T lymphocytes rapidly reposition their centrosome to the center of the immunological synapse (IS), the specialized interface between the T cell and the target cell, to drive the polarized secretion of effector molecules in the direction of the bound target cell. Using an optical trap to provide spatial and temporal control over target cell presentation and orientation, we show that centrosome repositioning in Jurkat T cells exhibits kinetically distinct Polarization and Docking phases (3 and 1 m/min, respectively), with a transition point between them occurring 2.2 m from the IS membrane. In contrast to previous studies, repositioning requires intracellular calcium flux and signaling through both the TCR and integrin to be robust. In terms of the force driving centrosome repositioning, we find that the center of the IS invaginates dramatically to approach the centrosome in frustrated conjugates where the centrosome is stuck behind the T cells nucleus, arguing that the force driving repositioning is focused at the center of the IS. Consistently, imaging of microtubules during normal centrosome repositioning events reveals the formation of a relatively straight microtubule bundle attached in end-on fashion at the center of the IS that then shrinks in length with time, drawing the centrosome to the IS. Consistent with such a microtubule end-on capture-shrinkage mechanism (Nguyen-Ngoc et al., 2007;Laan et al., 2012), centrosome repositioning is strongly impaired by inhibiting either microtubule depolymerization (using taxol) or dynein (using ciliobrevin, siRNA, DN constructs), and completely blocked by simultaneous inhibition of both. Moreover, dynein inhibition alone completely blocks the Docking phase, suggesting an interesting force: velocity relationship. We conclude that centrosome repositioning in T cells occurs via a microtubule end-on capture-shrinkage mechanism operating at the center of the IS, where the stepping of cortically-anchored dynein attached to the microtubule plus end, coupled with plus end depolymerization, creates in a mutual and interdependent fashion the pulling force on the centrosome. This mechanism drives a repositioning process possessing two kinetic phases that rapidly and robustly draws the centrosome to the IS membrane to support the microtubule-dependent vectorial delivery of effector molecules to the target cell. The contact area between a T cell and an antigen presenting cell (APC) is organized into a bulls eye arrangement of segregated concentric regions, collectively known as the immunological synapse (IS). The IS serves as the structural basis of signaling and secretion between the T cell and APC. The center area of the IS, termed the central supramolecular activation cluster (cSMAC), is marked by the accumulation of T cell receptor (TCR) microclusters (MCs). We recently showed that the centripetal movement of TCR MCs to the cSMAC is driven entirely by a combination of actin polymerization driven actin retrograde flow in the dSMAC (lamellipodial actin network) and actomyosin II driven actin arc contraction in the pSMAC (lamellar actin network). Saito and colleagues have, however, reported that the microtubule dependent transport of TCR MCs driven by cytoplasmic dynein contributes significantly to the centripetal movement of TCR MCs. Recently, a very effective membrane-permeable small molecule inhibitor of cytoplasmic dynein called Ciliobrevin was described. Here we show that the kinetics of centripetal TCR MCs movement are normal in Ciliobrevin treated cells, suggesting that their movement isindeed largely, if not entirely, driven by actin-dependent mechanisms. Finally, we show that Ciliobrevin D is cytotoxic to Jurkat T cells when the cells are imaged with blue light, such as when imaging GFP-chimeras, but not when imaging with green light and higher wavelengths, representing a cautionary tale for other investigators interested in using Ciliobrevin to inhibit dynein in living cells.

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National Heart, Lung, and Blood Institute
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Yi, Jason; Wu, Xufeng; Chung, Andrew H et al. (2013) Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage. J Cell Biol 202:779-92