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.
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