Biology subjects

Pal, D. S.

Publications and source records attributed to Pal, D. S..

8 recordsLinked to original sources

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↗

bioRxiv2024

PIP5K-Ras bistability initiates plasma membrane symmetry breaking to regulate cell polarity and migration

Symmetry breaking is a fundamental process that underlies key cellular behaviors such as cell polarity and migration, but the mechanism - how a uniform plasma membrane spontaneously transitions to an asymmetric state - is still unknown. Here in this study, using a combination of Dictyostelium amoeba, multiple mammalian leukocytes, and human cancer cells and 3D organoid systems, we monitored the localization dynamics of RasGTP and PIP5K, dissected the effects of eliminating, conditionally increasing, or optogenetically manipulating membrane PIP5K levels, screened for key regulators of Ras activation, and tracked single-molecules of PIP5K. Our data converge on a core biochemical circuit involving mutually inhibitory interactions between PIP5K and RasGTP that is both necessary and sufficient for symmetry breaking. The process is initiated by stochastic, localized Ras activation coupled with a decrease in the lifetime of the association of PIP5K with the membrane. A resulting localized reduction in PI(4,5)P2 facilitates the recruitment of a RasGEF to the corresponding domain, amplifying RasGTP production through a positive feedback loop. Dissociated PIP5K relocates to other membrane regions, where it suppresses Ras activation. These events separate the membrane into distinct active and inactive zones, even when receptor inputs or cytoskeletal activities are absent. This same core biochemical circuit controls the spatial organization of downstream PI3K/Akt/Rac signaling and actin/actomyosin activities, which generate the localized protrusions to define polarity and migration mode. While many models have been forwarded to explain polarity or symmetry breaking, the PIP5K-Ras mutually inhibitory bistable circuit we present here is the first universal molecular mechanism to explain the initiation of asymmetry as well as subsequent polarity and migration.

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

Ras-mediated homeostatic control of front-back signaling dictates cell polarity

Studies in the model systems, Dictyostelium amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation on the membrane is spatiotemporally regulated by its activators, RasGEFs, and inhibitors, RasGAPs, which might be expected to create a stable front and back, respectively, in migrating cells. Focusing on C2GAPB in amoebae and RASAL3 in neutrophils, we investigated how Ras activity along the cortex controls polarity. Since existing gene knockout and overexpression studies can be circumvented, we chose optogenetic approaches to assess the immediate, local effects of these Ras regulators on the cell cortex. In both cellular systems, optically targeting the respective RasGAPs to the cell front extinguished existing protrusions and changed the direction of migration, as might be expected. However, when the expression of C2GAPB was induced globally, amoebae polarized within hours. Furthermore, within minutes of globally recruiting either C2GAPB in amoebae or RASAL3 in neutrophils, each cell type polarized and moved more rapidly. Targeting the RasGAPs to the cell backs exaggerated these effects on migration and polarity. Overall, in both cell types, RasGAP-mediated polarization was brought about by increased actomyosin contractility at the back and sustained, localized F-actin polymerization at the front. These experimental results were accurately captured by computational simulations in which Ras levels control front and back feedback loops. The discovery that context-dependent Ras activity on the cell cortex has counterintuitive, unanticipated effects on cell polarity can have important implications for future drug-design strategies targeting oncogenic Ras.

cell biology↗

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

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↗

bioRxiv2020

Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major

Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this the parasite is heavily dependent on its gluconeogenic machinery. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial pyruvate carboxylase (PC) gene. PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funnelled into this pathway. Absence of PC in Leishmania thus raises question about the mechanism of pyruvate entry into the gluconeogenic route. We report here that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and cytosolic carbonic anhydrase (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we showed that both these enzymes are necessary in promoting gluconeogenesis and supporting parasite growth under glucose limiting condition. Functional crosstalk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that while LmCA1 exhibited constitutive expression, LmME protein level was strongly upregulated in low glucose condition. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through pyruvate-malate-oxaloacetate bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.

biochemistry↗