Biology subjects

Bear, J. E.

Publications and source records attributed to Bear, J. E..

13 recordsLinked to original sources

bioRxiv2026

Nuclear SUN2 coordinates endothelial cell-matrix interactions to regulate blood vessel homeostasis and barrier function

Vascular endothelial cells respond to environmental forces to remodel vessels during development and to achieve homeostasis, and mis-regulated responses lead to vascular dysfunction and disease. The nucleus participates in force transduction to cell-matrix junctions via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that resides in the nuclear envelope, but how these forces are regulated and relayed is incompletely understood. We found that the LINC complex protein SUN2 is required for proper endothelial cell-matrix interactions that occur far from the nucleus and affect angiogenic expansion, vascular responses to flow, and barrier integrity. Endothelial cells lacking SUN2 had inappropriate flow responses and reduced expression of flow-mediated transcription factors in vitro and in vivo. Expression of several matrix and adhesion genes was reduced in SUN2-depleted cells, leading to defective extracellular matrix, dysmorphic focal adhesions resistant to dynamic turnover, and disturbed cell-matrix force distribution. Mechanistically, nuclear SUN2 affected dynamic regulation of the microtubule cytoskeleton that correlated with matrix metalloprotease-dependent barrier dysfunction. These findings indicate that nuclear SUN2 establishes and maintains blood vessel homeostasis by controlling microtubule-mediated effects on focal adhesion turnover and extracellular matrix properties, with implications for cardiovascular aging and diseases such as Marfan syndrome that affect vessel wall integrity.

cell biology↗

bioRxiv2026

Quantitative analysis of fibroblast migration reveals migratory states characterized by force generation, cell shape and motion

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

cell biology↗

bioRxiv2026

Actin-membrane interface stress regulates Arp2/3-branched actin density during lamellipodial protrusion

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous branched actin in cells. Here, we utilize mouse CRISPR knock-in fibroblasts with labeled Arp2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of predicted interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that Arp2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of Arp2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility utilizing cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

cell biology↗

bioRxiv2025

SureCLIP and DrCLIP: Genetic manipulation-free, generalizable interactome mapping for cells and zebrafish

Unbiased interactome mapping currently relies on two types of approaches: co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and proximity-based tagging. Although co-IP/MS does not require genetic manipulation - thereby avoiding artifacts from exogenous protein expression and the time needed to generate stable cell or animal lines - it is increasingly being supplanted by proximity labeling techniques due to lower sensitivity and limited robustness. These drawbacks largely arise from the non-covalent nature of protein-protein interactions (PPIs) and carryover of bait and immunoglobulin (IgG) proteins in the final eluate. Here, we present a reversible crosslinker-mediated co-IP/MS workflow for endogenous proteins that (1) obviates the need for extensive lysis, wash, and elution optimization, (2) minimizes bait and IgG co-elution, and (3) captures several hundreds of interactors. We first describe subcellular-resolution, reversible crosslinker-mediated co-immunoprecipitation (SureCLIP), which enables PPI mapping of Hic-5, a focal adhesion protein, and lamin B1, a nuclear envelope protein, in cultured cells. We then introduce DrCLIP, a Danio rerio adaptation of SureCLIP, which allows comprehensive mapping of PPIs in zebrafish larvae. Using this approach, we profiled the kindlin-2 interactome in wild-type embryos without genetic manipulation. Both SureCLIP and DrCLIP exhibit high sensitivity and enable identification of novel interactors, providing a generalizable and powerful platform for defining native interactomes in 2D culture and whole animals.

cell biology↗

bioRxiv2025

Optogenetic control of PLC-γ1 activity polarizes cell motility

Phospholipase C-{gamma}1 (PLC-{gamma}1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-{gamma}1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type PLC-{gamma}1 (OptoPLC-{gamma}1) in Plcg1-null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-{gamma}1, we demonstrate that a hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-{gamma}1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-{gamma}1 S345F polarizes cell motility and migration on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-{gamma}1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-{gamma}1 regulation and demonstrate that local activation of PLC-{gamma}1 is sufficient to direct cell motility.

cell biology↗

bioRxiv2025

Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis

Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role for the actin binding protein Coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrindependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain enriched E3 ubiquitin ligase that interacts with a netrin receptor and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was not able to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.

cell biology↗

bioRxiv2025

The NHSL1-A complex interacts with the Arp2/3 complex and controls cell migration efficiency and chemotaxis.

