous tools now allow automated tracking from video, a significant challenge persists in compiling, analyzing, and exploring the large datasets generated by these approaches. Here, we introduce CellTracksColab, a platform tailored to simplify the exploration and analysis of cell tracking data. CellTracksColab facilitates the compiling and analysis of results across multiple fields of view, conditions, and repeats, ensuring a holistic dataset overview. CellTracksColab also harnesses the power of high-dimensional data reduction and clustering, enabling researchers to identify distinct behavioral patterns and trends without bias. Finally, CellTracksColab also includes specialized analysis modules enabling spatial analyses (clustering, proximity to specific regions of interest). We demonstrate CellTracksColab capabilities with 3 use cases, including T cells and cancer cell migration, as well as filopodia dynamics. CellTracksColab is available for the broader scientific community at https://github.com/CellMigrationLab/CellTracksColab.

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.

Deep learning enables the transformative analysis of large multidimensional microscopy datasets, but barriers remain in implementing these advanced techniques. Many researchers lack access to annotated data, high performance computing (HPC) resources and expertise to develop, train and deploy deep learning models. In recent years, several approaches have been developed to democratize deep learning usage in microscopy. Tools such as the BioImage Model Zoo facilitate sharing and reuse of pretrained models, distributing them as one-click image processing solutions. Yet often, deep learning models need to be trained or fine-tuned on the end-user dataset to perform well. We previously released ZeroCostDL4Mic, an online platform relying on Google Colab that helped democratize deep learning by providing a zero-code interface to train and evaluate models capable of performing various bioimage processing tasks, such as segmentation, object detection, denoising, super-resolution microscopy and image-to-image translation. Here, we introduce DL4MicEverywhere, an advancement of the ZeroCostDL4Mic framework.

Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results – particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed – AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.

Live imaging is a powerful tool, enabling scientists to observe living organisms in real time. In particular, when combined with fluorescence microscopy, live imaging allows the monitoring of cellular components with high sensitivity and specificity. Yet, due to critical challenges (i.e., drift, phototoxicity, dataset size), implementing live imaging and analyzing the resulting datasets is rarely straightforward. Over the past years, the development of bioimage analysis tools, including deep learning, is changing how we perform live imaging. Here we briefly cover important computational methods aiding live imaging and carrying out key tasks such as drift correction, denoising, super-resolution imaging, artificial labeling, tracking, and time series analysis. We also cover recent advances in self-driving microscopy.

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

The immune synapse (IS) is a cell-cell interaction platform critical in lymphocyte activation by specific antigens. Despite of B cells being able to also respond to soluble antigens, in particular the in vivo importance of the IS and surface-tethered antigen recognition has strongly emerged in the recent years. The IS serves as a dynamic hub for multiple cellular actions but the molecular details of these functions, especially beyond the B cell antigen receptor (BCR) signalling, remain poorly understood. Here, to address the lack in the systems level understanding of the IS, we setup methodology for comprehensive investigation of the composition of the primary mouse B cells’ IS at proteome level. Utilizing functionalized magnetic beads to mimic antigen presenting cells and trigger IS formation on them, we developed a method to specifically and robustly extract the cell adhesions on the beads, namely the IS or transferrin receptor mediated adhesion as a control. Our data revealed 661 proteins exclusively present in the IS at 15 minutes after BCR engagement, 13 exclusively in the control adhesions and 365 proteins shared between the samples. We got strong coverage of the known components of the IS as well as identified a plethora of unknown proteins and functional pathways with hitherto unknown roles in B cell IS. Thus, in this work, we validated the IS isolation method as a valuable tool to study early B cell activation by surface-bound antigens as well as unveil several novel proteins and pathways suggestive of new functional aspects in the IS.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

Filopodia are fingerlike membrane protrusions extended by cells to sense their environment. Filopodia are widely used by migrating cells in vivo and directly contribute to several physiological processes and diseases. Due to the essential roles of filopodia in sensing the extracellular environment, there is a need to characterize their composition and ultrastructure further. This chapter highlights FiloMap, an image analysis pipeline that utilizes Fiji and R to map the localization of proteins within filopodia from microscopy images. I provide step-by-step protocols on (a) setting up FiloMap in Fiji and R, (b) extracting line intensity profiles from filopodia stainings in Fiji, (c) further analyzing line intensity profiles in R, and (d) creating filopodia maps to compare the localization of multiple proteins within filopodia. Notably, while FiloMap was written to analyze filopodia, the analysis pipeline described here can also analyze and compile any line intensity profiles.

