The evolution and uses of the stories of the Prophets

This is a stable archival PDF of an open-access, peer-reviewed journal volume originally published at www.mizanproject.org/journal

Centrosome docking at the immunological synapse is controlled by Lck signaling.

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor-proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor-induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cellsThis work was supported by grants BFU2009-07886 and CONSOLIDER COAT CSD2009-00016 to M.A. Alonso, and BFU2011-22859 to I. Correas (all of them from the Ministerio de Economía y Competitividad, Spain), and grant S2010/BMD-2305 from the Comunidad de Madrid to I. Correa

A Model for the Interplay of Receptor Recycling and Receptor-Mediated Contact in T Cells

Orientation of organelles inside T cells (TC) toward antigen-presenting cells (APC) ensures that the immune response is properly directed, but the orientation mechanisms remain largely unknown. Structural dynamics of TC are coupled to dynamics of T-cell receptor (TCR), which recognizes antigen on the APC surface. Engagement of the TCR triggers its internalization followed by delayed polarized recycling to the plasma membrane through the submembrane recycling compartment (RC), which organelle shares intracellular location with the TC effector apparatus. TCR engagement also triggers TC-APC interface expansion enabling further receptor engagement. To analyze the interplay of the cell-cell contact and receptor dynamics, we constructed a new numerical model. The new model displays the experimentally observed selective stabilization of the contact initiated next to the RC, and only transient formation of contact diametrically opposed to the RC. In the general case wherein the TC-APC contact is initiated in an arbitrary orientation to the RC, the modeling predicts that the contact dynamics and receptor recycling can interact, resulting effectively in migration of the contact to the TC surface domain adjacent to the submembrane RC. Using three-dimensional live-cell confocal microscopy, we obtain data consistent with this unexpected behavior. We conclude that a TC can stabilize its contact with an APC by aligning it with the polarized intracellular traffic of TCR. The results also suggest that the orientation of TC organelles, such as the RC and the effector apparatus, toward the APC can be achieved without any intracellular translocation of the organelles

Fyn-Binding Protein (Fyb)/Slp-76–Associated Protein (Slap), Ena/Vasodilator-Stimulated Phosphoprotein (Vasp) Proteins and the Arp2/3 Complex Link T Cell Receptor (Tcr) Signaling to the Actin Cytoskeleton

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76–associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3–coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton

HIV-1 Nef Employs Two Distinct Mechanisms to Modulate Lck Subcellular Localization and TCR Induced Actin Remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host

An Experimental and Computational Study of Effects of Microtubule Stabilization on T-Cell Polarity

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-µM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the “synapse” with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies

Casein kinase I delta controls centrosome positioning during T cell activation

CK1delta binds and phosphorylates the microtubule plus-end–binding protein EB1 and promotes centrosome translocation to the immunological synapse in T cells

Synthetic Multivalent Ligands as Probes of Signal Transduction

Cell-surface receptors acquire information from the extracellular environment and coordinate intracellular responses. Many receptors do not operate as individual entities, but rather as part of dimeric or oligomeric complexes. Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses. Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades. Understanding the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication is critical; chemical synthesis can provide compounds to address the role of receptor assembly in signal transduction. Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements. This Review focuses on the use of synthetic multivalent ligands to characterize receptor function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50669/1/2348_ftp.pd