Biology subjects

Gopinathan, A.

Publications and source records attributed to Gopinathan, A..

14 recordsLinked to original sources

bioRxiv2026

From Flocks to Swarms: Rewriting the Rules of Collective Cell Migration

From flocking birds to swarming insects, collective motion enables groups to accomplish tasks beyond the capabilities of individuals. These behaviors emerge from local interactions among group members, but the interaction rules that organize collectives at the cellular scale remain poorly understood, in part because they are difficult to manipulate experimentally. Here we develop a cell-machine interface that enables us to rewrite the rules of collective migration. Cell-cell interaction rules are specified in software and implemented in living cells through real-time computer vision and closed-loop optogenetic control. Using a single engineered cell line, we program cells to self-organize into flocks, to propagate regenerative signal relays that recapitulate the interaction logic of immune-cell swarms, and to collectively navigate guidance cues that individual cells cannot. Our work shows that altering simple rules of cell-cell interaction is sufficient to generate new emergent collective capabilities. This approach provides an experimental framework for discovering the organizational principles of multicellular coordination and designing synthetic collectives with capabilities beyond those of their individual members.

synthetic biology↗

bioRxiv2026

Cell Cluster Geometry and Fluidity Control the Transition from Single-Cell Chemorepulsion to Collective Chemotaxis

Directed migration along chemical gradients controls immune surveillance, development, and cancer invasion. However, the same chemical cue can produce different responses depending on its concentration and whether cells move alone or in groups. For example, in steep gradients, isolated malignant lymphocyte cells migrate away from the chemoattractant source, whereas clusters of the same cells continue to migrate toward it. Here, combining computational modeling and experimental observations, we show that this reversal is governed by coupled mechanisms acting across molecular, cellular, and collective scales. At the single-cell level, our model predicts that receptor endocytosis generates a feedback that produces a nonmonotonic surface receptor density with increasing chemoattractant concentration. Above a critical concentration that depends on the cells volume-to-sensing-area ratio, receptor depletion reverses cell polarity and drives chemorepulsion. However, in clusters, cell-cell contacts reduce the membrane area exposed to ligand, increasing the volume-to-sensing-area ratio, thus increasing the critical concentration and preserving chemotaxis. An agent-based model incorporating these mechanisms quantitatively reproduces the sign reversal of the migration index across gradient steepness and cluster size. We show that collective rearrangements further stabilize chemoattraction with exchanges between the cluster rim and core helping remove chemorepulsive cells from the leading edge, keeping their fraction below the threshold required to reverse cluster migration. The model further predicts, and experiments confirm, that increasing ambient ligand concentration while keeping the gradient fixed reduces cluster chemoattraction. Our results identify receptor trafficking, cell geometry, and cluster fluidity as physical determinants of collective directional decision-making, with implications for immune cell homing, tissue morphogenesis, and cancer dissemination.

biophysics↗

bioRxiv2026

SARS-CoV-2 membrane protein conformations induce distinct membrane curvatures

The assembly and budding of enveloped viruses requires thousands of membrane proteins to collectively remodel host-cell membranes into highly curved virions. In SARS-CoV-2, this process is driven by interactions between viral structural proteins and the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane. The membrane (M) protein, an embedded homodimer and the most abundant viral component, exists in two conformations: a compact "short" form and an elongated "long" form. Although M is essential for virion assembly, how its conformations contribute to the generation and organization of the membrane curvature required for budding has remained unknown. Here, we used all-atom and Martini coarse-grained molecular dynamics simulations to show that individual M proteins can induce distinct membrane curvatures, depending on their conformation. The long form bends the membrane around its C-terminal, forming a valley-like depression, while the short form predominantly bends the membrane away from the C-terminal producing an anisotropic ridge. The induced curvatures correspond to the bulb and neck regions of a budding virion, respectively. Coarse-grained simulations of M protein pairs further reveal that curvature modulates long-range, membrane-mediated M-M interactions, leading to repulsion between dissimilar conformations. Together, these results suggest that the long and short forms of M naturally segregate to shape the virions bulb and neck, potentially facilitating genome encapsulation and membrane scission. This mechanism provides a physical basis for coronavirus budding and suggests that conformationally encoded curvature fields may represent a general principle underlying the formation of enveloped viruses. Significance StatementEnveloped viruses must bend host cell membranes to form new viral particles, but the driving force for membrane bending has been unclear. Using molecular dynamics simulations, we show that the SARS-CoV-2 membrane protein, the most abundant structural protein of the virus, drives membrane bending, and that its two natural conformations generate opposite signs of curvature which match the geometry of distinct regions of a budding virus. Moreover, these induced curvatures cause proteins in different conformations to repel one another providing a physical basis for their spatial segregation. Our results suggest that the membrane proteins conformations can contribute significantly to the membrane remodeling needed for virus assembly and release, revealing a simple mechanism that may apply to other enveloped viruses.

