SummaryNon-muscle myosin II (MyoII) contractility is central to the regulation of numerous cellular processes, including migration. Rho is a well-characterized modulator of actomyosin contractility, but the function of other GTPases, such as Rac, in regulating contractility is currently not well understood. Here, we show that activation of Rac by the guanine nucleotide exchange factor Asef2 (also known as SPATA13) impairs migration on type I collagen through a MyoII-dependent mechanism that enhances contractility. Knockdown of endogenous Rac or treatment of cells with a Rac-specific inhibitor decreases the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Moreover, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. In addition, Asef2 slows the turnover of adhesions in protrusive regions of cells by promoting large mature adhesions, which has been linked to actomyosin contractility, with increased amounts of active b1 integrin. Hence, our data reveal a new role for Rac activation, promoted by Asef2, in modulating actomyosin contractility, which is important for regulating cell migration and adhesion dynamics.

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

#VanguardSTEM is an online community and platform that centers women, girls, and non-binary people of color in science, technology, engineering, and mathematics (STEM) fields. We publish original and curated content, using cultural production to include a multiplicity of identities as worthy of recognition and thus redefine STEM identity and belonging. #VanguardSTEM is rooted firmly in Queer, Black feminisms which delineate the experiences and critiques of Black women matter and that these insights can foster a restorative and regenerative construction of the cultures in which we exist. In describing how #VanguardSTEM descended from counterspaces, we draw on speculative fiction to define a #VanguardSTEM hyperspace as a fluid “place-time” that is born digital and enabled by social media, but materializes in the physical world for specific purposes. As Black women in STEM, we consider how our situated knowledges and scientific expertise inform our process. We propose an intersectional scientific methodology to address the influence of embodied observation, embedded context and collective impact on scientific inquiry. Through #VanguardSTEM, we assert, without apology, the right of Black, Indigenous, women of color to self-advocate by fully representing ourselves, our STEM identities and interests, without assimilation.

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.