Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.

Septins Enable T Cell Contact Guidance via Amoeboid-Mesenchymal Switch.

Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes dynamically alternate and combine their amoeboid (non-adhesive) and mesenchymal (adhesive) migration modes. However, which mechanisms guide and balance different migration modes are largely unclear. Here we report that suppression of septins GTPase activity induces an abrupt amoeboid-to-mesenchymal transition of T cell migration mode, characterized by a distinct, highly deformable integrin-dependent immune cell contact guidance. Surprisingly, the T cell actomyosin cortex contractility becomes diminished, dispensable and antagonistic to mesenchymal-like migration mode. Instead, mesenchymal-like T cells rely on microtubule stabilization and their non-canonical dynein motor activity for high fidelity contact guidance. Our results establish septin's GTPase activity as an important on/off switch for integrin-dependent migration of T lymphocytes, enabling their dynein-driven fluid-like mesenchymal propulsion along the complex adhesion cues.

Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, the locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, a complementary mechanism of metastatic locomotion powered by dynein-generated forces is identified. These forces arise within a non-stretchable microtubule network and drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. It is also shown that the dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network formed by spatially confining granular hydrogel scaffolds (GHS) made up of microscale hydrogel particles (microgels). These results indicate that the complementary motricity mediated by dynein is always necessary and, in certain instances, sufficient for disseminating metastatic breast cancer cells. These findings advance the fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.

Profiling an integrated network of cellular senescence and immune resilience measures in natural aging: a prospective multi-cohort study.

Background: Biological aging begins decades before the onset of age-related clinical conditions and is mediated by both cellular senescence and declining adaptive immune function. These processes are functionally related with the rate of senescent cell accumulation dependent upon a balance between induction and immune clearance. We previously showed that biomarkers in these domains can identify patients at-risk of surgery-related adverse events. Here, we describe evidence of clinical relevance in early aging and metabolic phenotypes in a general adult population. Methods: We enrolled a total of 482 participants (ages 25-90) into two prospective, cross-sectional healthy aging cohorts. Expression of biomarkers of adaptive immune function and cellular senescence (SapereX) was measured in CD3+ T cells isolated from peripheral blood. Findings: We established a network of biomarkers of adaptive immune function that correlate with cellular senescence and associate with early aging phenotypes. SapereX immune components associated with a decrease in CD4+ T cells, an increase in cytotoxic CD8+ T cells, and a loss of CD8+ naïve T cells (Pearson correlation 0.3-0.6). These components also associated with a metric of immune resilience, an ability to withstand antigen challenge and inflammation. In contrast, SapereX components were only weakly associated with GlycanAge (Pearson correlation 0.03-0.15) and commonly used DNA methylation clocks (Pearson correlation 0-0.25). Finally, SapereX biomarkers, in particular p16, were associated with chronic inflammation and metabolic dysregulation. Interpretation: Measurement of SapereX biomarkers may capture essential elements of the relationship between cellular senescence and dysregulated adaptive immune function and may provide a benchmark for clinically relevant health decisions.

Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling.

Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

Dynein-Powered Cell Locomotion Guides Metastasis of Breast Cancer.

Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.

Spatiotemporal coordination of Rac1 and Cdc42 at the whole cell level during cell ruffling.

Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

Coordinated regulation of Cdc42ep1, actin, and septin filaments during neural crest cell migration.

The septin cytoskeleton has been demonstrated to interact with other cytoskeletal components to regulate various cellular processes, including cell migration. However, the mechanisms of how septin regulates cell migration are not fully understood. In this study, we use the highly migratory neural crest cells of frog embryos to examine the role of septin filaments in cell migration. We found that septin filaments are required for the proper migration of neural crest cells by controlling both the speed and the direction of cell migration. We further determined that septin filaments regulate these features of cell migration by interacting with actin stress fibers. In neural crest cells, septin filaments co-align with actin stress fibers, and the loss of septin filaments leads to impaired stability and contractility of actin stress fibers. In addition, we showed that a partial loss of septin filaments leads to drastic changes in the orientations of newly formed actin stress fibers, suggesting that septin filaments help maintain the persistent orientation of actin stress fibers during directed cell migration. Lastly, our study revealed that these activities of septin filaments depend on Cdc42ep1, which colocalizes with septin filaments in the center of neural crest cells. Cdc42ep1 interacts with septin filaments in a reciprocal manner, with septin filaments recruiting Cdc42ep1 to the cell center and Cdc42ep1 supporting the formation of septin filaments.