biophysics↗

bioRxiv2026

Mechanisms of viral budding through cellular membranes

Budding is a fundamental membrane-remodeling process central to many cellular functions and is exploited by numerous enveloped viruses to acquire their lipid envelopes. Despite extensive molecular characterization, the physical mechanisms that determine whether budding proceeds to completion or becomes stalled remain unclear. Here, we develop a theoretical model based on the Helfrich elastic formalism to investigate how membrane geometry and boundary conditions regulate the elastic energy of viral budding. We analyze two representative cases: budding from a flat membrane, characteristic of HIV-1 and alphaviruses, and budding from a vesicle, as observed for SARS-CoV-2 in the ER-Golgi intermediate compartment (ERGIC). Our results reveal distinct energetic pathways: vesicle-like geometries exhibit a stronger energetic bias toward closure, whereas flat membranes develop extended low-slope regions in the energy landscape that can hinder completion. Relaxing far-field boundary constraints reduces the energetic cost associated with membrane area conservation and renders the flat-membrane case energetically comparable to the vesicle case, providing a physical explanation for why viruses frequently bud adjacent to one another or within pre-curved membrane regions. Comparison with thin-section TEM images of alphavirus budding shows results consistent with the theoretical membrane profiles. Together, these findings establish how curvature coupling, boundary flexibility, and local membrane geometry cooperate to control the efficiency and completion of membrane budding.

biophysics↗

bioRxiv2025

Clustering of SARS-CoV-2 membrane proteins in lipid bilayer membranes

The accumulation of viral structural proteins along the ER-Golgi intermediate compartment (ERGIC) membrane leads to SARS-CoV-2 self-assembly and budding, driven by the interactions between these proteins, RNA and the ERGIC membrane. The membrane protein (M) is believed to interact with other structural proteins and form clusters needed for the induction of membrane curvature that facilitates virion formation. However, the role played by direct and membrane-mediated interactions between M proteins and their interactions with other proteins in the clustering process remains unclear. Here, we utilize a combination of all-atom molecular dynamics (MD) simulations, continuum modeling and experiments to show that M-M interactions are sufficient to drive clustering in ERGIC-like lipid bilayers in the absence of other proteins or RNA. Using all-atom MD simulations we were able to estimate the membrane thinning induced by M proteins and the resulting membrane-mediated M-M interaction. Combining this with a continuum model that describes the evolution of M protein density in a planar lipid membrane, we identified the existence of a critical, direct M-M interaction energy needed for cluster assembly at a given density. By comparing the model predictions with analysis of atomic force microscopy images of M protein clusters in supported lipid bilayers, we were able to estimate the direct M-M interaction energy and found it to be significantly larger than the membrane mediated interaction energy. Our work therefore establishes that M protein interactions are sufficient to drive clustering and provides a quantitative understanding of the role played by direct and membrane-mediated interactions of M proteins in viral assembly and budding.

biophysics↗

bioRxiv2025

Competitive binding and geometric changes allow fast, complete translocation of intact HIV-1 capsids through the nuclear pore complex

