Biology subjects

Ginsberg, M. H.

Publications and source records attributed to Ginsberg, M. H..

4 recordsLinked to original sources

bioRxiv2026

Talin-1 determines the direction of primary mouse neutrophils migrating in vivo

Talin-1 is essential for {beta}2 integrin activation in neutrophils, yet its dynamic behavior during neutrophil trafficking in vivo remains poorly understood. Here, we generated EGFP-talin1 knock-in mice, enabling real-time visualization of talin-1 dynamics under physiological conditions. EGFP-talin1 is robustly expressed and preserves without altering {beta}2 integrin expression and activation. Using total internal reflection fluorescence (TIRF) microscopy under flow, we found that talin-1 was rapidly recruited to the plasma membrane during rolling and accumulates further during neutrophil arrest. Intravital microscopy revealed highly dynamic and stage-specific talin-1 redistribution during luminal crawling, transendothelial migration, and interstitial migration. Talin-1 preferentially accumulated at endothelial contact sites during crawling and polarized toward the leading edge during directional migration. These findings establish EGFP-talin1 knock-in mice as platform for visualizing integrin-associated cytoskeletal dynamics in vivo and identify dynamic talin-1 polarization as a feature of neutrophil trafficking.

immunology↗

bioRxiv2025

RAP1-RHO small GTPase cross-talk mediates integrin-dependent and -independent platelet procoagulant response

Platelet adhesion and procoagulant activity are critical for primary and secondary hemostasis, respectively. The small GTPase RAP1 is a central regulator of platelet aggregation as it controls IIb{beta}3 integrin activation through direct interaction with the integrin adapter protein, TALIN-1 (Tln-1). In addition to their aggregation defect, activated platelets lacking RAP1 (Rap1mKO) exhibited a marked impairment in surface exposure of phosphatidylserine (PtdSer), a negatively charged phospholipid with procoagulant activity. However, the mechanisms by which RAP1 regulates PtdSer exposure are unclear. Here we investigated the hypothesis that RAP1 regulates platelet PtdSer exposure through cross-talk with small GTPases of the Rho family. Consistent with their defect in PtdSer exposure, Rap1mKO platelets showed reduced procoagulant activity in vitro and in vivo when compared to controls. Stimulated Rap1mKO platelets exhibited elevated RHOA-GTP levels, and inhibition of the RHOA effector, Rho associated coiled-coil kinase (ROCK), partially restored PtdSer exposure in these cells. A milder defect in PtdSer exposure was observed for platelets from Tln-1mR35/118E mice, i.e. mice with impaired RAP1-Tln-1 interaction but otherwise intact RAP1 signaling. ROCK inhibition fully restored PtdSer exposure in Tln-1mR35/118E platelets. Opening of the mitochondrial permeability transition pore, a cellular response critical to PtdSer exposure, was impaired in Rap1mKO platelets and restored by pretreatment of cells with the ROCK inhibitor. Our study provides first evidence that platelet RAP1 signaling affects hemostatic plug formation independent of its key role in platelet adhesion. Additionally, our studies strongly suggest that RAP1 regulates PtdSer exposure and procoagulant activity in a RHOA/integrin-dependent and -independent manner.

cell biology↗

bioRxiv2025

A CD25-CCR7 complex initiates non-canonical IL-2 signaling

IL-2, a central regulator of immune function, binds to its receptor subunit CD25 (IL-2R), promoting IL-2 interaction with {beta} and {gamma} subunits to trigger the canonical IL-2 signaling pathway. An anti-mouse CD25 antibody, PC61, triggers alternative IL-2 signaling, leading to integrin activation. PC61 induces a complex formed by the IL-2-dependent association of CD25 with CCR7, suggesting that the formation of this complex initiates alternative IL-2 signaling. Here, we used structure-based design together with combinatorial screening to identify an IL-2 mutant (denoted IL-2(E52K)) that spares canonical IL-2 signaling but disrupts both PC61-induced complex formation and integrin activation while retaining the full CD25 affinity of the parent molecule. We also report that heparan sulfate (HS), a physiological ligand of IL-2 that triggers alternative signaling, induced IL-2-dependent CD25-CCR7 association, whereas IL-2(E52K) failed to support both HS-induced CD25-CCR7 complex formation and integrin activation. Thus, both anti-CD25 antibody and HS require common features of IL-2 needed for CD25-CCR7 complex assembly and resulting integrin activation. Collectively, these data show that IL-2 promotes CD25 interaction with CCR7, thereby forming the signal initiating complex. Furthermore, canonical and alternative IL-2 signaling can be decoupled by an IL-2 mutation, creating a tool to specify the biological role of alternative IL-2 signaling in immune responses.

cell biology↗

bioRxiv2024

Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

How genetic lesions drive cell transformation and whether they can be circumvented without compromising function of non-transformed cells are enduring questions in oncology. Here we show that in mature T cells--in which physiologic clonal proliferation is a cardinal feature-- constitutive MYC transcription and Tsc1 loss in mice modeled aggressive human malignancy by reinforcing each others oncogenic programs. This cooperation was supported by MYC-induced large neutral amino acid transporter chaperone SLC3A2 and dietary leucine, which in synergy with Tsc1 deletion overstimulated mTORC1 to promote mitochondrial fitness and MYC protein overexpression in a positive feedback circuit. A low leucine diet was therapeutic even in late-stage disease but did not hinder T cell immunity to infectious challenge, nor impede T cell transformation driven by constitutive nutrient mTORC1 signaling via Depdc5 loss. Thus, mTORC1 signaling hypersensitivity to leucine as an onco-nutrient enables an onco-circuit, decoupling pathologic from physiologic utilization of nutrient acquisition pathways.

cancer biology↗