Methods for assessment of membrane protrusion dynamics.

Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.

Spatiotemporal development of coexisting wave domains of Rho activity in the cell cortex.

The Rho family GTPases are molecular switches that regulate cytoskeletal dynamics and cell movement through a complex spatiotemporal organization of their activity. In Patiria miniata (starfish) oocytes under in vitro experimental conditions (with overexpressed Ect2, induced expression of Δ90 cyclin B, and roscovitine treatment), such activity generates multiple co-existing regions of coherent propagation of actin waves. Here we use computational modeling to investigate the development and properties of such wave domains. The model reveals that the formation of wave domains requires a balance between the activation and inhibition in the Rho signaling motif. Intriguingly, the development of the wave domains is preceded by a stage of low-activity quasi-static patterns, which may not be readily observed in experiments. Spatiotemporal patterns of this stage and the different paths of their destabilization define the behavior of the system in the later high-activity (observable) stage. Accounting for a strong intrinsic noise allowed us to achieve good quantitative agreement between simulated dynamics in different parameter regimes of the model and different wave dynamics in Patiria miniata and wild type Xenopus laevis (frog) data. For quantitative comparison of simulated and experimental results, we developed an automated method of wave domain detection, which revealed a sharp reversal in the process of pattern formation in starfish oocytes. Overall, our findings provide an insight into spatiotemporal regulation of complex and diverse but still computationally reproducible cell-level actin dynamics.

Correcting Artifacts in Ratiometric Biosensor Imaging; an Improved Approach for Dividing Noisy Signals.

The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of the method first by examining two noninteracting fluorophores distributed evenly in cells. This generated a uniform ratio that could provide a ground truth. We then analyzed actual protein activities reported by a single chain biosensor for the guanine exchange factor (GEF) Asef, and a dual chain biosensor for the GTPase Cdc42. The reduction of edge artifacts revealed persistent Asef activity in a narrow band (∼640 nm wide) immediately adjacent to the cell edge. For Cdc42, the NCF method revealed an artifact that would have been obscured by traditional background subtraction approaches.

Bistability in the polarity circuit of yeast.

Cells polarize their growth or movement in many different physiological contexts. A key driver of polarity is the Rho GTPase Cdc42, which when activated becomes clustered or concentrated at polar sites. Multiple models for polarity establishment have been proposed. All of them rely on positive feedback to reinforce regions of high Cdc42 activity. Positive feedback can lead to bistability, a scenario in which cells can exist in either a polarized or unpolarized state under identical external conditions. Determining if the signaling circuit that drives Cdc42 polarity is bistable would provide important information about the mechanism that underlies polarity establishment and insights into the design features required for proper cellular function. We studied polarity establishment during the mating response of yeast. Using microfluidics to precisely control the temporal profile of mating pheromone and live-cell imaging to monitor the polarity process in single living cells, we found that the polarity circuit of yeast shows hysteresis, a characteristic feature of bistable systems. Our analysis also revealed that cells exposed to high pheromone concentrations rapidly lose polarity following a precipitous removal of pheromone. We used a reaction-diffusion model for polarity establishment to demonstrate that delayed negative regulation is sufficient to explain our experimental results. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Shape-to-graph mapping method for efficient characterization and classification of complex geometries in biological images.

With the ever-increasing quality and quantity of imaging data in biomedical research comes the demand for computational methodologies that enable efficient and reliable automated extraction of the quantitative information contained within these images. One of the challenges in providing such methodology is the need for tailoring algorithms to the specifics of the data, limiting their areas of application. Here we present a broadly applicable approach to quantification and classification of complex shapes and patterns in biological or other multi-component formations. This approach integrates the mapping of all shape boundaries within an image onto a global information-rich graph and machine learning on the multidimensional measures of the graph. We demonstrated the power of this method by (1) extracting subtle structural differences from visually indistinguishable images in our phenotype rescue experiments using the endothelial tube formations assay, (2) training the algorithm to identify biophysical parameters underlying the formation of different multicellular networks in our simulation model of collective cell behavior, and (3) analyzing the response of U2OS cell cultures to a broad array of small molecule perturbations.

Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity.

Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell. However, in many diseases, including cancer, these signaling proteins are switched on for too long or are active in the wrong place. To better understand why this is the case, researchers manipulate proteins to identify the processes they regulate. One way to do this is to engineer proteins so that they can be controlled by light, turning them either on or off. Ideally, a light-controlled tool can activate proteins at defined times, control proteins in specific locations within the cell and regulate any protein of interest. However, current methods do not combine all of these requirements in one tool, and scientists often have to use different methods, depending on the topic they are researching. Now, Shaaya et al. set out to develop a single tool that combines all required features. The researchers engineered a light-sensitive 'switch' that allowed them to activate a specific protein by illuminating it with blue light and to deactivate it by turning the light off. Unlike other methods, the new tool uses a light-sensitive switch that works like a clamp. In the dark, the clamp is open, which 'stretches' and distorts the protein, rendering it inactive. In light, however, the clamp closes and the structure of the protein and its activity are restored. Moreover, it can activate proteins multiple times, control proteins in specific locations within the cell and it can be applied to a variety of proteins. This specific design makes it possible to combine multiple features in one tool that will both simplify and broaden its use to investigate specific proteins and signaling pathways in a broad range of diseases.

Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

Cell-cycle control of cell polarity in yeast.

In many cells, morphogenetic events are coordinated with the cell cycle by cyclin-dependent kinases (CDKs). For example, many mammalian cells display extended morphologies during interphase but round up into more spherical shapes during mitosis (high CDK activity) and constrict a furrow during cytokinesis (low CDK activity). In the budding yeast Saccharomyces cerevisiae, bud formation reproducibly initiates near the G1/S transition and requires activation of CDKs at a point called "start" in G1. Previous work suggested that CDKs acted by controlling the ability of cells to polarize Cdc42, a conserved Rho-family GTPase that regulates cell polarity and the actin cytoskeleton in many systems. However, we report that yeast daughter cells can polarize Cdc42 before CDK activation at start. This polarization operates via a positive feedback loop mediated by the Cdc42 effector Ste20. We further identify a major and novel locus of CDK action downstream of Cdc42 polarization, affecting the ability of several other Cdc42 effectors to localize to the polarity site.

Biomechanics of Endothelial Tubule Formation Differentially Modulated by Cerebral Cavernous Malformation Proteins.

At early stages of organismal development, endothelial cells self-organize into complex networks subsequently giving rise to mature blood vessels. The compromised collective behavior of endothelial cells leads to the development of a number of vascular diseases, many of which can be life-threatening. Cerebral cavernous malformation is an example of vascular diseases caused by abnormal development of blood vessels in the brain. Despite numerous efforts to date, enlarged blood vessels (cavernomas) can be effectively treated only by risky and complex brain surgery. In this work, we use a comprehensive simulation model to dissect the mechanisms contributing to an emergent behavior of the multicellular system. By tightly integrating computational and experimental approaches we gain a systems-level understanding of the basic mechanisms of vascular tubule formation, its destabilization, and pharmacological rescue, which may facilitate the development of new strategies for manipulating collective endothelial cell behavior in the disease context.

EdgeProps: A Computational Platform for Correlative Analysis of Cell Dynamics and Near-Edge Protein Activity.

Developing molecular tools to visualize and control Rho GTPase signaling in living cells has been instrumental in elucidating the mechanisms of cytoskeletal reorganization and causal relationships between activation events in cell function. An indispensable part of such studies is the quantitative characterization of the spatiotemporal GTPase activity. Here we present a computational pipeline, EdgeProps, designed for comparative/correlative analysis of cell dynamics (edge velocity) and near-edge protein activity (intensity of a fluorescent signal). The tool offers a user-friendly interface with three functional modules for processing, visualization, and statistical characterization of single-cell imaging data.

Temporal regulation of morphogenetic events in Saccharomyces cerevisiae.

Tip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented toward that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single-cell level. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin "priming" facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently for the cell to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell-wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.