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中程序性细胞死亡的分子的裂解颗粒来杀死靶细胞。 在这里，我们使用快速的全内反射显微镜对来自与刺激表面接触的人CD 8 + CTL的溶解颗粒的胞吐进行成像。 使用mGFP标记的Lamp-1跟踪裂解颗粒的界膜的命运，而使用用mRFP标记的颗粒酶A、颗粒酶B或丝甘氨酸跟踪裂解颗粒货物的命运。 成像显示，溶解颗粒通过与质膜完全融合而释放，使得可视化的所有三种货物的颗粒的全部内容物以亚秒的时间尺度释放到培养基中。 然而，GFP-Lamp-1的行为更为复杂。 具体而言，虽然它进入质膜在所有情况下，它然后扩散远离的网站胞吐的程度从几乎完全高度限制。 后一种行为见于大多数病例，可能有助于代偿性内吞作用的过程。 最后，我们观察到的三种货物释放时的扩散性质对呈现给靶细胞的颗粒酶和丝甘蛋白的大分子复合物的大小设置了上限。 肌动蛋白逆行流动和肌动蛋白-肌球蛋白II收缩都与TCR微簇的向内运动和免疫突触形成有关，但没有研究整合和量化它们的相对贡献。 使用Jurkat T细胞表达荧光肌球蛋白IIA重链和F-Tractin，一种新的报告F-肌动蛋白，我们现在提供直接的证据，dSMAC和pSMAC对应于lamelipodial（LP）和lamellar（LM）肌动蛋白网络，分别如前所述。 重要的是，我们的图像显示了LM/pSMAC处的同心和收缩的肌动蛋白II弧/环。 此外，向心性移动TCR微簇的速度非常密切地对应于肌动蛋白逆行流在LP/dSMAC和肌动蛋白-肌球蛋白II弧收缩在LM/pSMAC的速率。 使用细胞松弛素D和jasplakinalin选择性地抑制肌动蛋白逆行流动的LP/dSMAC，和blebbistatin选择性地抑制肌动蛋白-肌球蛋白II弧收缩的LM/pSMAC，我们证明，这两个力都需要向心TCR微簇运输。 最后，我们表明，LFA-1簇积累随着时间的推移，在内部方面的LM/pSMAC，这种积累是依赖于肌动蛋白II收缩。 因此，肌动蛋白逆行流动和肌动蛋白-肌球蛋白II弧收缩协调驱动受体簇动力学在免疫突触。 T细胞和抗原呈递细胞（APC）之间的接触区域被组织成分隔的同心区域的牛眼排列，统称为免疫突触（IS）。IS作为T细胞和APC之间的信号传导和分泌的结构基础。IS的中心区域，称为中央超分子活化簇（cSMAC），以T细胞受体（TCR）微簇（MC）的积累为标志。我们最近发现，TCR MCs向cSMAC的向心运动完全由dSMAC（层状肌动蛋白网络）中肌动蛋白聚合驱动的肌动蛋白逆行流动和pSMAC（层状肌动蛋白网络）中肌动球蛋白II驱动的肌动蛋白弧收缩的组合驱动。然而，Saito及其同事报道了由细胞质动力蛋白驱动的TCR MCs的微管依赖性转运对TCR MCs的向心运动有显著贡献。 最近，描述了一种非常有效的细胞质动力蛋白的膜渗透性小分子抑制剂，称为Ciliobrevin。在这里，我们表明，向心TCR MCs运动的动力学是正常的纤毛霉素处理的细胞，这表明他们的运动确实是很大程度上，如果不是完全，由肌动蛋白依赖性机制驱动。最后，我们表明，纤毛菌素D是细胞毒性的Jurkat T细胞时，细胞用蓝光成像，如当成像GFP嵌合体，但不是当成像与绿色光和更高的波长，代表一个警示故事，为其他研究人员有兴趣使用纤毛菌素抑制动力蛋白在活细胞。

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.