Biology subjects

Devreotes, P. N.

Publications and source records attributed to Devreotes, P. N..

13 recordsLinked to original sources

bioRxiv2026

Membrane Kymograph Generator: A cross-platform GUI software for automated generation and analysis of kymographs along dynamic cell boundaries

SummaryThe plasma membrane and accompanying cortex serve as one of the major hubs of the signal transduction and cytoskeletal activities that collectively regulate numerous cell physiological processes such as migration, polarity, macropinocytosis, phagocytosis, cytokinesis, etc. Yet, dynamically tracking membrane-cortex associated protein or lipid kinetics over time from live-cell image series remains a challenging task, primarily due to the difficulty of accurately extracting and aligning the cell boundary between consecutive frames, as the cell continuously deforms and moves. Here, we present Membrane Kymograph Generator, a cross-platform software that accepts multichannel time-lapse live-cell fluorescent imaging datasets as input and automates the cumbersome, heuristic process of boundary tracking, inter-frame alignment, and intensity sampling along the boundary. The software implements a rotational offset minimization algorithm that circularly aligns boundaries across consecutive frames by exhaustively searching for the optimal angular shift that minimizes point-to-point distances, while handling variations in boundary point counts due to cell shape changes. The software outputs kymographs that represent spatiotemporal dynamics of different membrane-associated proteins or biosensors, allows users to fine-tune visualization parameters through an interactive interface, and provides built-in correlation analysis tools for multi-channel datasets. Furthermore, the software allows advanced programmatic usage for batch processing and further analysis via a native API. Our validation tests demonstrated that the Membrane Kymograph Generator can be used to accurately track, visualize, and quantitate the spatial kinetics of a wide array of membrane proteins and lipid biosensors over extended time periods, in a variety of cell types, including Dictyostelium amoeba, human neutrophils, mouse macrophages, and different mammalian cancer cells. The GUI-based software is user-friendly, does not require any technical expertise from users, and significantly reduces the manual effort and time required for kymograph generation and downstream analysis, while ensuring high accuracy and reproducibility. Availability and ImplementationMembrane Kymograph Generator is a free and open-source software, licensed under GNU General Public License 3.0 or later. This software is cross-platform: It can be graphically installed on both x86-64 and AArch64/ARM64 computers, running either Windows, macOS, or any standard Linux distribution. The software is distributed as single installer files (and portable executables) targeting specific hardware architectures and operating systems, and hence, it can be installed natively without any dependency resolution. The source code, detailed documentation, specific installers, portable binaries, and test data are freely available at https://github.com/tatsatb/membrane-kymograph-generator. Additionally, since the software is written in Python, it can also be installed inside any Python environment using PIP package manager (package ID: https://pypi.org/project/membrane-kymograph) and can also be interacted via a built-in Python API.

bioinformatics↗

bioRxiv2025

Spatial distribution of cytoskeleton-mediated feedback controls cell polarization: a computational study

In the social amoeba Dictyostelium, cell motility is regulated through a signal transduction excitable network that interfaces with the cytoskeleton to control actin polymerization patterns. In turn, the cytoskeleton influences the signaling machinery via several feedback loops, but the nature and function of this feedback remain poorly understood. In this study, we use computational models to discern the essential role of complementary positive and negative feedback loops in polarizing cells. We contrast two potential mechanisms for the negative feedback: local inhibition and global inhibition. Our results indicate that both mechanisms can stabilize the leading edge and inhibit actin polymerization in other sites, preventing multipolarity. While some experimental perturbations align more closely with the local inhibition model, statistical analyses reveal its limited polarization potential within a wide excitability range. Conversely, global inhibition more effectively suppresses secondary and tertiary leading-edge formation, making it a more robust polarization mechanism. This raises an intriguing question: if local inhibition better replicates experimental observations but is less effective for polarization than local excitation and global inhibition, could there be a supplementary mechanism enhancing its polarization potential? To address this, we propose a novel mechanism involving the dynamic partitioning of back molecules which enhances communication between the front and back of the cell and can be leveraged by local feedback interactions between the cytoskeleton and signal transduction to improve polarization efficiency.

cell biology↗

bioRxiv2025

Dynamic and Biphasic Regulation of Cell Migration by Ras

Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, non-transcriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that RasGEFs generally increase cortical tension while RasGAPs decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration. SignificanceThis study challenges the traditional view of Ras as solely a positive regulator of cell functions by controlling of gene expression. Using optogenetics to rapidly modulate Ras activity in Dictyostelium, we demonstrate a biphasic relationship between Ras activity and migration: both excessive and insufficient Ras activity impair cell movement. Importantly, these effects occur rapidly, independent of transcriptional changes, revealing the mechanism by which Ras controls cell migration. The findings suggest that optimal Ras activity and cortical tension are crucial for efficient migration, and that targeting Ras in cancer therapy should consider the cells initial state, aiming to push Ras activity outside the optimal range for migration. This nuanced understanding of the role of Ras in migration has significant implications for developing more effective cancer treatments, as simply inhibiting Ras might inadvertently promote metastasis in certain contexts.

