Tubulin and Actin Interplay at the T Cell and Antigen-Presenting Cell Interface

T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off

The swing of lipids at peroxisomes and endolysosomes in t cell activation

The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.This review was funded by grant SAF2017-82886-R from the Spanish Ministry of Economy and Competitiveness (MINECO), grant S2017/BMD-3671-INFLAMUNE-CM from the Comunidad de Madrid, a grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud” (CIVP19A5941 XIX Concurso-2018) and a grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (BIOMEDICINA-2018), the Fundació Marató TV3 (grant 122/C/2015) and “La Caixa” Banking Foundation (HR17-00016). BIOIMID (PIE13/041) from Instituto de Salud Carlos III, CIBER Cardiovascular (CB16/11/00272, Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). SGD and ARG are funded by fellowship FPU and FPI programs, from Ministry of Science and Universities, respectively

Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav

7 FiguresRho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell–derived factor-1 (SDF-1α). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1α–activated human lymphocytes, where it transiently interacts with the SDF-1α receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1α–mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.From the Servicio de Inmunología, Hospital Universitario de la Princesa,Madrid, Spain; Centro de Investigación del Cáncer (CIC), Campus Miguel deUnamuno, Salamanca, Spain; and Facultad de Medicina, Universidad Autónoma de San Luis Potosí (UASLP), San Luis Potosí, Mexico.Peer reviewe

MTOC translocation modulates IS formation and controls sustained T cell signaling

The translocation of the microtubule-organizing center (MTOC) toward the nascent immune synapse (IS) is an early step in lymphocyte activation initiated by T cell receptor (TCR) signaling. The molecular mechanisms that control the physical movement of the lymphocyte MTOC remain largely unknown. We have studied the role of the dynein–dynactin complex, a microtubule-based molecular motor, in the process of T cell activation during T cell antigen–presenting cell cognate immune interactions. Impairment of dynein–dynactin complex activity, either by overexpressing the p50-dynamitin component of dynactin to disrupt the complex or by knocking down dynein heavy chain expression to prevent its formation, inhibited MTOC translocation after TCR antigen priming. This resulted in a strong reduction in the phosphorylation of molecules such as ζ chain–associated protein kinase 70 (ZAP70), linker of activated T cells (LAT), and Vav1; prevented the supply of molecules to the IS from intracellular pools, resulting in a disorganized and dysfunctional IS architecture; and impaired interleukin-2 production. Together, these data reveal MTOC translocation as an important mechanism underlying IS formation and sustained T cell signaling

Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav

7 FiguresRho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell–derived factor-1 (SDF-1α). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1α–activated human lymphocytes, where it transiently interacts with the SDF-1α receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1α–mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.From the Servicio de Inmunología, Hospital Universitario de la Princesa,Madrid, Spain; Centro de Investigación del Cáncer (CIC), Campus Miguel deUnamuno, Salamanca, Spain; and Facultad de Medicina, Universidad Autónoma de San Luis Potosí (UASLP), San Luis Potosí, Mexico.Peer reviewe

Integrin and CD3/TCR activation are regulated by the scaffold protein AKAP450

During antigen recognition by T cells, membrane receptors and cytoskeletal molecules form a specialized structure at the T cell–antigen-presenting cell junction called the immune synapse (IS). We report a role for the scaffolding protein A-kinase anchoring protein-450 (AKAP450), a member of the A-kinase anchoring protein family, in IS formation and T-cell signaling in antigen- and superantigen-dependent T-cell activation. Suppression of AKAP450 by overexpression of a dominant-negative form or siRNA knockdown disrupted the positioning and conformational activation of lymphocyte function-associated antigen 1 at the IS and impaired associated signaling events, including phosphorylation of phospholipase C-γ1 and protein kinase C-θ. AKAP450 was also required for correct activation and phosphorylation of CD3, LAT, and Vav1, key T-cell receptor-activated intracellular signaling molecules. Consistently, antigen-triggered reorientation of the microtubule-organizing center at the IS and interleukin-2 secretion were diminished in AKAP450-disrupted T cells. These results indicate key roles for AKAP450 in the organization and activation of receptor molecules at the IS during T-cell signaling events.Ministerio de Educación y CienciaComunidad de MadridFundación CNIC del Instituto Carlos IIIRed RECAVADepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.This study was supported by grants S2022/BMD-7209- INTEGRAMUNE-CM to NBMC from the Madrid Regional Government, PDC2021-121719-I00 and PLEC2022-009298 (AEI/ FEDER, UE) from the Spanish Ministry of Economy and Competitiveness (MINECO). Work in the MI lab is funded by grant PID2020-114148RB-I00 from the Spanish Ministry of Science and Innovation MCIN/AEI/ 10.13039/501100011033, which was in part granted with FEDER funding (EC). CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). ÁG-M is supported by an Investigo Grant by SEPE (Fondos de Resiliencia), Gobierno de España. SR is supported by a Sara Borrell fellowship from ISCIII and DC-F is supported by a fellowship from “la Caixa” Foundation (LCF/BQ/DR19/11740010). CS is supported by PEJ-2021-TL/BMD-21204 “Garantı́ a Juvenil” grant form Comunidad de Madrid. ML-P is a FPI fellowship (PRE2021- 097478) from the Spanish Ministry of Science and Innovation. The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation. The CNIC is a Severo Ochoa Center of Excellence (MINECO award CEX2020-001041-S). Funding agencies have not intervened in the design of the studies, with no copyright over the study.S

Role of Fyn in the rearrangement of tubulin cytoskeleton induced through TCR.

The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, and the secretory apparatus is the most characteristic early event that involves the tubulin cytoskeleton of T or NK cells after their interaction with APC or target cells. Our results show that Fyn kinase activity is essential for MTOC reorientation in an Ag-dependent system. Moreover, T cells from Fyn-deficient mice are unable to rearrange their tubulin cytoskeleton in response to anti-CD3-coated beads. Analysis of conjugates of T cells from transgenic OT-I mice with dendritic cells revealed that an antagonist peptide induces translocation of the MTOC, and that this process is impaired in T cells from Fyn(-/-) OT-I mice. In addition, Fyn deficiency significantly affects the MTOC relocation mediated by agonist peptide stimulation. These results reveal Fyn to be a key regulator of tubulin cytoskeleton reorganization in T cells