Fibroblastic migration is of key importance in wound healing. While the intermediate filament (IF) protein vimentin is required for normal wound healing, we examined whether vimentin-mediated regulation of fibroblast migration could be involved. In wound healing assays triggering cell polarization and directed migration, we observed that vimentin-deficient mouse and rat embryonic fibroblasts lost their directional persistence. We show that vimentin maintains directionality by guiding focal adhesions (FAs) in fibroblasts. Detailed analysis showed that vimentin stabilizes FAs and regulates their disassembly rate. The destabilization of Vim-/- FAs was reflected by smaller FAs. Live cell and super-resolution imaging demonstrate that vimentin interacts dynamically with the key molecules of FAs and, importantly, with FAK, which is crucial for the maturation of FAs. These results demonstrate that vimentin IFs control the maturation, stability, dynamics, arrangement, and overall orientation of FAs, with a net effect on FA coordination during migration.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

This work demonstrates and guides how to use a range of state-of-the-art artificial neural-networks to analyse bacterial microscopy images using the recently developed ZeroCostDL4Mic platform. We generated a database of image datasets used to train networks for various image analysis tasks and present strategies for data acquisition and curation, as well as model training. We showcase different deep learning (DL) approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the ability of DL to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. Our purposefully-built database of training and testing data aids in novice users’ training, enabling them to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of tools for bacterial cell biology and antibiotic research.

Deep learning-based approaches are revolutionizing imaging-driven scientific research. However, the accessibility and reproducibility of deep learning-based workflows for imaging scientists remain far from sufficient. Several tools have recently risen to the challenge of democratizing deep learning by providing user-friendly interfaces to analyze new data with pre-trained or fine-tuned models.

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.

Fluorescence microscopy enables the direct observation of previously hidden dynamic processes of life, allowing profound insights into mechanisms of health and disease. However, imaging of live samples is fundamentally limited by the toxicity of the illuminating light and images are often acquired using low light conditions. As a consequence, images can become very noisy which severely complicates their interpretation. In recent years, deep learning (DL) has emerged as a very successful approach to remove this noise while retaining the useful signal. Unlike classical algorithms which use well-defined mathematical functions to remove noise, DL methods learn to denoise from example data, providing a powerful content-aware approach. In this review, we first describe the different types of noise that typically corrupt fluorescence microscopy images and introduce the denoising task. We then present the main DL-based denoising methods and their relative advantages and disadvantages. We aim to provide insights into how DL-based denoising methods operate and help users choose the most appropriate tools for their applications.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

Deep learning algorithms are powerful tools for analyzing, restoring and transforming bioimaging data. One promise of deep learning is parameter-free one-click image analysis with expert-level performance in a fraction of the time previously required. However, as with most emerging technologies, the potential for inappropriate use is raising concerns among the research community. In this Comment, we discuss key concepts that we believe are important for researchers to consider when using deep learning for their microscopy studies. We describe how results obtained using deep learning can be validated and propose what should, in our view, be considered when choosing a suitable tool. We also suggest what aspects of a deep learning analysis should be reported in publications to ensure reproducibility. We hope this perspective will foster further discussion among developers, image analysis specialists, users and journal editors to define adequate guidelines and ensure the appropriate use of this transformative technology.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its β1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

Artificial intelligence (AI)-powered algorithms are now influencing many aspects of our day-to-day life, from providing movies/music recommendations to controlling self-driving cars. These algorithms are also increasingly used in the lab to aid biomedical research. In particular, the ability to analyse and process images using AI is slowly revolutionizing the quality and quantity of data we collect from microscopy images. In fact, AI-based algorithms can now be applied to perform virtually any high-performance image analysis tasks such as classifying images, detecting and segmenting objects, aligning images or improving image quality by removing noise or increasing image resolution. This short feature article briefly underlies the principles behind using AI algorithms to analyse microscopy images with a specific focus on segmentation and denoising.