cell biology↗

bioRxiv2024

A negative feedback loop between small GTPase Rap1 and mammalian tumor suppressor homolog KrsB regulates cell-substrate adhesion in Dictyostelium

Cell adhesion to the substrate influences a variety of cell behaviors and its proper regulation is essential for migration, although details of the molecular pathways regulating cell adhesion during migration are lacking. Rap1 is a small GTPase that regulates adhesion in mammalian cells, as well as in Dictyostelium discoideum social amoeba, which is an established model for studying directed cell migration. In Dictyostelium, Rap1 controls adhesion via its effects on adhesion mediator talin and Ser/Thr kinase Phg2, which inhibits myosin II function. Kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and Drosophila Hippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Since KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased adhesion to the substrate, which leads to reduced movement. Expression of constitutively active Rap1 G12V increased cell spreading and adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. In contrast, dominant negative Rap1 S17N completely reversed the over-adhesive phenotype of KrsB-null cells and impaired KrsB phosphorylation, suggesting that KrsB activation and function in adhesion requires Rap1. Even though Rap1 did not require KrsB for its function in adhesion, KrsB negatively regulates Rap1 function as seen by increased cortical localization of Rap1 in KrsB-null cells. Consistent with this, chemoattractant-induced activation of downstream effectors of Rap1, TalB and Phg2, was increased in the absence of KrsB. Taken together, these findings suggest that Rap1 leads to activation of KrsB, which inhibits Rap1 and its downstream targets, shutting off adhesion. The existence of a negative feedback loop between Rap1 and KrsB may contribute to the dynamic regulation of cell adhesion that is necessary for rapid amoeboid-type migration.

cell biology↗

bioRxiv2024

Machine learning sparse reaction-diffusion models from stochastic dynamics and spatiotemporal patterns

Pattern-forming stochastic systems arise throughout biology, with dynamic molecular waves observed in biochemical networks regulating critical cellular processes. Modeling these reaction-diffusion systems using handcrafted stochastic partial differential equations (PDEs) requires extensive trial-and-error tuning. Data-driven approaches for improved modeling are needed but have been hindered by data scarcity and noise. Here, we present a solution to the inverse problem of learning stochastic reaction-diffusion models from limited data by optimizing two spatiotemporal features: (1) stochastic dynamics and (2) spatiotemporal patterns. Combined with sparsity enforcement, this method identifies novel activator-inhibitor models with interpretable structure. We demonstrate robust learning from simulations of excitable systems with varying data scarcity, as well as noisy live-cell imaging data with low temporal resolution and a single observed biomolecule. This generalizable approach to learning governing stochastic PDEs enhances our ability to model and understand complex spatiotemporal systems from limited, real-world data. TeaserThis machine learning approach estimates stochastic PDE models using noisy, scarce data from simulations and live-cell imaging.

systems biology↗

bioRxiv2024

Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity

To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in Dictyostelium and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.

cell biology↗

bioRxiv2024

Self-organizing glycolytic waves fuel cell migration and cancer progression

Glycolysis has traditionally been thought to take place in the cytosol but we observed the enrichment of glycolytic enzymes in propagating waves of the cell cortex in human epithelial cells. These waves reflect excitable Ras/PI3K signal transduction and F-actin/actomyosin networks that drive cellular protrusions, suggesting that localized glycolysis at the cortex provides ATP for cell morphological events such as migration, phagocytosis, and cytokinesis. Perturbations that altered cortical waves caused corresponding changes in enzyme localization and ATP production whereas synthetic recruitment of glycolytic enzymes to the cell cortex enhanced cell spreading and motility. Interestingly, the cortical waves and ATP levels were positively correlated with the metastatic potential of cancer cells. The coordinated signal transduction, cytoskeletal, and glycolytic waves in cancer cells may explain their increased motility and their greater reliance on glycolysis, often referred to as the Warburg effect.

biochemistry↗

bioRxiv2023

A dynamic partitioning mechanism polarizes membrane protein distribution

The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins in Dictyostelium and mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate "dynamic partitioning" as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes.

cell biology↗

bioRxiv2022

Direct activation of the Ras-Akt network mediates polarity and organizes protrusions in human neutrophil migration

Functions of Ras oncogenes and their downstream effectors are typically associated with cell proliferation and growth control while their role in immune cell migration has been largely unexplored. Although Ras-mediated signaling cascades have been implicated in immune response, there is no conclusive evidence to show local activation of these pathways on the plasma membrane directly regulates cell motility or polarity. Using spatiotemporally precise, cryptochrome-based optogenetic systems in human neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant-sensing receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or the guanine nucleotide exchange factor, RasGRP4, immediately increased spreading and random motility in neutrophils. Second, creating Ras activity at the cell rear generated new protrusions at the site and reversed pre-existing polarity, similar to the effects of steep chemoattractant gradients. Third, recruiting GTPase activating protein, RASAL3, at cell fronts abrogated existing protrusions and changed the direction of motility whereas dynamically inhibiting nascent fronts stopped migration completely. Fourth, combining pharmacological inhibition studies with optogenetics revealed that mTorC2 is more important than PI3K for Ras-mediated polarity and migration. Finally, local recruitment of Ras-mTorC2 effector, Akt, also generated new protrusions, rearranged pre-existing polarity, and triggered migration, even in absence of PI3K signaling. We propose that actin assembly, cell shape, and migration modes in immune cells are promptly controlled by rapid, local activities of established components of classical growth-control pathways independently of receptor activation.

