Biology subjects

Oakes, P. W.

Publications and source records attributed to Oakes, P. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Disease-associated MYH11 tail variants disrupt vascular smooth muscle myosin II filament dynamics and cell mechanics

Smooth muscle myosin II (SMII), encoded by MYH11, is the principal motor driving vascular smooth muscle cell (VSMC) contraction. How disease-associated MYH11 variants alter SMII behavior in living cells remains poorly understood. We directly visualized two human MYH11 tail variants in VSMCs: pathogenic K1256del and a variant of uncertain significance A1839V, identified in a patient with severe aortoiliac aneurysmal disease. Both variants reduced SMII filament assembly and VSMC contractile output, but with distinct severity. A1839V predominantly retained filamentous organization and displayed partial agonist-induced stabilization, consistent with an intermediate cellular phenotype. In contrast, K1256del severely disrupted filament architecture and behavior, including reduced focal adhesion organization, increased SMII network flow and accelerated filament exchange. Furthermore, K1256del abolished the filament stabilization response to induced contraction. These findings identify failure of SMII filament stabilization as a cellular mechanism linking MYH11 variation to impaired VSMC mechanics. More broadly, they show that direct live-cell phenotyping can distinguish variant-specific defects in the contractile machinery, providing a scalable functional strategy to complement genetic classification and prioritize MYH11 variants for deeper mechanistic and in vivo study.

cell biology↗

bioRxiv2026

Mechanically-induced Septin Networks Protect Nuclear Integrity

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remains unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases the likelihood of nuclear membrane rupture during confinement. Together these data suggest that septins act as a dynamic mechanosensitive protective mechanism to buffer mechanical forces on the nucleus.

cell biology↗

bioRxiv2025

A mechanical origin for implantation defects in embryos from aged females

Women over 35 experience a marked reduction in fertility. The origin of these fertility defects appears to reside in the implantation capacity of the embryo itself, but the mechanistic basis of this impairment is not well-understood. Here, we identify a core mechanical defect in embryos from aged mothers that impairs the process of implantation. Using mouse models, we find that reproductive aging yields increased contractility in the extra-embryonic trophectoderm, the outer epithelial tissue responsible for mediating uterine attachment and embryo implantation. This hypercontractile state elevates tissue surface tension and viscosity in the blastocyst, culminating in defective spreading during implantation. Enhanced contractility is necessary and sufficient for this age-related defect in implantation, and early embryo mechanics can be used to predict successful implantation for embryos from both young and aged mothers. Our work represents a potential foundation for improving embryo selection in Assisted Reproductive Technologies to resolve age-related defects in female fertility.

developmental biology↗

bioRxiv2025

Local RhoA activation induces septin recruitment

The regulation of the actin cytoskeleton is key for controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA, to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.

cell biology↗

bioRxiv2025

LIM Domain Proteins link molecular and global tension by recognizing strained actin in adhesions

Mechanotransduction is fundamental to cell signaling and depends on force-sensitive adhesion proteins. How these proteins differentiate and integrate their responses to tension remains an open question. We show mechanosensitive LIM domain proteins like zyxin detect global adhesion tension by recognizing strained actin within these structures. In sharp contrast, vinculin localization and intramolecular tension remain unchanged, despite vinculins well-documented role in mechanotransduction. This reveals a stark disconnect between molecular tension and global tension in adhesions. We further show tension-dependent localization is specific to LIM domain proteins that recognize strained actin and extends to LIM proteins at cell-cell junctions, suggesting a common mechanotransduction mechanism. Finally, we show zyxins tension-dependent adhesion localization stabilizes actin and recruits VASP to promote stress fiber polymerization, identical to its role in stress fiber repair. Our findings reveal a fundamental role for LIM domain protein force-sensing in adhesions and highlight the non-linear connection between molecular and global tension.

cell biology↗

bioRxiv2023

T cells Use Focal Adhesions to Pull Themselves Through Confined Environments

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical composition. Their migration has classically been defined as amoeboid under the assumption that it is integrin-independent. Here we show that activated primary Th1 T cells require both confinement and extracellular matrix protein to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cell preferentially follows tracks of other T cells, suggesting that these adhesions are modifying the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated adhesions play a key role in T cell motility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/562587v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@8ba84aorg.highwire.dtl.DTLVardef@d5a504org.highwire.dtl.DTLVardef@44d104org.highwire.dtl.DTLVardef@1f68df5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Limiting Pool and Actin Architecture Controls Myosin Cluster Sizes in Adherent Cells