Recent experiments on HIV-1 capsid translocation through the nuclear pore complex (NPC) have demonstrated the docking of intact or nearly intact capsids at the pore, followed by translocation, with capsid disassembly occurring only within the nucleus near the site of integration. Given that the size of the capsid is comparable to the pore dimensions, these new findings raise questions regarding the energetics and dynamics of capsid passage across the significant entropic barrier created by the disordered FG nucleoporins (FG nups) in the NPC central channel. Here, we develop an analytical model for the transport of the HIV-1 capsid that considers the geometry of the capsid and pore, the free energy barrier due to the FG nups, and capsid-nup interactions. Our results show that capsid entry into the pore is favorable if the narrow end enters first, consistent with experimental observations, and that increasing the capsid-nup interaction strength enhances inward capsid flux. However, capsid-nup interaction alone is insufficient for complete capsid nuclear import and competitive binding by the nuclear factor CPSF6 to FG-binding sites on the capsid can serve as a ratcheting mechanism for full nuclear import. We show that nuclear import is arrested below a minimum CPSF6 concentration, consistent with experimental observations, and that pore dilation or capsid deformation can accelerate the translocation process. Our work explores the physics underlying a new perspective on HIV-1 viral genome nuclear import and provides quantitative explanations for experimentally observed phenomena while making testable predictions. Significance statementHIV-1 remains a devastating global health threat, with millions of new infections each year. However, the mechanism by which the viral genome enters the host nucleus is still not fully understood. Recent experiments show that the HIV-1 capsid can remain intact - or nearly so - during nuclear import, challenging long-held assumptions about the limits on size for transport through the nuclear pore. Here, we present a biophysical model that explains the directional transport of the capsid, the role of capsid and pore geometry and host factors such as CPSF6, and elucidates a ratcheting mechanism that allows entry of intact capsids within physiological timescales. This work provides a mechanistic framework for a critical step in the early stages of HIV-1 infection.

biophysics↗

bioRxiv2025

Membrane-bound cargo carried by teams of motors with heterogeneous velocities go faster and further than rigid cargo

Intracellular transport by teams of molecular motors is an essential cell-biological process that ensures the proper distribution of organelles, and other materials within cells. These teams of motors cooperate and compete in complex ways to achieve desired transport velocity and runlength. In-vitro experiments have observed that coupling motors through a lipid membrane that mimics in vivo membrane-bound cargoes leads to a higher cargo velocity. However, the mechanisms behind this increase in lipid cargo velocity are unclear. Here we seek to understand these mechanisms using Brownian dynamics simulations. We show that an underlying heterogeneity in single motor velocity is essential for the increased velocity of lipid cargoes. Our simulations also show that while the runlengths of both rigid and lipid cargoes increase, and the velocities decrease, with an increase in the fraction of slower motors, lipid cargoes can travel faster and substantially further with the same degree of heterogeneity, suggesting functional advantages of motor velocity heterogeneity. Together, our work explains mechanisms behind previous experimental observations and generates new experimentally testable predictions on velocities and runlengths relevant for in vivo transport.

biophysics↗

bioRxiv2024

Sensing the shape of a surface by intracellular filaments

Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as Escherichia coli by directing cell-wall synthesis according to local curvature cues. However, the basis of MreBs curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of [~]50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization, and suggest new design principles for synthetic shape sensing nanomaterials. Significance statementThe protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like Escherichia coli by guiding new cell-wall insertion based on local curvature cues. However, the mechanism by which a nanometer-scale MreB filament "senses" the micron-scale curvature of the cell wall has remained a mystery. We introduce a biophysical model of the energetics of twisted and bent filaments bound to curved surfaces, which predicts that localization of filaments like MreB is sensitive to both mean and Gaussian curvature. The model captures experimentally measured curvature enrichment patterns and explains how MreB naturally localizes to saddle-shaped regions without energy-consuming processes. Beyond cell shape regulation, our work suggests design principles for synthetic systems that can sense and respond to surface shape.

biophysics↗

bioRxiv2023

Synthesis, Insertion and Characterization of SARS-CoV-2 Membrane Protein Within Lipid Bilayers