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.

In this study, direct and effective intracellular delivery of CRISPR/Cas9 plasmids for homology‐directed repair is achieved by functionalized mesoporous silica nanoparticles (MSNs). The functionalized MSNs (Cy5.5‐MSNs‐NLS) are synthesized by in situ labeling of a fluorescent dye (Cy5.5) and surface conjugation of nuclear localization sequence (NLS, PKKKRKV), showing a high loading efficiency (50%) toward the plasmids (PXN cutdown plasmid: GFP‐Cas9‐paxillin_gRNA and repair plasmid: AICSDP‐1: PXN‐EGFP). Subsequently, a polymeric coating of the poly(dimethyldiallylammonium chloride) (PDDA) is electrostatically deposited onto the plasmid‐loaded Cy5.5‐MSNs‐NLS by microfluidic nanoprecipitation. The coating layer offers effective protection against the denaturation of plasmids by EcoRV restriction enzymes, and is shown to prevent premature release. Moreover, owing to the positive charge and pH‐responsive disaggregation of PDDA, enhanced cellular internalization (16 h) and endosomal escape (4 h) of the nanocarrier are observed. After escape of nanocarrier system into the cytoplasm, the NLS on the surface of MSNs facilitates nuclear transport of the CRISPR/Cas9 plasmids, achieving successful GFP‐tag knock‐in of the PXN genomic sequence in U2OS cells. This intracellular delivery system thus offers an attractive method to overcome physiological barriers for CRISPR/Cas9 delivery, showing considerable promise for paxillin‐associated focal adhesion and signaling regulator investigation.

A rapid and convenient method was developed to load gene molecules into nanostructures, exploring structure–function relationship for efficient intracellular delivery and gene editing, opening up new avenues for the MOF-based non-viral vectors.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

Fluorescence microscopy has become a ubiquitous method to observe the location of specific molecular components within cells. However, the resolution of light microscopy is limited by the laws of diffraction to a few hundred nanometers, blurring most cellular details. Over the last two decades, several techniques – grouped under the ‘super-resolution microscopy’ moniker – have been designed to bypass this limitation, revealing the cellular organization down to the nanoscale. The number and variety of these techniques have steadily increased, to the point that it has become difficult for cell biologists and seasoned microscopists alike to identify the specific technique best suited to their needs. Available techniques include image processing strategies that generate super-resolved images, optical imaging schemes that overcome the diffraction limit and sample manipulations that expand the size of the biological sample. In this Cell Science at a Glance article and the accompanying poster, we provide key pointers to help users navigate through the various super-resolution methods by briefly summarizing the principles behind each technique, highlighting both critical strengths and weaknesses, as well as providing example images.

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance and wound healing. In order for cells to migrate, they must interact with their environment using adhesion receptors, such as integrins, and form specialized adhesion complexes that mediate responses to different extracellular cues. In this review, we discuss the role of integrin adhesion complexes (IACs) in cell migration, highlighting the layers of regulation that are involved, including intracellular signalling cascades, mechanosensing and reciprocal feedback to the extracellular environment. We also discuss the role of IACs in extracellular matrix remodeling and how they impact upon cell migration.

Filopodia are adhesive cellular protrusions specialized in the detection of extracellular matrix (ECM)-derived cues. Although ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins ("adhesome") to fine-tune cellular behavior, the components of the filopodia adhesions remain undefined. Here, we performed a structured-illumination-microscopy-based screen to map the localization of 80 target proteins, linked to cell adhesion and migration, within myosin-X-induced filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains, suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phosphoinositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localization of PI(3,4)P 2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions, and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia.

