Biology subjects

Burkhardt, J. K.

Publications and source records attributed to Burkhardt, J. K..

7 recordsLinked to original sources

bioRxiv2025

S1P induces bleb-based T cell motility via S1PR1-dependent activation of RhoA and WNK1

In vivo, the chemokine CCL19 and its receptor CCR7 control T cell retention in lymph nodes, while the lipid chemoattractant spingosine-1-phosphate (S1P) drives T cell egress from lymphoid organs. CCL19 is known to activate actin polymerization at the leading edge of migrating cells, generating a mode of motility driven by lamellipodial protrusions. In contrast, we showed recently that S1P induces a transient lamellipodial response, followed by pressure-driven bleb-based motility. Here, we elucidate the mechanisms controlling S1P responses in naive T cells. We show that S1P signals through S1PR1, with coupling through Gai. In contrast to CCR7, which signals through Gai to induce sustained Rac1 activation, S1PR1 engagement yields only weak and transient Rac1 activation; the dominant response is sustained activation of RhoA. This pathway, together with a pathway involving phospholipase C and myosin light chain kinase, results in phosphorylation of myosin regulatory light chain (MLC) and enhanced myosin contractility. Inhibition of mTORC2 blocks MLC phosphorylation, consistent with evidence that tension sensing by mTORC2 can couple Rac1 and RhoA signaling during leukocyte migration. Surprisingly, although RhoA pathway inhibitors blocked S1P-induced MLC phosphorylation and blebbing, they failed to block S1P-dependent chemotaxis. This led to the identification of a second arm of the S1P response: WNK1-dependent phosphorylation of SPAK1 and OSXR1, proteins that regulate ion channels and water influx. Partial WNK1 inhibition, together with inhibition of myosin contractility, was sufficient to block S1P-induced blebbing and chemotaxis, indicating that S1P-driven T cell migration involves coordinate activation of myosin contractility and water influx. One sentence summaryS1P signals elicit sustained RhoA activation and water influx to drive bleb-based T cell motility.\

cell biology↗

bioRxiv2024

Long range mutual activation establishes Rho and Rac polarity during cell migration

In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are appropriately positioned at the opposite poles of migrating cells is unknown. Here we leverage optogenetics, manipulation of cell mechanics, and mathematical modeling to reveal a surprising mechanochemical long-range mutual activation of the front and back polarity programs that complements their well-known local mutual inhibition. Rac-based protrusion stimulates Rho activation at the opposite side of the cell via membrane tension-based activation of mTORC2. Conversely, Rho-based contraction induces cortical-flow-based regulation of phosphoinositide signaling to trigger Rac activation at the opposite side of the cell. We develop a minimal unifying mechanochemical model of the cell to explain how this long-range facilitation complements local inhibition to enable robust Rho and Rac partitioning. We show that this long-range mutual activation of Rac and Rho is conserved in epithelial cells and is also essential for efficient polarity and migration of primary human T cells, indicating the generality of this circuit. Our findings demonstrate that the actin cortex and plasma membrane function as an integrated mechanochemical system for long-range partitioning of Rac and Rho during cell migration and likely other cellular contexts.

cell biology↗

bioRxiv2024

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion

Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including fertilisation and the formation of placenta, muscle and osteoclasts, multinucleated cells that are unique in their ability to resorb bone. The mechanisms of osteoclast multinucleation involve dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. Here, we found that moesin, a cytoskeletal linker protein member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role in both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin inhibition favors their ability to fuse into multinucleated osteoclasts. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes (TNTs), F-actin-based intercellular bridges that we reveal here to trigger cell-cell fusion. Moesin also controls HIV-1- and inflammation-induced cell fusion. In addition, moesin regulates the formation of the sealing zone, the adhesive structure determining osteoclast bone resorption area, and thus controls bone degradation, via a {beta}3-integrin/RhoA/SLK pathway. Supporting our results, moesin-deficient mice present a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclast activity in bone disease therapy.

cell biology↗

bioRxiv2020

LFA-1 signals to promote actin polymerization and upstream migration in T cells

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the PI3K and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, while VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl, failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior. Summary StatementInflammatory responses require leukocyte migration along the vascular wall. We show that signaling from {beta}2, but not {beta}1, integrins induces cytoskeletal changes needed for upstream migration under shear flow.

cell biology↗

bioRxiv2019

CrkL is required for donor T cell migration to GvHD Target organs

The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

immunology↗

bioRxiv2019

Genetic immunodeficiency and autoimmune disease reveal distinct roles of Hem1 in the WAVE2 and mTORC2 complexes

Immunodeficiency often coincides with immune hyperresponsiveness such as autoimmunity, lymphoproliferation, or atopy, but the molecular basis of this paradox is typically unknown. We describe four families with immunodeficiency coupled with atopy, lymphoproliferation, cytokine overproduction, hemophagocytic lymphohistocytosis, and autoimmunity. We discovered loss-of-function variants in the gene NCKAP1L, encoding the hematopoietic-specific Hem1 protein. Three mutations cause Hem1 protein and WAVE regulatory complex (WRC) loss, thereby disrupting actin polymerization, synapse formation, and immune cell migration. Another mutant, M371V encodes a stable Hem1 protein but abrogates binding of the Arf1 GTPase and identifies Arf1 as a critical Hem1 regulator. All mutations reduce the cortical actin barrier to cytokine release explaining immune hyperresponsiveness. Finally, Hem1 loss blocked mTORC2-dependent AKT phosphorylation, T cell proliferation, and effector cytokine production during T cell activation. Thus, our data show that Hem1 independently governs two key regulatory complexes, the WRC and mTORC2, and how Hem1 loss causes a combined immunodeficiency and immune hyperresponsiveness disease.\n\nOne sentence summaryHem1 loss of function mutations cause a congenital immunodysregulatory disease and reveal its role regulating WAVE2 and mTORC2 signaling.

immunology↗

bioRxiv2019

Mouse T cell priming is enhanced by maturation-dependent stiffening of the dendritic cell cortex

T cell activation by dendritic cells (DCs) involves forces exerted by the T cell actin cytoskeleton, which are opposed by the cortical cytoskeleton of the interacting APC. During an immune response, DCs undergo a maturation process that optimizes their ability to efficiently prime naive T cells. Using atomic force microscopy, we find that during maturation, DC cortical stiffness increases via process that involves actin polymerization. Using stimulatory hydrogels and DCs expressing mutant cytoskeletal proteins, we find that increasing stiffness lowers the agonist dose needed for T cell activation. CD4+ T cells exhibit much more profound stiffness-dependency than CD8+ T cells. Finally, stiffness responses are most robust when T cells are stimulated with pMHC rather than anti-CD3{varepsilon}, consistent with a mechanosensing mechanism involving receptor deformation. Taken together, our data reveal that maturation-associated cytoskeletal changes alter the biophysical properties of DCs, providing mechanical cues that costimulate T cell activation.

immunology↗