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.

biophysics↗

bioRxiv2023

Local Monomer Levels and Established Filaments Potentiate Non-Muscle Myosin 2 Assembly

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

cell biology↗

bioRxiv2023

Non-muscle myosin 2 filaments are processive in cells

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

cell biology↗

bioRxiv2022

Smooth Muscle Myosin 2 Filaments Dynamically Assemble and Stabilize During Induced Contractility

BackgroundVascular smooth muscle cells (SMCs) dynamically tune blood vessel diameter to regulate blood pressure, provide vessel wall structural integrity, and absorb shock on a beat-to-beat timescale. Smooth muscle myosin 2 (SMII) is the dominant motor protein driving SMC contraction. To function, SMII monomers dynamically assemble into filaments, which associate with the actin cytoskeleton to drive contractility. Precisely how SMII filaments assemble and exchange in living SMCs, however, both at steady-state and during induced contractility, remains poorly defined. MethodsWe used a single-cell filament assembly assay to determine SMII assembly into filaments at steady-state and upon induced contractility in rat aortic SMCs (A7R5) transiently-expressing EGFP-tagged SM1A isoform of SMII. We then used fluorescence recovery after photobleaching (FRAP) to characterize SMII exchange kinetics at steady-state and upon induced contractility, and measured changes in force production using traction force microscopy. Finally, we developed a CRISPR knock-in EGFP-SMII murine model to quantify SMII dynamics at endogenous expression in primary SMCs and intact arterioles. ResultsWhile predominantly filamentous at baseline, induced contraction rapidly increased SMII filament assembly. FRAP revealed rapid SMII exchange kinetics, more similar to non-muscle myosin II than striated myosin II, and induced contractility consistently stabilized SMII filaments. Super-resolution imaging revealed SMII and non-muscle myosin II filament structures consistent with co-assembly. Endogenous EGFP-SMII in primary SMCs and intact arterioles paralleled cell culture studies with similar baseline exchange kinetics and activation-dependent stabilization. ConclusionsTogether, these data support a model in which SMII is surprisingly dynamic and co-assembles with non-muscle myosin II. Vascular SMC activation further increases SMII filament assembly while reducing filament exchange, consistent with stabilization of a dynamic SMII pool during force generation, which allows cells to dynamically adapt their overall contractility in response to environmental conditions. CLINICAL IMPLICATIONSSmooth muscle myosin II (SMII) is the principal contractile motor protein of vascular smooth muscle tissue, but its molecular behavior in living differentiated SMCs remains poorly defined. Here, we show that SMII is not a static contractile scaffold but highly dynamic. SMII exhibits dynamic exchange between the filament and monomeric forms at baseline, but becomes acutely stabilized during agonist-induced contraction, including in primary SMCs and intact arterioles at endogenous Myh11 expression. These findings suggest that vascular tone is tuned not only by activating pre-existing myosin filaments, but also by rapidly shifting SMII filament assembly levels and exchange rates. This framework is relevant to diseases in which SMC contractility and arterial wall mechanics are abnormal, including MYH11-associated thoracic aortic disease and related disorders of arterial stiffness or maladaptive vasomotor regulation. Although this study is mechanistic, it identifies SMII filament dynamics as a measurable layer of vascular regulation and a potential readout for future studies of pathogenic MYH11 variants, disease modeling, and therapies that alter myosin activation and/or cytoskeletal stability. O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/511341v2_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@d64a8forg.highwire.dtl.DTLVardef@b9000corg.highwire.dtl.DTLVardef@1b24a91org.highwire.dtl.DTLVardef@3d81be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Atypical peripheral actin band formation via overactivation of RhoA and Non-muscle myosin II in Mitofusin 2 deficient cells