immunology↗

bioRxiv2022

Nanotopography modulates intracellular excitable systems through cytoskeleton actuation

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust, and acts cooperatively with multiple other guidance cues in complex microenvironments. One-Sentence SummaryCells sense nanotopography directly through their cytoskeletal dynamics, in a reversal of traditional signaling pathway hierarchies.

biophysics↗

bioRxiv2022

Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration

During cell migration and polarization, hundreds of signal transduction and cytoskeletal components self-organize to generate localized protrusions. Although biochemical and genetic analyses have delineated many specific interactions, how the activation and localization of so many different molecules are spatiotemporally orchestrated at the subcellular level has remained unclear. Here we show that the regulation of negative surface charge on the inner leaflet of the plasma membrane plays an integrative role in the molecular interactions. Surface charge, or zeta potential, is transiently lowered at new protrusions and within cortical waves of Ras/PI3K/TORC2/F-actin network activation. Rapid alterations of inner leaflet anionic phospholipids, such as PI(4,5)P2, PI(3,4)P2, phosphatidylserine, and phosphatidic acid, collectively contribute to the surface charge changes. Abruptly reducing the surface charge by recruiting positively charged optogenetic actuators was sufficient to trigger the entire biochemical network, initiate de novo protrusions, and abrogate pre-existing polarity. These effects were blocked by genetic or pharmacological inhibitions of key signaling components such as Akt and PI3K/TORC2. Conversely, increasing the negative surface deactivated the network and locally suppressed chemoattractant-induced protrusions or subverted EGF-induced ERK activation. Computational simulations involving excitable biochemical networks demonstrated that slight changes in feedback loops, induced by recruitment of the actuators, could lead to outsized effects on system activation. We propose that key signaling network components act on, and are in turn acted upon, by surface charge, closing feedback loops which bring about the global-scale molecular self-organization required for spontaneous protrusion formation, cell migration, and polarity establishment.

cell biology↗

bioRxiv2021

Cortical waves mediate the cellular response to electric fields

Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis share both similarities and differences with chemical-gradient-sensing pathways. We examined a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 minutes after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display "U-turn" behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

cell biology↗

bioRxiv2021

3D stochastic simulation of chemoattractant-mediated excitability in cells

During the last decade, a consensus has emerged that the stochastic triggering of an excitable system drives pseudopod formation and subsequent migration of amoeboid cells. The presence of chemoattractant stimuli alters the threshold for triggering this activity and can bias the direction of migration. Though noise plays an important role in these behaviors, mathematical models have typically ignored its origin and merely introduced it as an external signal into a series of reaction-diffusion equations. Here we consider a more realistic description based on a reaction-diffusion master equation formalism to implement these networks. In this scheme, noise arises naturally from a stochastic description of the various reaction and diffusion terms. Working on a three-dimensional geometry in which separate compartments are divided into a tetrahedral mesh, we implement a modular description of the system, consisting of G-protein coupled receptor signaling (GPCR), a local excitation-global inhibition mechanism (LEGI), and signal transduction excitable network (STEN). Our models implement detailed biochemical descriptions whenever this information is available, such as in the GPCR and G-protein interactions. In contrast, where the biochemical entities are less certain, such as the LEGI mechanism, we consider various possible schemes and highlight the differences between them. Our stimulations show that even when the LEGI mechanism displays perfect adaptation in terms of the mean level of proteins, the variance shows a dose-dependence. This differs between the various models considered, suggesting a possible means for determining experimentally among the various potential networks. Overall, our simulations recreate temporal and spatial patterns observed experimentally in both wild-type and perturbed cells, providing further evidence for the excitable system paradigm. Moreover, because of the overall importance and ubiquity of the modules we consider, including GPCR signaling and adaptation, our results will be of interest beyond the field of directed migration. Author summaryThough the term noise usually carries negative connotations, it can also contribute positively to the characteristic dynamics of a system. In biological systems, where noise arises from the stochastic interactions between molecules, its study is usually confined to genetic regulatory systems in which copy numbers are small and fluctuations large. However, noise can have important roles when the number of signaling molecules is large. The extension of pseudopods and the subsequent motion of amoeboid cells arises from the noise-induced trigger of an excitable system. Chemoattractant signals bias this triggering thereby directing cell motion. To date, this paradigm has not been tested by mathematical models that account accurately for the noise that arises in the corresponding reactions. In this study, we employ a reaction-diffusion master equation approach to investigate the effects of noise. Using a modular approach and a three-dimensional cell model with specific subdomains attributed to the cell membrane and cortex, we explore the spatiotemporal dynamics of the system. Our simulations recreate many experimentally-observed cell behaviors thereby supporting the biased-excitable hypothesis.

cell biology↗