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RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localizes in growth cones of undifferentiated neurites, while in developing axons it displays a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevents axon initiation but has no effect on elongation, while formin inhibition reduces axon extension without significantly altering initial outgrowth. Besides, RhoA-mDia promotes axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK that restrains growth cone microtubule assembly and protrusion. Finally, we show that similar mechanisms might operate during axonal regeneration, with RhoA-ROCK slowing axon regrowth after axotomy and RhoA-mDia favoring extension of regenerated axons.

The incidence of melanoma, the most fatal dermatological cancer, has dramatically increased over the last few decades. Modern targeted therapy with kinase inhibitors induces potent clinical responses, but drug resistance quickly develops. Combination therapy improves treatment outcomes. Therefore, novel inhibitors targeting aberrant proliferative signaling in melanoma via the MAPK/ERK and PI3K/AKT pathways are urgently needed. Biosensors were combined that report on ERK/AKT activity with image-based high-content screening and HPLC-based activity profiling. An in-house library of 2576 plant extracts was screened on two melanoma cell lines with different oncogenic mutations leading to pathological ERK/AKT activity. Out of 140 plant extract hits, 44 were selected for HPLC activity profiling. Active thymol derivatives and piperamides from Arnica montana and Piper nigrum were identified that inhibited pathological ERK and/or AKT activity. The pipeline used enabled an efficient identification of natural products targeting oncogenic signaling in melanoma.

Cell mechanical interactions play a fundamental role in the self-organisation of organisms. How these interactions drive coordinated cell movement in three-dimensions remains unclear. Here we report that cell doublets embedded in a 3D extracellular matrix undergo spontaneous rotations and we investigate the rotation mechanism using live cell imaging, quantitative measurements, mechanical perturbations, and theory. We find that rotation is driven by a polarized distribution of myosin within cell cortices. The mismatched orientation of this polarized distribution breaks the doublet mirror symmetry. In addition, cells adhere at their interface through adherens junctions and with the extracellular matrix through focal contacts near myosin clusters. Using a physical theory describing the doublet as two interacting active surfaces, we find that rotation is driven by myosin-generated gradients of active tension, whose profiles are dictated by interacting cell polarity axes. We show that interface three-dimensional shapes can be understood from the Curie principle: shapes symmetries are related to broken symmetries of myosin distribution in cortices. To test for the rotation mechanism, we suppress myosin clusters using laser ablation and we generate new myosin clusters by optogenetics. Our work clarifies how polarity-oriented active mechanical forces drive collective cell motion in three dimensions.

Live-cell imaging allows the study of apoptosis at cellular level, highlighting morphological hallmarks such as nuclear shrinkage, membrane blebbing, and cell disruption. Identifying the exact location and timing of this process is essential to foster the understanding of its spatial-temporal regulation. However, the analysis of live-cell imaging datasets is complex, whereas computational tools tailored to this task are yet scarce. Therefore, we developed ADeS, an Apoptosis Detection System based on deep learning and activity recognition. ADeS uses morpho-dynamic hallmarks to detect the exact location and timing of apoptotic events in different cell types, reaching an accuracy above 97% in the classification of our validation datasets acquiredin vitroandin vivo. Moreover, ADeS is the first successful implementation of a deep learning network for the automatic detection of apoptotic cells in full microscopy movies in an end-to-end fashion, outperforming human in the same task. As a case study, we employed ADeS for the analysis of cell survivalin vitro, and for tissue damage assessmentin vivo, showing its potential application for toxicity assays, treatments evaluation and measuring of tissue dynamics.

A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of

The PI3K/AKT and MAPK/ERK pathways are frequently mutated in metastatic melanoma. In a screen of over 2500 plant extracts, the dichloromethane extract of Ericameria nauseosa significantly inhibited oncogenic activity of AKT in MM121224 human melanoma cells. This extract was analyzed by analytical HPLC, and the column effluent was fractionated and tested for activity to generate the so-called HPLC-based activity profile. Compounds eluting within active time-windows of the chromatogram were subsequently isolated in a larger scale to afford 11 flavones (1-11), four flavanones (12-15), two diterpenes (16, 17), and a seco-caryophyllene (18). All isolated compounds were tested for activity, whereby only flavonoids were found active. Of these, flavones were shown to be more active than the flavanones. The most potent flavone was compound 9, that was displaying an IC

A library of more than 2’500 plant extracts were screened for their activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and a total of 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3-5), 12 known sesquiterpenoids (6-17), as well as four known flavonoids (19-22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3-18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 of 8.9 ± 0.9 μM

An increasing experimental evidence points to physiological importance of space-time correlations in signalling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of bio-molecules between cells allows the collectives to perform more complex tasks and better tackle environmental challenges. To understand this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signalling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We make a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells induce ERK activity waves with different size, duration, and frequency. The open-source implementations of ARCOS are available as R and Python packages, and as a plugin for napari image viewer to interactively quantify collective phenomena without prior programming experience.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.

Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these experiments and the heterogenous nature of signaling responses due to cell-cell variability hinder the exploration of such datasets. The population averages insufficiently describe the dynamics, yet finding prototypic dynamic patterns that relate to different cell fates is difficult when mining thousands of time-series. Here we demonstrate a protocol where we identify such dynamic phenotypes in a population of PC-12 cells that respond to a range of sustained growth factor perturbations. We use Time-Course Inspector, a free R/Shiny web application to explore and cluster single-cell time-series.

The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation, commonly observed in breast cancer, display increased ERK pulse frequency, inner cell survival and loss of lumen formation. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.

Directed flows of cellsin vivoare essential in morphogenesis. They shape living matter in phenomena involving cell mechanics and regulations of the acto-myosin cytoskeleton. However the onset of coherent motion is still poorly understood. Here we show that coherence is associated with spontaneous alignments of cell polarity by designing cellular rings of controlled dimensions. A tug-of-war between polarities dictates the onset of coherence, as assessed by tracking live cellular shapes and motions in various experimental conditions. In addition, we identify an internally driven constraint set by cellular acto-myosin cables at boundaries as essential to ensure coherence, and active force is generated as evaluated by the high RhoA activity. The cables are required to trigger coherence as shown by our numerical simulations based on a novel Vicsek-type model including free active boundaries. We quantitatively reproducein silicocoherence onsets and we predict criteria leading to coherence. Altogether, spontaneous coherent motion results from basic competitions between cell orientations and active cables at boundaries.

Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedbacks operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs and its perturbation can enhance the efficiency of MAPK inhibitors.

Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF.

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to ‘posterior-ize’ 88% of the embryo, profoundly altering gene expression and morphogenetic movements in all cells within this region. Posterior fate adoption requires at least 1 h of signaling, whereas a 30 min Erk pulse specifies a distinct cell type, intermediate neuroblasts. In contrast to the persistence detector model of Erk interpretation, cell fates are controlled by the cumulative load of signaling, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.

Stimulation of PC‐12 cells with epidermal (EGF) versus nerve (NGF) growth factors (GFs) biases the distribution between transient and sustained single‐cell ERK activity states, and between proliferation and differentiation fates within a cell population. We report that fibroblast GF (FGF2) evokes a distinct behavior that consists of a gradually changing population distribution of transient/sustained ERK signaling states in response to increasing inputs in a dose response. Temporally controlled GF perturbations of MAPK signaling dynamics applied using microfluidics reveal that this wider mix of ERK states emerges through the combination of an intracellular feedback, and competition of FGF2 binding to FGF receptors (FGFRs) and heparan sulfate proteoglycan (HSPG) co‐receptors. We show that the latter experimental modality is instructive for model selection using a Bayesian parameter inference. Our results provide novel insights into how different receptor tyrosine kinase (RTK) systems differentially wire the MAPK network to fine‐tune fate decisions at the cell population level., Analyses of single‐cell MAPK/ERK signaling dynamics in response to temporally controlled EGF, NGF, FGF2 stimulations show that FGF2 evokes distinct signaling dynamics compared to EGF/NGF. A mathematical model that accounts for these responses is presented.

Cell death events continuously challenge epithelial barrier function, yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of Extracellular Signal-Regulated Kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires Epidermal Growth Factor Receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

NeuriteTracker is a computer vision approach used to analyze neuronal morphodynamics and to examine spatiotemporal Rho GTPase signaling networks regulating neurite outgrowth., Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

Summary Thanks to recent advances in live cell imaging of biosensors, microscopy experiments can generate thousands of single-cell time-series. To identify sub-populations with distinct temporal behaviours that correspond to different cell fates, we developed Time Course Inspector (TCI)—a unique tool written in R/Shiny to combine time-series analysis with clustering. With TCI it is convenient to inspect time-series, plot different data views and remove outliers. TCI facilitates interactive exploration of various hierarchical clustering and cluster validation methods. We showcase TCI by analysing a single-cell signalling time-series dataset acquired using a fluorescent biosensor. Availability and implementation https://github.com/pertzlab/shiny-timecourse-inspector. Supplementary information Supplementary data are available at Bioinformatics online.

