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(F) Jurkat cells were transfected as in A, stained on ice with anti-CD3-PE, cross-linked for the indicated times at 37☌, incubated in cold stripping buffer (removing surface antibodies), and analyzed by flow cytometry for TCR internalization in GFP + cells. (E) Jurkat cells were transfected as in A, stained on ice with anti-CD3-PE and cell surface expression of the TCR was analyzed on GFP + cells. The percent adhesion (GFP-negative, low, and high) in each sample was determined by flow cytometry. (D) Jurkat cells were transfected as in A and analyzed for fibronectin binding. Conjugation efficiency was determined by flow cytometry. (C) Jurkat cells were transfected with control or with both shArp2-GFP and shArp3-GFP vectors (green), and incubated with unpulsed or SEE-pulsed/PKH26-stained NALM6 B cells (red). Each population was conjugated with unpulsed or SEE-pulsed/CMAC-stained Raji cells (blue), labeled with anti-αTubulin (red), and scored for MTOC polarization (B). (A) Jurkat cells were transfected with control, shArp2-GFP, or shArp3-GFP vectors (green). Altogether, our results have identified Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing.
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Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Keys org safe Activator Lifetime OS (Win-Mac) VolumeLock 2.3 Patch / Win. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. We therefore examined the requirement for Arp2/3- and formin-dependent F-actin nucleation during T cell activation. A band of soldiers in search of a secret Japanese ammunition store face off against deadly saltwater crocodiles in the swamps of Ramree Island. With Charlie Bond, Steven Dolton, Ryan Harvey, David Hon Ma Chu. T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. Saltwater: The Battle for Ramree Island: Directed by Steve Lawson.