Filopodia are fingerlike membrane protrusions that are extended by cells in vitro and in vivo. Due to important roles in sensing the extracellular microenvironment, filopodia and filopodia-like protrusions have been implicated in numerous biological processes including epithelial sheet zippering in development and wound healing and in cancer progression. Recently, there has been an explosion in the number of software available to analyze specific features of cell protrusions with the aim of gaining mechanistic insights into the action of filopodia and filopodia-like structures. In this methods chapter, we highlight an open-access software called FiloQuant that has been developed to specifically quantify the length, density, and dynamics of filopodia and filopodia-like structures from in vitro and in vivo generated samples. We provide step-by-step protocols on (i) how to install FiloQuant in the ImageJ platform (Fiji), (ii) how to quantify filopodia and filopodia-like protrusions from single images using FiloQuant, and (iii) how to track filopodial protrusions from live-cell imaging experiments using FiloQuant and TrackMate.

Significance Compromised vascular integrity is associated with capillary leakage in sepsis, but effective therapies stabilizing the vasculature are lacking. Here, we show that targeting β1-integrin in vivo with inhibitory antibodies or deletion of a single allele of endothelial β1-integrin inhibits lipopolysaccharide (LPS)-induced vascular leakage in murine endotoxemia. The inflammatory agents IL-1β, thrombin, and LPS induced changes in endothelial cell–extracellular matrix (ECM) adhesion via β1-integrin, angiopoietin-2, and the adapter protein tensin-1, leading to increased endothelial cell contractility and permeability. These results indicate that β1-integrin actively promotes vascular leakage and that targeting β1-integrin signaling could be a novel means of achieving vascular stabilization in pathological vascular leak.

Intravascular ATP and adenosine have emerged as important regulators of endothelial barrier function, vascular remodeling and neovascularization at various pathological states, including hypoxia, inflammation and oxidative stress. By using human umbilical vein endothelial cells (HUVEC) and bovine vasa vasorum endothelial cells (VVEC) as representatives of macro- and microvessel phenotypes, this study was undertaken to evaluate cellular mechanisms contributing to physiological adaptation of vascular endothelium to hypoxia, with a particular emphasis on ectoenzymatic purine-converting activities and their link to intracellular ATP homeostasis and signaling pathways. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), ecto-5'-nucleotidase/CD73 and ecto-adenylate kinase activities were determined by thin-layer chromatography (TLC) with 3 H-labelled nucleotide substrates. Exposure of HUVEC and VVEC to 1% O 2 for 4-24 h triggered rather moderate activation of ATP breakdown into adenosine via the CD39-CD73 axis. Additional TLC analysis of salvage pathways revealed the enhanced ability of hypoxic HUVEC to convert cell-incorporated [ 3 H]adenosine into [ 3 H]ADP/ATP. Furthermore, following a period of hypoxia, HUVEC underwent concurrent changes in intracellular signaling manifested in the depletion of putative ATP stores and targeted up-regulation of phospho-p53, p70S6K/mTOR and other tyrosine kinases. The revealed complex implication of both extrinsic and intrinsic mechanisms into a tuned hypoxia-induced control of purine homeostasis and signaling may open up further research for the development of pharmacological treatments to improve endothelial cell function under disease conditions associated with a loss of cellular ATP during oxygen deprivation.

During mitosis, animal cells disassemble focal adhesions and round up while remaining attached to the substrata via actin cables and unknown adhesive structures. In this issue of Developmental Cell, Dix et al. (2018) describe integrin-positive adhesions, devoid of classical focal adhesion components, that persist throughout mitosis to contribute to re-spreading.

2D surfaces offer simple analysis of cells in culture, yet these often yield different cell morphologies and responses from those observed in vivo. Considerable effort has therefore been expended on the generation of more tissue-like environments for the study of cell behavior in vitro. Purified matrix proteins provide a 3D scaffold that better mimics the in vivo situation; however, these are far removed from the complex tissue composition seen in vivo. Cell-derived matrices (CDMs) offer a more physiologically relevant alternative for studying in vivo-like cell behavior in vitro. In the protocol described here, fibroblasts cultured on gelatin-coated surfaces are maintained in the presence of ascorbic acid to strengthen matrix deposition over 1-3 weeks. The resulting fibrillar CDMs are denuded of cells, and other cells are subsequently cultured on them, after which their behavior is monitored. We also demonstrate how to use CDMs as an in vivo-relevant reductionist model for studying tumor-stroma-induced changes in carcinoma cell proliferation and migration.