SUMMARY/ABSTRACTThe membrane protein (M) is the most abundant structural protein in the SARS-CoV-2 virus and functions exclusively as a membrane-embedded homodimer. M protein is required for the formation of the SARS-CoV-2 virus particle and has been shown to interact with the Spike and Envelope proteins, as well as the RNA-packaging Nucleocapsid protein. Our knowledge of M protein is very limited due to its small size and challenges in expressing enough protein for use in structural and biophysical experiments. We report the successful development of a SUMO tag-based expression system to produce and purify significant quantities of M protein, and a method to insert the synthesized dimers into a suspended lipid membrane in a homogeneous orientation. We used AFM and Cryo-EM to image individual membrane-bound M protein dimers and characterize the configurations that they can assume. Our experimental results are in agreement with our molecular dynamics simulations which predict thinning of the membrane around the M protein and a propensity to induce local membrane curvature. Taken together, our results shed new light on M protein properties within the lipid bilayer and suggest mechanisms that could contribute to viral assembly and budding.

biophysics↗

bioRxiv2022

Feedback linking cell envelope stiffness, curvature, and synthesis enables robust rod-shaped bacterial growth

Bacterial growth is remarkably robust to environmental fluctuations, yet the mechanisms of growth-rate homeostasis are poorly understood. Here, we combine theory and experiment to infer mechanisms by which Escherichia coli adapts its growth rate in response to changes in osmolarity, a fundamental physicochemical property of the environment. The central tenet of our theoretical model is that cell-envelope expansion is only sensitive to local information such as enzyme concentrations, cell-envelope curvature, and mechanical strain in the envelope. We constrained this model with quantitative measurements of the dynamics of E. coli elongation rate and cell width after hyperosmotic shock. Our analysis demonstrated that adaptive cell-envelope softening is a key process underlying growth-rate homeostasis. Furthermore, our model correctly predicted that softening does not occur above a critical hyperosmotic shock magnitude and precisely recapitulated the elongation-rate dynamics in response to shocks with magnitude larger than this threshold. Finally, we found that to coordinately achieve growth-rate and cell-width homeostasis, cells employ direct feedback between cell-envelope curvature and envelope expansion. In sum, our analysis points to new cellular mechanisms of bacterial growth-rate homeostasis and provides a practical theoretical framework for understanding this process. Significance StatementThe bacterial cell envelope is the critical structure that defines cell size and shape, and its expansion therefore defines cell growth. Although size, shape, and growth rate are important cellular variables that are robust to environmental fluctuations, the feedback mechanisms by which these variables influence cell-envelope expansion are unknown. Here, we explore how E. coli cells achieve growth-rate and cell-width homeostasis during fluctuations in osmolarity, a key environmental property. A biophysical model in which the cell envelope softens after an osmotic shock and envelope expansion depends directly on local curvature quantitatively recapitulated all experimental observations. Our study elucidates new mechanisms of bacterial cell morphogenesis and highlights the deep interplay between global cellular variables and the mechanisms of cell-envelope expansion.

microbiology↗

bioRxiv2021

Cargo surface fluidity reduces inter-motor interference, promotes load-sharing and enhances run-lengths in an ATP dependent manner

In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work as teams is unclear. Here we explored the possibility that purely physical mechanisms may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a 3D dynamical simulation of cargo transport along microtubules by teams of canonically non-cooperative kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. We showed explicitly, for the first time, that surface fluidity leads to the reduction of negative mechanical interference between kinesins, enhancing load sharing thereby decreasing single motor off-rates and increasing processivity. Remarkably, we also showed that, independent of fluidity, increasing numbers of bound motors pull the cargo closer to the microtubule, increasing the on-rates of individual unbound motors, resulting in a cooperative increase in bound motor numbers that depends on 3D cargo geometry. At the cargo level, surface fluidity makes more motors available for binding, though this effect is significant only at low ATP or high motor density. Interestingly, we find that the fluidity induced reduction in mechanical interference dominates over the increased availability of motors for typical physiological and in vitro conditions, this allowing us to reconcile different experimental results in different regimes. Finally, we show that these effects can altogether result in enhanced mechanical efficiency and tunable cargo run-lengths for teams of molecular motors over physiological ranges of fluidity with implications for new experimental validation efforts, transport in vivo, artificial cargo design and multi-motor-driven mechanical processes in general. Author summaryIn cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of protein filaments. How do intrinsically non-cooperative motors function as teams within cells? In this paper, we show, using computer simulations, that the fluid surfaces of cellular cargo reduce the mechanical interference between motors allowing better load sharing and decreasing their detachment rates, thereby increasing the distance over which they can carry cargo. We additionally found that motors pull cargo closer to the filament increasing the attachment rates of other unbound motors. These effects act synergistically with an increased availability of motors due to fluidity to further increase travel distances. Our simulation model accounted for the dynamics of the motors on the cargo surface and their load and ATP dependent kinetics, allowing us to connect single motor properties to overall transport. Our work on understanding how teamwork arises in mechanically coupled motors sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving mechanical efficiency and transport of artificial cargo powered by motor teams.

