Biology subjects

Fowler, V. M.

Publications and source records attributed to Fowler, V. M..

11 recordsLinked to original sources

bioRxiv2025

Cyclase associated actin cytoskeleton regulatory protein 2 (CAP2) organizes the actin cytoskeleton to influence the biomechanical properties of the ocular lens

Cyclase-associated actin cytoskeleton regulatory protein 2 (CAP2) is a conserved actin-binding protein that promotes actin filament (F-actin) turnover by disassembling ADF/cofilin-decorated filaments, supporting F-actin remodeling in differentiating cells and tissues. In the ocular lens, the actin cytoskeleton is critical for maintaining tissue biomechanical properties during fiber cell maturation. To assess CAP2s role in the lens, we examined lens-specific CAP2 knockout (CAP2cKO) mice at cellular and tissue levels. CAP2cKO lenses were normal in size, shape, and transparency but exhibited increased stiffness under compression and enhanced recovery after load removal. While total actin levels and F-actin were unchanged, immunofluorescence revealed higher levels of Tropomodulin 1 (Tmod1, F-actin pointed-end capping), Tropomyosin3.5 (Tpm3.5, F-actin stabilizing), and T-plastin (F-actin bundling) in F-actin-rich membrane protrusions of mature fibers, while -actinin-1 (F-actin cross-linking) was reduced. These findings suggest that CAP2 loss disrupts F-actin remodeling, promoting filament stabilization through Tpm3.5 binding, Tmod1 capping and T-plastin bundling. Consequently, F-actin networks become stiffer and more resilient, altering lens biomechanics. This study provides the first evidence that CAP2 regulates cell biomechanical properties in a non-muscle tissue through modulation of F-actin-associated proteins. Summary statementCAP2 loss alters the actin cytoskeleton through increased F-actin stabilization by Tmod1 and Tpm3.5, and filament bundling by T-plastin, leading to stiffer lenses.

cell biology↗

bioRxiv2025

MYOSIN IIA MOTOR REGULATES ATTACHING-EFFACING BACTERIA INTERACTIONS WITH INTESTINAL EPITHELIUM

Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium, colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton.

cell biology↗

bioRxiv2024

A novel isoform of Tensin1 promotes actin filament assembly for efficient erythroblast enucleation

Mammalian red blood cells are generated via a terminal erythroid differentiation pathway culminating in cell polarization and enucleation. Actin filament polymerization is critical for enucleation, but the molecular regulatory mechanisms remain poorly understood. We utilized publicly available RNA-seq and proteomics datasets to mine for actin-binding proteins and actin- nucleation factors differentially expressed during human erythroid differentiation and discovered that a focal adhesion protein--Tensin-1--dramatically increases in expression late in differentiation. Remarkably, we found that differentiating human CD34+ cells express a novel truncated form of Tensin-1 (eTNS1; Mr [~]125 kDa) missing the N-terminal half of the protein, due to an internal mRNA translation start site resulting in a unique exon 1. eTNS1 localized to the cytoplasm during terminal erythroid differentiation, with no apparent membrane association or focal adhesion formation. Knocking out eTNS1 had no effect on assembly of the spectrin membrane skeleton but led to impaired enucleation and absent or mis-localized actin filament foci in enucleating erythroblasts. We conclude that eTNS1 is a novel regulator of actin filament assembly during human erythroid terminal differentiation required for efficient enucleation.

cell biology↗

bioRxiv2021

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

cell biology↗

bioRxiv2021

Nanoscale organization of Actin Filaments in the Red Blood Cell Membrane Skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes interconnected by long spectrin molecules at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modelling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization of F-actin nodes in the network in situ is not understood. Here, we examined F-actin distribution in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci [~]200-300 nm apart interspersed with dimmer F-actin staining regions. Live cell imaging reveals dynamic lateral movements, appearance and disappearance of F-actin foci. Single molecule STORM imaging and computational cluster analysis of experimental and synthetic data sets indicate that individual filaments are non-randomly distributed, with the majority as multiple filaments, and the remainder sparsely distributed as single filaments. These data indicate that F-actin nodes are non-uniformly distributed in the spectrin-F-actin network and necessitate reconsideration of current models of forces accounting for RBC shape and membrane deformability, predicated upon uniform distribution of F-actin nodes and associated proteins across the micron-scale RBC membrane.

