Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28
June 10, 2016 by larryhbern
Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28
Curator: Larry H. Bernstein, MD, FCAP
New connections: T cell actin dynamics
Nancy R. Gough
Sci. Signal. 19 Apr 2016; 9(424): ec95 http://dx.doi.org:/10.1126/scisignal.aaf8940
When T cells receive the appropriate signals through the T cell receptor (TCR) complex and the costimulatory receptor CD28, a complex rearrangement of the cytoskeleton occurs that enables the formation of the immunological synapse, a specialized structure that forms between the antigen-presenting cell and the T cell. In this week’s issue, Roybal et al. used sophisticated imaging of live cells and computational image analysis to visualize the dynamic rearrangements of actin and various regulatory proteins in T cells activated through the TCR in the absence or presence of CD28 signaling. The regulatory proteins WAVE2 and cofilin were efficiently recruited to the immunological synapse only when both TCR and CD28 signaled. Fried et al. used a different fluorescence imaging approach, triple-color FRET (fluorescence resonance energy transfer), to visualize not the movement of proteins with the cell, but the interactions between the actin-regulatory proteins WASp and WIP in live cells. The WASp-WIP interaction is required for T cell activation. The triple-color FRET analysis revealed how changes in the interaction between WASp and WIP resulted in WASp functioning as both an on and off switch. Although most studies’ focus of T cell cytoskeletal dynamics is on the changes that occur at the immunological synapse, as González-Granado et al. showed, the nuclear cytoskeleton is also important for the immune response. In this study, live-cell imaging and immunofluorescence analysis revealed that nuclear lamin-A contributed to the polymerization of actin and, thus, immunological synapse formation. These studies highlight the insights that can be obtained about molecular dynamics from live-cell image analysis.
K. T. Roybal, T. E. Buck, X. Ruan, B. H. Cho, D. J. Clark, R. Ambler, H. M. Tunbridge, J. Zhang, P. Verkade, C. Wülfing, R. F. Murphy, Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics. Sci. Signal.9, rs3 (2016). [Abstract]
Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics
Kole T. Roybal1,2,*,†, et al. Sci. Signal. 19 Apr 2016; 9(424): rs3. http://dx.doi.org:/10.1126/scisignal.aad4149
Imaging T cell actin dynamics
T cells must receive signals through the T cell receptor (TCR) and the costimulatory receptor CD28 to become fully activated. Critical to this process is the reorganization of plasma membrane actin at the immunological synapse, the interface between a T cell and an antigen-presenting cell. Roybal et al.imaged actin and fluorescently tagged actin regulatory proteins in T cells activated through the TCR in the absence or presence of CD28 signaling. Computational image processing to normalize differences in cell shape enabled tracking of the fluorescent proteins. The regulatory proteins WAVE2 and cofilin were efficiently recruited to the immunological synapse only when both TCR and CD28 signaled. Constitutive activation of either protein in TCR-stimulated T cells enabled normal actin reorganization even when CD28 signaling was blocked. This combination of imaging and computational analysis could be applied to other systems to determine the spatiotemporal dynamics of signaling molecules.
Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.
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S. Fried, B. Reicher, M. H. Pauker, S. Eliyahu, O. Matalon, E. Noy, J. Chill, M. Barda-Saad, Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex. Sci. Signal.7, ra60 (2014). [Abstract]
Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex
Sophia Fried1,*, et al. Sci. Signal. 24 Jun 2014; 7(331): ra60 http://dx.doi.org:/10.1126/scisignal.2005198
Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.
J. M. González-Granado, C. Silvestre-Roig, V. Rocha-Perugini, L. Trigueros-Motos, D. Cibrián, G. Morlino, M. Blanco-Berrocal, F. G. Osorio, J. M. P. Freije, C. López-Otín, F. Sánchez-Madrid, V. Andrés, Nuclear envelope Lamin-a couples actin dynamics with immunological synapse architecture and T cell activation.Sci. Signal.7, ra37 (2014). [Abstract]
Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation
José M. González-Granado1, et al. Sci. Signal. 22 Apr 2014; 7(322): ra37 http://dx.doi.org:/10.1126/scisignal.2004872
In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal–regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A–dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.
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RESEARCH ARTICLE
A large Rab GTPase encoded byCRACR2Ais a component of subsynaptic vesicles that transmit T cell activation signals
T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.
More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.
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Posted in Autoimmune Inflammatory DIseases, Bacterial Resistance, Biological Networks, Cell Biology, Signaling & Cell Circuits, Curation, Cytoskeleton, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Genetics & Pharmaceutical, Genomic Expression, Human Immune System in Health and in Disease, Immunology, Microvesicle, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Viral diseases | Tagged Actin-Regulating Complex, cell-mediated immunity, Nuclear Envelope Lamin-A, subsynaptic vesicles, T cell actin dynamics, T cells, T-cell activation, T-cell receptor signaling, T-cell/actin dynamics, WAVE2 and cofilin, Wiskott-Aldrich syndrome protein (WASp) | Leave a Comment
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