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T-cell activation is necessary for producing an adaptive immune response. For T-cell activation to occur, an antigen-presenting cell (APC) must make direct contact with it. This contact region has been shown to control many T-cell functions, and it is from this contact point that protrusions have been observed. To further characterize these protrusions, this study investigated the effect of various APC-mimicking forces on protrusion dynamics.  To mimic APC-induced T-cell activation, a micropipette force probe attached to an activating microbead was used. To evaluate micropipette flexibility, the bending stiffness was measured against a microindenter calibrated with Aurora’s 406A force probe. T-cell activation was induced once the microbead would make contact with the cell at a desired compressive or pulling force. To determine if protrusion was affected by external forces produced by APCs during activation, protrusion lengths were monitored following the application of various forces. It was found that the higher the compressive force, the shorter the protrusion was. Conversely, the larger the absolute pulling force was, the longer the protrusion grew. It was also observed longer protrusions were smaller in diameter, at all forces. To determine whether protrusion dynamics were affected by Arp2/3, a protein complex regulating actin polymerization, T-cells were treated with an Arp2/3-specific inhibitor. Not only were protrusion maximum lengths reduced following treatment, but they began growth later in time and at slower speeds. Finally, confocal imaging was used to assess F actin localization within protrusions. The results showed that the walls were rich with F actin whereas the center was depleted. A similar analysis of T cells and model APCs showed the same F actin localization. This study suggests that protrusion growth is set by an intracellular constant time and that protrusion dynamics are influenced by external forces. In addition to this, actin assembly within protrusions is facilitated by the Arp2/3 complex.

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