Biology subjects

Bear, J.

Publications and source records attributed to Bear, J..

6 recordsLinked to original sources

bioRxiv2026

Arp2/3-mediated turnover of large clathrin lattices is regulated through the tyrosine kinase ACK

Clathrin mediated endocytosis (CME) is a vital cellular process that mediates cell signaling by controlling the internalization of extracellular cargo and activated receptors. Arp2/3-branched actin provides force to assist in membrane invagination and scission in CME. Loss of Arp2/3-branched actin in conditional Arpc2 KO fibroblasts results in an increase of large arrested clathrin lattices (LACLs) visualized by live-cell TIRF imaging. Additional structural details of LACLs were revealed using electron microscopy and include an increase in arrested clathrin lattices of various curvatures. Numerous CME proteins have heightened levels of tyrosine phosphorylation at these LACLs in Arpc2 KO cells. We identified the non-receptor tyrosine kinase ACK (Activated Cdc42-Associated Kinase) as a key upstream regulator of LACL turnover. CRISPR KO of ACK abrogates LACL tyrosine phosphorylation and impairs cells capacity to resolve LACLs. Our results support a model where ACK recruitment and activation at LACLs drives branched actin formation to help resolve large accumulations of arrested CME structures.

Cell Biology↗

bioRxiv2026

TRIM9 switches the morphological phenotype of melanoma cells

Melanoma is a highly plastic cancer characterized by distinct cellular phenotypes associated with broadly unique gene expression profiles. TRIM9 is a brain-enriched E3 ubiquitin ligase detected in melanoma, but how TRIM9 expression regulates melanoma phenotype is unknown. Using two metastatic human melanoma cell lines and a mouse melanoma model, we found that TRIM9 promoted melanoma proliferation and altered cell morphology. In cell lines, TRIM9 promoted cellular blebbing and negatively regulated adhesion, secretion, and mesenchymal motility. TRIM9 interacted with VASP in melanoma cells, altering VASP modification, localization, and dynamics. In the absence of TRIM9, cells had an altered actin organization and more focal adhesions, where VASP accumulated and exhibited rapid turnover. We find the alterations in actin architecture and adhesion associated with TRIM9 deletion were coincident with increased motile and contractile mesenchymal behavior in vitro. In vivo loss of TRIM9 in melanoma slowed tumor growth and altered metastasis frequency, size, and destination. Our findings indicate TRIM9 alters the proliferative and morphological phenotypes of metastatic melanoma cells to influence disease progression.

cancer biology↗

bioRxiv2022

Zyxin and non-muscle myosin are required for single fibroblast durotaxis, but Rho-kinase activity and the Arp2/3 complex are dispensable

Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.

cell biology↗

bioRxiv2021

Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Proteinco-immunization in rhesus macaques

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The DNA-only vaccine regimens were compared to a regimen that included co- immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques. Author summaryAnti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2.

immunology↗

bioRxiv2020

An optogenetic switch for the Set2 methyltransferase provides evidence for rapid transcription-dependent and independent dynamics of H3K36 methylation

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the short-term genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light activated nuclear shuttle (LANS) domain. Early H3K36me3 dynamics identified rapid methylation in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes exhibited disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting a rate-limiting mechanism for H3K36me3 deposition. Removal H3K36me2/3 was also rapid and highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner.

molecular biology↗

bioRxiv2018

Lysophosphatidic acid provokes fibroblast chemotaxis through combinatorial regulation of myosin II

Lysophophatidic acid (LPA), a biologically active phospholipid that is ubiquitously present in tissues and organs, provokes cellular responses such as proliferation, apoptosis, differentiation and migration via activation of G-protein coupled receptors. These receptors activate a broad range of intracellular signaling cascades to mediate these responses. Using microfluidic chambers that generate and maintain stable gradients, we observed that chemotaxis of fibroblasts to LPA has higher directional fidelity than chemotaxis provoked by the receptor tyrosine kinase (RTK) ligand platelet-derived growth factor (PDGF). Unlike fast moving amoeboid cells, mesenchymal cells such as fibroblasts do not require PI3K for chemotaxis to a GPCR ligand. In addition, the Arp2/3 complex is not required for fibroblast GPCR-based chemotaxis in either 2D or 3D environments. Our data indicate that combinatorial regulation of myosin II involving global activation by RhoA/ROCK and local inhibition of myosin II at the leading edge by PKC results in highly efficient chemotaxis of fibroblasts to LPA. Based on these observations, we develop a simple mathematical model to explain how dual regulation of myosin II is responsible for enhanced chemotaxis in LPA gradients relative to PDGF. Using pharmacological approaches, we test predictions of this model and modulate the fidelity of LPA and PDGF chemotaxis.

cell biology↗