A RNA interference, also called a gene knockdown, is a biological technique which consists of inhibiting a targeted gene in a cell. By doing so, one can identify statistical dependencies between a gene and a cell phenotype. However, during such a gene inhibition process, additional genes may also be modified. This is called the "off-target effect". The consequence is that there are some additional phenotype perturbations which are "off-target". In this paper, we study new machine learning tools that both model the cell phenotypes and remove the "off-target effect". We propose two new automatic methods to remove the "off-target" components from a data sample. The first method is based on vector quantization (VQ). The second method we propose relies on a classification forest. Both methods rely on analyzing the homogeneity of several repetitions of a gene knockdown. The baseline we consider is a Gaussian mixture model whose parameters are learned under constraints with a standard Expectation---Maximization algorithm. We evaluate these methods on a real data set, a semi-synthetic data set, and a synthetic toy data set. The real data set and the semi-synthetic data set are composed of cell growth dynamic quantities measured in time laps movies. The main result is that we obtain the best recognition performance with the probabilistic version of the VQ-based method.

To better understand Rho GTPase signaling in neurite outgrowth, Bagonis et al. overcome persisting algorithmic limitations that have, thus far, stymied comprehensive studies of morphological variation among growth cones. They introduce Growth Cone Analyzer—a versatile computer vision tool for the quantification of filopodia-rich cell morphologies., Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.

Kinase Translocation Reporters (KTRs) are genetically encoded fluorescent activity sensors that convert kinase activity into a nucleocytoplasmic shuttling equilibrium for visualizing single cell signaling dynamics. Here, we adapt the first generation KTR for extracellular signal-regulated kinase (ERK) to allow easy implementation in vivo. This sensor, “ERK-nKTR,” allows quantitative and qualitative assessment of ERK activity by analysis of individual nuclei, and faithfully reports ERK activity during development and neural function in diverse cell contexts in C. elegans. Analysis of ERK activity over time in the Vulval Precursor Cells (VPCs), a well-characterized paradigm of EGFR-Ras-ERK signaling, has identified dynamic features not evident from analysis of developmental endpoints alone, including pulsatile, frequency-modulated signaling associated with proximity to the EGF source. The toolkit described here will facilitate studies of ERK signaling in other C. elegans contexts, and the design features will enable implementation of this technology in other multicellular organisms.

Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.

Directional migration requires robust front/back polarity. We find that fibroblasts treated with platelet-derived growth factor (PDGF) and pre-polarized by plating on a fibronectin line substrate, exhibit persistent migration for hours. This does not occur in the absence of PDGF, or on uniformly-coated fibronectin substrates. Persistent migration arises from establishment of two functional modules at cell front and back. At the front, formation of a zone containing podosome-like structures (PLS), dynamically correlates with low RhoA and myosin activity, and absence of a contractile lamella. At the back, myosin contractility specifically controls tail retraction with minimal crosstalk to the front module. The PLS zone is maintained in a dynamic steady state that preserves size and position relative to the cell front, allowing for long term coordination of front and back modules. We propose that front/back uncoupling achieved by the PLS zone is crucial for persistent migration in absence of directional cues.

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton.

Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity.

The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility., Scientific Reports, 6, ISSN:2045-2322

Rho GTPases are key regulators of the cytoskeleton during the process of neurite outgrowth. Based on overexpression of dominant-positive and negative Rho GTPase constructs, the classic view is that Rac1 and Cdc42 are important for neurite elongation whereas RhoA regulates neurite retraction in response to collapsing agents. However, recent work has suggested a much finer control of spatiotemporal Rho GTPase signaling in this process. Understanding this complexity level necessitates a panel of more sensitive tools than previously used. Here, we discuss a novel assay that enables the biochemical fractionation of the neurite from the soma of differentiating N1E-115 neuronal-like cells. This allows for spatiotemporal characterization of a large number of protein components, interactions, and post-translational modifications using classic biochemical and also proteomics approaches. We also provide protocols for siRNA-mediated knockdown of genes and sensitive assays that allow quantitative analysis of the neurite outgrowth process.

Rho-family GTPases are molecular switches that transmit extracellular cues to intracellular signaling pathways. Their regulation is likely to be highly regulated in space and in time, but most of what is known about Rho-family GTPase signaling has been derived from techniques that do not resolve these dimensions. New imaging technologies now allow the visualization of Rho GTPase signaling with high spatio-temporal resolution. This has led to insights that significantly extend classic models and call for a novel conceptual framework. These approaches clearly show three things. First, Rho GTPase signaling dynamics occur on micrometer length scales and subminute timescales. Second, multiple subcellular pools of one given Rho GTPase can operate simultaneously in time and space to regulate a wide variety of morphogenetic events (e.g. leading-edge membrane protrusion, tail retraction, membrane ruffling). These different Rho GTPase subcellular pools might be described as ‘spatio-temporal signaling modules’ and might involve the specific interaction of one GTPase with different guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and effectors. Third, complex spatio-temporal signaling programs that involve precise crosstalk between multiple Rho GTPase signaling modules regulate specific morphogenetic events. The next challenge is to decipher the molecular circuitry underlying this complex spatio-temporal modularity to produce integrated models of Rho GTPase signaling.