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophillike cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After the initial spreading period, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and ER to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and Myosin light-chain kinase, causing an over-activation of non-muscle myosin II and a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The formation of the peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

cell biology↗

bioRxiv2021

A Workflow for Rapid Unbiased Quantification of Fibrillar Feature Alignment in Biological Images

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT - Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

biophysics↗

bioRxiv2021

RhoA mediates stress fiber strain site recognition by the LIM protein testin

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testins mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state.

cell biology↗

bioRxiv2020

TRM Integrins CD103 and CD49a Differentially Support Adherence and Motility After Resolution of Influenza Virus Infection

Tissue resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon re-exposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/{beta}7 and CD49a/CD29({beta}1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by two-weeks post-infection and that each integrin contributes a distinct function regulating CD8 T cell motility both in vitro and in vivo, with CD49a facilitating migration and CD103 limiting motility through tethering. These results demonstrate for the first time how CD103 and CD49a differentially impact adherence and migration in the tissue, likely affecting overall retention, maintenance of TRM, and host protection. Significance StatementCurrent influenza vaccination strategies require annual immunizations, with fairly low efficacy rates. One technique to improve protection against a greater breadth of influenza viruses is to elicit broadly cross-reactive cell-mediated immunity and generate a local population of cytotoxic T cells to respond to conserved regions of circulating viruses. However, this approach requires improved understanding of how these cells migrate within and attach to the tissue, in order to persist and offer long-term immunity. This study investigates how receptors on the T cell surface impact the cells ability to interact with the tissue and provide evidence for which of these receptors are essential for protection. Furthermore, these studies reveal functional in vivo mechanisms of cellular markers used to characterize TRM.

immunology↗

bioRxiv2019

Multiscale model of integrin adhesion assembly

The ability of adherent cells to form adhesions is critical to several phases of their physiology. The assembly of adhesions is mediated by several types of integrins. These integrins differ in physical properties, including rate of diffusion on the plasma membrane, rapidity of changing conformation from bent to extended, affinity for extracellular matrix ligands, and lifetimes of their ligand-bound states. However, the way in which nanoscale physical properties of integrins ensure proper adhesion assembly remains elusive. We observe experimentally that both beta-1 and beta-3 integrins localize in nascent adhesions at the cell leading edge. In order to understand how different nanoscale parameters of beta-1 and beta-3 integrins mediate proper adhesion assembly, we therefore develop a coarse-grained computational model. Results from the model demonstrate that morphology and distribution of nascent adhesions depend on ligand binding affinity and strength of pairwise interactions. Organization of nascent adhesions depends on the relative amounts of integrins with different bond kinetics. Moreover, the model shows that the architecture of an actin filament network does not perturb the total amount of integrin clustering and ligand binding; however, only bundled actin architectures favor adhesion stability and ultimately maturation. Together, our results support the view that cells can finely tune the expression of different integrin types to determine both structural and dynamic properties of adhesions.

biophysics↗

bioRxiv2018

Membrane-cytoskeleton mechanical feedback mediated by myosin-I controls phagocytic efficiency

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We have found that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we found that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a novel role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a model for a membrane-tension based feedback mechanism for phagocytic cup closure.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2017

The lamellipodium is a myosin independent mechanosensor

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Youngs modulus of [~]7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.\n\nSignificance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

biophysics↗

bioRxiv2017

Bimodal Spindle Orientation Drives Tissue Regularity in a Proliferating Epithelium

We investigated the relationship between proliferation and tissue topology in an epithelial tissue undergoing elongation. We found that cell division is not required for elongation of the early Drosophila follicular epithelium, but does drive the tissue towards optimal geometric packing. To increase tissue regularity, cell divisions are oriented in the planar axis, along the direction of tissue expansion. Planar division orientation is governed by apico-cortical tension, which aligns with tissue expansion but not with interphase cell shape elongation. Hertwigs Rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. We tested whether this observation could be explained by anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis. We found that Pins/Mud does not participate in planar division orientation. Rather, tension translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes - apical-basal and planar - is controlled by distinct, independent mechanisms in a proliferating epithelium.\n\nSummary StatementRegularity in a proliferating epithelium requires cells to divorce division orientation from interphase shape, which they accomplish by using distinct mechanisms to orient divisions in the apical-basal and planar axes.

developmental biology↗