biophysics↗

bioRxiv2021

Microstructural determinants of lens stiffness in rat versus guinea pig lenses

Proper ocular lens function requires lens biomechanical flexibility which is lost in presbyopia during aging. As increasing lens size has been shown previously to correlate with lens biomechanical stiffness in aging, we tested the hypothesis that whole lens size determines gross biomechanical stiffness. We used an allometric approach to evaluate this hypothesis by comparing lenses from three rodent species (mouse, rats and guinea pigs) of varying size. While rat lenses are larger and stiffer than mouse lenses, guinea pig lenses are even larger than rat lenses but are softer than the rat lens. This indicates that lens size is not a sole determinant of lens stiffness and disproves our hypothesis. Therefore, we investigated the scaling of lens microstructural features that could potentially explain the differences in biomechanical stiffness between rat and guinea pig lenses, including lens capsule thickness, epithelial cell area, fiber cell widths, suture organization, and nuclear size. Capsule thickness, epithelial cell area, and fiber cell widths scaled with lens size (i.e., greater in guinea pig lenses than rats), indicating that sizes of these features do not correlate with the stiffness of rat lenses, while suture organization was similar between rats and guinea pigs. However, we found that the hard rat lens nucleus occupies a greater fraction of the lens than the guinea pig lens nucleus, suggesting a role for nuclear size in determining whole lens stiffness. Therefore, while many features contribute to lens biomechanical properties, the size of the lens nucleus with respect to the size of the lens could be a major determinant of lens stiffness in rats versus guinea pigs.

cell biology↗

bioRxiv2020

Proteomic and functional analyses of the periodic membrane skeleton in neurons

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of candidate MPS-interacting proteins that span diverse functional categories. We validated representative proteins in several of these categories, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, demonstrating periodic distributions of [~]20 proteins in neurons using super-resolution imaging. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide new insights into the interactome of the MPS, and suggest new functions of the MPS in neurons.

neuroscience↗

bioRxiv2019

Tpm3.1 association with actin stress fibers is required for lens epithelial to mesenchymal transition

PurposeEpithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Targeting specific, stress fiber associated tropomyosin in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT.\n\nMethodsWe identified Tpm isoforms in mouse immortalized lens epithelial cells and isolated whole lenses by semi-quantitative PCR followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To stimulate EMT, we cultured cells or native lenses in TGF{beta}2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by sma mRNA (qPCR) and protein (WES immunoassay) analysis.\n\nResultsLens epithelial cells express eight tropomyosin isoforms. Cell culture studies showed that TGF{beta}2 treatment results in an upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces sma in TGF{beta}2 treated cells. We confirmed the role of Tpm3.1 in lens epithelial cells in the native lens ex vivo. Culture of whole lenses in the presence of TGF{beta}2 results in stress fiber formation at the basal regions of epithelial cells. Knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial sma levels.\n\nConclusionTargeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to represses lens EMT.

cell biology↗

bioRxiv2018

Tropomyosin 3.5 protects F-actin networks required for tissue biomechanical properties

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to fine focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. The major mouse lens Tpm is Tpm3.5 (TM5NM5), a previously unstudied isoform. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and -actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that Tpm is essential for cell biomechanical stability in a load-bearing non-muscle tissue and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivo.\n\nSummaryTropomyosin 3.5 stabilizes F-actin in eye lens fiber cells and promotes normal tissue biomechanical properties. Tpm3.5 deficiency leads to F-actin network rearrangements and decreased lens stiffness and resilience.

cell biology↗

bioRxiv2018

The effects of mechanical strain on mouse eye lens capsule and cellular microstructure

The understanding of multiscale load transfer within complex soft tissues is incomplete. The eye lens is ideal for multiscale tissue mechanics studies as its principal function is to fine focus light at different distances onto the retina via mechanical shape changes. The biomechanical function, resiliency, and intricate microstructure of the lens make it an excellent non-connective soft tissue model. We hypothesized that compressive strain applied onto whole lens tissue leads to deformation of specific microstructures and that this deformation is reversible following removal of load. For this examination, mouse lenses were compressed by sequential application of increasing load. Using confocal microscopy and quantitative image analysis, we determined that axial strain [≥]10% reduces capsule thickness, expands epithelial cell area, and separates fiber cell tips at the anterior region of the lenses. At the equatorial region, strain [≥]6% increases fiber cell widths. The effects of strain on lens epithelial cell area, capsule thickness, and equatorial fiber cell widths are reversible following the release of lenses from strain. However, although fiber cell tip separation following the removal of low loads is reversible, the separation becomes irreversible with application of higher loads. This irreversible separation between fiber cell tips leads to incomplete bulk lens resiliency. The lens is an accessible biomechanical model system that provides new insights on multiscale transfer of loads in soft tissues.

cell biology↗

bioRxiv2017

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

The biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylated in vivo in RBCs, indicating active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disc cell shape. As structures similar to the RBC membrane skeleton are conserved in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.\n\nSignificance statementThe biconcave disc shape and deformability of the mammalian RBC is vital to its circulatory function, relying upon a 2D viscoelastic spectrin-F-actin network attached to the membrane. A role for myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has never been tested. We show that NMIIA forms phosphorylated bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity is required for interactions with the spectrin-F-actin network, and regulates RBC biconcave shape and deformability. These results provide a novel mechanism for actomyosin force generation at the plasma membrane, and may be applicable to other cell types such as neurons and polarized epithelial cells with a spectrin-F-actin-based membrane skeleton.

cell biology↗