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. Actin cytoskeletal organization and dynamics play fundamental roles in T cell immunological synapse (IS) formation. The T cells actin cytoskeleton undergoes striking rearrangements upon contact with an antigen presenting cell (APC) to form radially symmetric segregated domains named the distal, peripheral and central supramolecular activation clusters (dSMAC, pSMAC, and cSMAC). The outer dSMAC corresponds to a lamellipodial (LP) actin network composed of branched actin arrays that undergo robust, polymerization-driven actin retrograde flow. The central pSMAC corresponds to a lamellar (LM) actin network composed of concentric actin arcs that undergo actomyosin II-driven contraction. Finally, the inner cSMAC is largely devoid of actin. This bulls-eye pattern of SMACs generates a centripetal flow of actin that drives the inward movement of signaling and adhesion molecules to create a mature IS. Here we addressed how the concentric, linear actomyosin II arcs in the pSMAC are assembled. Current thinking would have it that the branched actin network in the dSMAC, which is created by Arp2/3-dependent nucleation, is converted by debranching and crosslinking into the concentric arcs. Using structured illumination microscopy, we identified for the first time linear actin filaments that are arranged perpendicular to the plasma membrane and that are embedded in the branched actin network in the dSMAC. As these filaments exit the inner aspect of the dSMAC, they splay out and reorient into concentric arcs with an inherent antiparallel organization required for myosin II-dependent contraction. The perpendicular actin filaments in the dSMAC are highlighted and accentuated by inhibiting Arp2/3-dependent actin polymerization using CK666, which collapses the branched actin array in between the perpendicular filaments. Importantly, inhibition of myosin II using blebbistatin results in loose, disorganized filaments in the pSMAC that fail to reorient into concentric arcs. These observations suggest that arc assembly can occur independently of Arp2/3-dependent nucleation, and that myosin II contractility is required for reorienting the perpendicular filaments emanating from the dSMAC into the concentric arcs in the pSMAC. We are now attempting to define the relative contributions of debranching/rearrangement of the branched actin network versus nucleation of linear filaments, possibly by the formin INF2 present on the plasma membrane at the tips of the perpendicular filaments, to the formation of the actin arcs in the pSMAC.
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