Cell migration is crucial for development and deregulation causes diseases. The Scar/WAVE complex promotes mesenchymal cell migration through Arp2/3 mediated lamellipodia protrusion. We previously discovered that all isoforms of Nance-Horan Syndrome-like 1 (NHSL1) protein interact directly with the Scar/WAVE complex and the NHSL1-F1 isoform negatively regulates Scar/WAVE-Arp2/3 activity thereby inhibiting 2D random cell migration. Here, we investigate the NHSL1-A1 isoform, which contains a Scar homology domain (SHD). The SHD in Scar/WAVE mediates the formation of the Scar/WAVE complex. We found that the SHD of NHLS1-A is sufficient for the formation of an NHSL1-A complex composed of the same proteins as the Scar/WAVE complex, but NHSL1-A replaces Scar/WAVE. NHSL1-A SHD recruits the NHSL1-A complex to lamellipodia, where also the Scar/WAVE complex resides. Scar/WAVE contains a WCA domain, which is phosphorylated by CK2 and recruits and activates the Arp2/3 complex to nucleate branched actin networks supporting lamellipodial protrusion. We identified a WCA domain in NHSL1 which interacts with the Arp2/3 complex. The NHSL1 WCA domain is phosphorylated by GSK3, and this increases the interaction with the Arp2/3 complex. In contrast to NHSL1-F1, the NHSL1-A complex promotes cell migration speed but not cell persistence via the Scar/WAVE complex and potentially via its WCA domain. In addition, the NHSL1-A complex is required for chemotaxis. Mechanistically, the NHSL1-A complex may increase lamellipodial Arp2/3 activity and lamellipodial speed while reducing lamellipodial persistence. Our findings reveal an additional layer of Arp2/3 complex control essential for mesenchymal cell migration highly relevant for development and disease.

cell biology↗

bioRxiv2024

Measurement of cellular traction forces during confined migration

To migrate efficiently through tissues, cells must transit through small constrictions within the extracellular matrix. However, in vivo environments are geometrically, mechanically, and chemically complex, and it has been difficult to understand how each of these parameters contribute to the propulsive strategy utilized by cells in these diverse settings. To address this, we employed a sacrificial micromolding approach to generate polymer substrates with tunable stiffness, controlled adhesivity, and user-defined microscale geometries. We combined this together with live-cell imaging and three-dimensional traction force microscopy (TFM) to quantify the forces that cells use to transit through constricting channels. Surprisingly, we observe that cells migrating through compliant constrictions take longer to transit and experience greater nuclear deformation than those migrating through more rigid constrictions. TFM reveals that this deformation is generated by inwardly directed contractile forces that decrease the size of the opening and pull the walls closed around the nucleus. These findings show that nuclear deformation during confined migration can be accomplished by internal cytoskeletal machinery rather than by reactive forces from the substrate, and our approach provides a mechanism to test between different models for how cells translocate their nucleus through narrow constrictions. The methods, analysis, and results presented here will be useful to understand how cells choose between propulsive strategies in different physical environments. Significance StatementCell migration is critical for both physiological events like wound healing and pathological events like metastasis. Understanding how cells move through complex environments will assist efforts to enhance or inhibit such processes. We developed a method to quantify the forces that cells use to move through multidimensional environments, including through narrow constrictions like those in tissues. Surprisingly, we find that cells transiting through soft constrictions take longer and deform more than those transiting through rigid constrictions, and we connect this finding to inwardly directed contractile forces generated by migrating cells. Together, this work reveals a key role for substrate rigidity to regulate cell transit through confining geometries and provides a quantitative platform to investigate similar processes in other settings.

biophysics↗

bioRxiv2023

Control of microtubules in neuronal processes by profilin 1 and actomyosin

Microtubules, intermediate filaments, and actin are cytoskeletal polymer networks found within the cell. While each has unique functions, all the cytoskeletal elements must work together for cellular mechanics to be fully operative. This is achieved through crosstalk mechanisms whereby the different networks influence each other through signaling pathways and direct interactions. Because crosstalk can be complex, it is possible for perturbations in one cytoskeletal element to affect the others in ways that are difficult to predict. Here we investigated how long-term changes to the actin cytoskeleton affect microtubules and intermediate filaments. Reducing F-actin or actomyosin contractility increased acetylated microtubules and intermediate filament expression, with the effect being significantly more pronounced in neuronal processes. Changes to microtubules were completely reversible if F-actin and myosin activity is restored. Moreover, the altered microtubules in neuronal processes resulting from F-actin depletion caused significant changes to microtubule-based transport, mimicking phenotypes that are linked to neurodegenerative disease. Thus, defects in actin dynamics cause a compensatory response in other cytoskeleton components which profoundly alters cellular function.