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first ‘Sharpin interactome’, which identifies a large number of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome, we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We have identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin–Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion. This article has an associated First Person interview with the first author of the paper.

Defective filopodia formation is linked to pathologies such as cancer, wherein actively protruding filopodia, at the invasive front, accompany cancer cell dissemination. Despite wide biological significance, delineating filopodia function in complex systems remains challenging and is particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia-like protrusions in both fixed- and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information, including protrusion dynamics, density, and length, from multiple cell types and in a range of microenvironments. In cellular models of breast ductal carcinoma in situ, we reveal a link between filopodia formation at the cell–matrix interface, in collectively invading cells and 3D tumor spheroids, and the in vitro invasive capacity of the carcinoma. Finally, using intravital microscopy, we observe that tumor spheroids display filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin–ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin– and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin cpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAKY397 phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals.

Integrins are a family of heterodimeric receptors that bind to components of the extracellular matrix and influence cellular processes as varied as proliferation and migration. These effects are achieved by tight spatiotemporal control over intracellular signalling pathways, including those that mediate cytoskeletal reorganisation. The ability of integrins to bind to ligands is governed by integrin conformation, or activity, and this is widely acknowledged to be an important route to the regulation of integrin function. Over the last 15 years, however, the pathways that regulate endocytosis and recycling of integrins have emerged as major players in controlling integrin action, and studying integrin trafficking has revealed fresh insight into the function of this fascinating class of extracellular matrix receptors, in particular in the context of cell migration and invasion. Here, we review our current understanding of the contribution of integrin trafficking to cell motility.

Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis.

This review discusses recent advances in our understanding of the role filopodia and filopodia-like structures in cell adhesion and three dimensional (3D) cell migration both in vitro and in vivo. In particular, we focus on recent advances demonstrating that filopodia are involved in substrate tethering and environment sensing in vivo. We further discuss the emerging role of filopodia and filopodial proteins in tumor dissemination as mounting in vitro, in vivo and clinical evidence suggest that filopodia drive cancer cell invasion and highlight filopodia proteins as attractive therapeutic targets. Finally, we outline outstanding questions that remain to be addressed to elucidate the role of filopodia during 3D cell migration.

Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.

Cell–extracellular matrix (ECM) adhesion is a fundamental requirement for multicellular existence due to roles in positioning, proliferation and differentiation. Phosphorylation plays a major role in adhesion signalling; however, a full understanding of the phosphorylation events that occur at sites of adhesion is lacking. Here we report a proteomic and phosphoproteomic analysis of adhesion complexes isolated from cells spread on fibronectin. We identify 1,174 proteins, 499 of which are phosphorylated (1,109 phosphorylation sites), including both well-characterized and novel adhesion-regulated phosphorylation events. Immunoblotting suggests that two classes of phosphorylated residues are found at adhesion sites—those induced by adhesion and those constitutively phosphorylated but recruited in response to adhesion. Kinase prediction analysis identifies novel kinases with putative roles in adhesion signalling including CDK1, inhibition of which reduces adhesion complex formation. This phospho-adhesome data set constitutes a valuable resource to improve our understanding of the signalling mechanisms through which cell–ECM interactions control cell behaviour.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.

Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling.

Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.

This review discusses recent advances in our understanding of adhesion receptor trafficking in vitro, and extrapolates them as far as what is currently possible towards an understanding of migration in three dimensions in vivo. Our specific focus is the mechanisms for endocytosis and recycling of the two major classes of cell-matrix adhesion receptors, integrins and syndecans. We review the signalling networks that are employed to regulate trafficking and conversely the effects of trafficking on signalling itself. We then define the contribution that this element of the migration process makes to processes such as wound healing and tumour invasion.

Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. While the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analyses of activated integrin-associated complexes suggested filamin-A and IQ motif-containing GTPase activating protein 1 (IQGAP1) as candidates that might link β1 integrin to Rac1. siRNA-mediated knock down of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active β1 integrin. Mass spectrometric analyses of individual filamin-A, IQGAP1 and Rac1 pull-downs, following by biochemical analyses, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated RacGAP1 recruitment to IQGAP1 and to active β1 integrin, and suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings lead to a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby co-ordinate directional cell migration.