Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In “frustrated” conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.     

Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.     

Th1 and Th2 cells form morphologically distinct immunological synapses

The arrangement of molecules at the interface between T cells and APCs is known as the immunological synapse (IS). We conducted experiments with supported planar bilayers and transfected fibroblast APC to examine the IS formed by polarized Th1 and Th2 cells. Th1 cells formed typical "bull's-eye" IS with a ring of adhesion molecules surrounding MHC/TCR interactions at all Ag concentrations tested, while Th2 cells formed multifocal IS at high concentrations of Ag. At low Ag concentrations, the majority of Th2 cells formed IS with a compact, central accumulation of MHC/TCR, but ICAM-1 was not excluded from the center of the IS. Additionally, CD45 was excluded from the center of the interface between Th1 cells and APC, while CD45 was found at the center of the multifocal IS formed by Th2 cells. Finally, phosphorylated signaling molecules colocalized with MHC/TCR to a greater extent in Th2 IS. Together, our results indicate that the IS formed by Th1 and Th2 cells are distinct in structure, with Th2 cells failing to form bull's-eye IS.     

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.     

Cytoplasmic dynein nucleates microtubules to organize them into radial arrays in vivo

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.     

A role for Rab7 in the movement of secretory granules in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.     

Dynein binds to beta-catenin and may tether microtubules at adherens junctions

Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.     

Microtubules' interaction with cell cortex is required for their radial organization, but not for centrosome positioning

Microtubules in interphase mammalian cells usually form a radial array with minus-ends concentrated in the central region and plus-ends placed at the periphery. This is accepted as correct, that two factors determinate the radial organization of microtubules - the centrosome, which nucleate and anchor the microtubules minus-ends, and the interaction of microtubules with cortical dynein, which positions centrosome in the cell center. However, it looks as if there are additional factors, affecting the radial structure of microtubule system. We show here that in aged Vero cytoplasts (17 h after enucleation) microtubule system lost radial organization and became chaotic. To clear up the reasons of that, we studied centrosome activity, its position in the cytoplasts and microtubule dynamics. We found that centrosome in aged cytoplasts was still active and placed in the central region of the cytoplasm, while after total disruption of the microtubules it was displaced from the center. Microtubules in aged cytoplasts were not stabilized, but they lost their ability to stop to grow near cell cortex and continued to grow reaching it. Aged cytoplast lamellae was partially depleted with dynactin though Golgi remained compact indicating dynein activity. We conclude that microtubule stoppage at cell cortex is mediated by some (protein) factors, and these factors influence radial structure of microtubule system. It seems that the key role in centrosome positioning is played by dynein complexes anchored everywhere in the cytoplasm rather than anchored in cell cortex.     

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal “basic” domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly–promoting factors with complementary activities.     

Rapid Lytic Granule Convergence to the MTOC in Natural Killer Cells Is Dependent on Dynein But Not Cytolytic Commitment

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules, we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization, lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization, dependent on dynein motor function, occurred before MTOC polarization, and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion, one that may prepare, but not commit cells for precision secretory function.     

Cargo Transport by Cytoplasmic Dynein Can Center Embryonic Centrosomes

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell’s center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Role of mitotic motors, dynein and kinesin, in the induction of abnormal centrosome integrity and multipolar spindles in cultured V79 cells exposed to dimethylarsinic acid.

The role of microtubule-based motors in the induction of abnormal centrosome integrity by dimethylarsinic acid (DMAA) was investigated with the use of monastrol, a specific inhibitor of mitotic kinesin, and vanadate, an inhibitor of dynein ATPase. Cytoplasmic dynein co-localized with multiple foci of gamma-tubulin in mitotic cells arrested by DMAA. Disruption of microtubules caused dispersion of dynein while multiple foci of gamma-tubulin were coalesced to a single dot. Vanadate also caused dispersion of dynein, which had been co-localized with multiple foci of gamma-tubulin by DMAA, without affecting spindle organization. However, the dispersion of dynein did not prohibit the induction of abnormal centrosome integrity by DMAA. Inhibition of mitotic kinesin by monastrol resulted in monoastral cells with non-migrated centrosomes in the cell center. Monastrol, when applied to mitotic cells with abnormal centrosome integrity, rapidly reduced the incidence of cells with the centrosome abnormality. Moreover, monastrol completely inhibited reorganization of abnormal centrosomes that had been coalesced to a single dot by microtubule disruption. These results suggest that abnormal centrosome integrity caused by DMAA is not simply due to dispersion of fragments of microtubule-organizing centers, but is dependent on the action of kinesin. In addition, the results suggest that kinesin plays a role not only in the induction of mitotic centrosome abnormality, but also in maintenance.     

Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.     

A ZYG-12–dynein interaction at the nuclear envelope defines cytoskeletal architecture in the C. elegans gonad

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12–mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined γ-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. γ-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules around zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.     

The many faces of the bouquet centrosome MTOC in meiosis and germ cell development

Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.     

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.     

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.     

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.     

Activation outcomes induced in naïve CD8 T-cells by macrophages primed via "phagocytic" and nonphagocytic pathways

The array of phagocytic receptors expressed by macrophages make them very efficient at pathogen clearance, and the phagocytic process links innate with adaptive immunity. Primary macrophages modulate antigen cross-presentation and T-cell activation. We assessed ex vivo the putative role of different phagocytic receptors in immune synapse formation with CD8 naïve T-cells from OT-I transgenic mice and compared this with the administration of antigen as a soluble peptide. Macrophages that have phagocytosed antigen induce T-cell microtubule-organizing center and F-actin cytoskeleton relocalization to the contact site, as well as the recruitment of proximal T-cell receptor signals such as activated Vav1 and PKCθ. At the same doses of loaded antigen (1 μM), “phagocytic” macrophages were more efficient than peptide-antigen–loaded macrophages at forming productive immune synapses with T-cells, as indicated by active T-cell TCR/CD3 conformation, LAT phosphorylation, IL-2 production, and T-cell proliferation. Similar T-cell proliferation efficiency was obtained when low doses of soluble peptide (3–30 nM) were loaded on macrophages. These results suggest that the pathway used for antigen uptake may modulate the antigen density presented on MHC-I, resulting in different signals induced in naïve CD8 T-cells, leading either to CD8 T-cell activation or anergy.