biophysics↗

bioRxiv2020

Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Cells across all kingdoms of life actively partition molecules between discrete cellular compartments. In Gram-positive bacteria, a thick and highly cross-linked peptidoglycan cell wall separates the bacterial membrane from the extracellular space, imposing a barrier that must be crossed by proteins whose functions require that they be exposed on the bacterial cell surface1,2. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA3, remain associated with the bacterial membrane yet somehow thread through tens of nanometers of dense, cross-linked cell wall to expose their N-terminus on the outer surface4,5. Here, we show that entropy can drive the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We develop a physical model predicting that the entropic constraint imposed by a thin periplasm is sufficient to drive translocation of an intrinsically disordered protein like ActA across a porous barrier similar to the cell wall. Consistent with this scenario, we demonstrate experimentally that translocation depends on both the dimensions of the cell envelope and the length of the disordered protein, and that translocation is reversible. We also show that disordered regions from eukaryotic nuclear pore complex proteins are capable of entropy-driven translocation through Gram-positive cell walls. These observations suggest that entropic forces alone, rather than chaperones or chemical energy, are sufficient to drive translocation of certain Gram-positive surface proteins for exposure on the outer surface of the cell wall.

biophysics↗

bioRxiv2018

Chiral twisting in cytoskeletal polymers regulates filament size and orientation

While cytoskeletal proteins in the actin family are structurally similar, as filaments they act as critical components of diverse cellular processes across all kingdoms of life. In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its physiological function. To bridge this gap, we performed molecular dynamics simulations for Caulobacter crescentus MreB and then utilized a coarse-grained biophysical model to successfully predict MreB filament properties in vivo. We discovered that MreB double protofilaments exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting determines the limit length and orientation of MreB filaments in vivo. Membrane binding of MreB also induces a stable membrane curvature that is physiologically relevant. Together, our data empower the prediction of cytoskeletal filament size from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular functions.

biophysics↗

bioRxiv2018

Cargo diffusion shortens single-kinesin runs at low viscous drag

Molecular motors are mechanoenzymes that actively drive long-range transport in cells. Thermal diffusion of the cargo can result in mechanical load on the motor carrying the cargo; the direction of this diffusion-based load is not correlated with motor motion. Recent single molecule-based experiments highlighted a strong asymmetric dependence of the run length of the single kinesin-1 motor on load direction, raising the intriguing possibility that thermal diffusion of the cargo may non-trivially influence the run length of the motor carrying the cargo. To test this possibility, here we employed Monte Carlo-based stochastic simulations to evaluate the transport of single-kinesin cargos over a large parameter space of physiologically relevant solution viscosities, cargo sizes, and motor velocities. Our simulations uncovered a previously unexplored, significant shortening effect of cargo diffusion on single-kinesin run length. This effect is non-monotonically influenced by viscous drag force on the cargo, which biases the effect of cargo diffusion toward the hindering direction. The non-monotonic variation of cargo run length with drag force is the direct result of the asymmetric response of kinesins run length to load direction. Our findings may be important for understanding the diverse characteristics of cargo transport, including run length, observed in living cells.

biophysics↗