Roles of cytoskektal dynamics in T lymphocyte function

细胞毒性T淋巴细胞（ctl）通过极化分泌含有触发靶细胞程序性死亡分子的溶解颗粒来杀死靶细胞。在这里，我们使用快速，多色全内反射显微镜对人CD8+ ctl与刺激表面接触的溶解颗粒的胞吐进行了成像。使用mRFP标记的Lamp-1跟踪裂解颗粒的限制膜的命运，而使用mRFP标记的颗粒酶A，颗粒酶B或serglycin跟踪裂解颗粒货物的命运。成像显示，溶解颗粒通过与质膜的完全融合而释放出来，因此可见的所有三种货物的颗粒的全部内容在亚秒的时间尺度上释放到介质中。然而，GFP-Lamp-1的行为更为复杂。具体地说，在所有病例中，当它进入质膜时，它从胞吐部位向外扩散的程度从几乎完全到高度限制不等。后一种行为在大多数情况下可见，可能促进代偿性内吞过程。最后，我们可视化的三种物质释放时的扩散特性对颗粒酶和serglycin大分子复合物的大小提出了上限，这些大分子复合物呈现在目标细胞中。

Cytotoxic T lymphocytes (CTLs) kill target cells by the polarized secretion of lytic granules containing molecules that trigger programmed cell death in the target cell. Here we imaged the exocytosis of lytic granules from human CD8+ CTLs in contact with stimulatory surfaces using rapid, multicolor Total Internal Refection microscopy. The fate of the limiting membrane of the lytic granule was followed using mGFP-tagged Lamp-1, while the fate of lytic granule cargo was followed using granzyme A, granzyme B or serglycin tagged with mRFP. Imaging revealed that lytic granules are released by full fusion with the plasma membrane such that the entire content of the granule for all three cargos visualized was released into the media on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. Specifically, while it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. This latter behavior was seen in the majority of cases and may facilitate a process of compensatory endocytosis. Finally, the diffusion properties upon release of the three cargos we visualized put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell. Actin retrograde flow and acto-myosin II contraction have both been implicated in the inward movement of TCR microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-Tractin, a novel reporter for F-actin, we now provide direct evidence that the dSMAC and pSMAC correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Importantly, our images reveal concentric and contracting acto-myosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and acto-myosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC, and blebbistatin to selectively inhibit acto-myosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that LFA-1 clusters accumulate over time at the inner aspect of the LM/pSMAC, and that this accumulation is dependent on acto-myosin II contraction. Thus, actin retrograde flow and acto-myosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse. 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 is indeed 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.