cell biology↗

bioRxiv2023

The actin binding protein profilin 1 is critical for mitochondria function

Profilin 1 (PFN1) is an actin binding protein that is vital for the polymerization of monomeric actin into filaments. Here we screened knockout cells for novel functions of PFN1 and discovered that mitophagy, a type of selective autophagy that removes defective or damaged mitochondria from the cell, was significantly upregulated in the absence of PFN1. Despite successful autophagosome formation and fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells still accumulate damaged, dysfunctional mitochondria. Subsequent imaging and functional assays showed that loss of PFN1 significantly affects mitochondria morphology, dynamics, and respiration. Further experiments revealed that PFN1 is located to the mitochondria matrix and is likely regulating mitochondria function from within rather than through polymerizing actin at the mitochondria surface. Finally, PFN1 mutants associated with amyotrophic lateral sclerosis (ALS) fail to rescue PFN1 knockout mitochondrial phenotypes and form aggregates within mitochondria, further perturbing them. Together, these results suggest a novel function for PFN1 in regulating mitochondria and identify a potential pathogenic mechanism of ALS-linked PFN1 variants.

cell biology↗

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↗

bioRxiv2022

Viral Airway Injury Promotes Cell Engraftment in an In Vitro Model of Cystic Fibrosis Cell Therapy

Cell therapy is a potential treatment for cystic fibrosis (CF). However, cell engraftment into the airway epithelium is challenging. Here, we model cell engraftment in vitro using the air-liquid interface (ALI) culture system by injuring well-differentiated CF ALI cultures and delivering non-CF cells at the time of peak injury. Engraftment efficiency was quantified by measuring chimerism by droplet digital PCR and functional ion transport in Ussing chambers. Using this model, we found that human bronchial epithelial cells (HBECs) engraft more efficiently when they are cultured by conditionally reprogrammed cell (CRC) culture methods. Cell engraftment into the airway epithelium requires airway injury, but the extent of injury needed is unknown. We compared three injury models and determined that severe injury with partial epithelial denudation facilitates long-term cell engraftment and functional CFTR recovery up to 20% of wildtype function. The airway epithelium promptly regenerates in response to injury, creating competition for space and posing a barrier to effective engraftment. We examined competition dynamics by time-lapse confocal imaging and found that delivered cells accelerate airway regeneration by incorporating into the epithelium. Irradiating the repairing epithelium granted engrafting cells a competitive advantage by diminishing resident stem cell proliferation. Intentionally causing severe injury to the lungs of people with CF would be dangerous. However, naturally occurring events like viral infection can induce similar epithelial damage with patches of denuded epithelium. We found that viral preconditioning promoted effective engraftment of cells primed for viral resistance.

cell biology↗

bioRxiv2020

What the Zebrafish's Eye Tells the Zebrafish's Brain: Retinal Ganglion Cells for Prey Capture and Colour Vision

In vertebrate vision, the tetrachromatic larval zebrafish permits non-invasive monitoring and manipulating of neural activity across the nervous system in vivo during ongoing behaviour. However, despite a perhaps unparalleled understanding of links between zebrafish brain circuits and visual behaviours, comparatively little is known about what their eyes send to the brain in the first place via retinal ganglion cells (RGCs). Major gaps in knowledge include any information on spectral coding, and information on potentially critical variations in RGC properties across the retinal surface to acknowledge asymmetries in the statistics of natural visual space and behavioural demands. Here, we use in vivo two photon (2P) imaging during hyperspectral visual stimulation as well as photolabeling of RGCs to provide the first eye-wide functional and anatomical census of RGCs in larval zebrafish. We find that RGCs functional and structural properties differ across the eye and include a notable population of UV-responsive On-sustained RGCs that are only found in the acute zone, likely to support visual prey capture of UV-bright zooplankton. Next, approximately half of RGCs display diverse forms of colour opponency - long in excess of what would be required to satisfy traditional models of colour vision. However, most information on spectral contrast was intermixed with temporal information. To consolidate this series of unexpected findings, we propose that zebrafish may use a novel "dual-achromatic" strategy segregated by a spectrally intermediate background subtraction system. Specifically, our data is consistent with a model where traditional achromatic image-forming vision is mainly driven by long-wavelength sensitive circuits, while in parallel UV-sensitive circuits serve a second achromatic system of foreground-vision that serves prey capture and, potentially, predator evasion.

neuroscience↗