Influence of external forces on actin-dependent T cell protrusions during immune synapse formation
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.
Optogenetic activation of muscle contraction in vivo
Optogenetics is a relatively new and exciting field of research aimed at activating excitable cells and bypassing the need for electrical stimulation both ex-vivo and in-vivo. By utilizing Channelrhodopsin-2, researchers can simply activate these cells with blue light, something that has commonly been done in neurons, but limited work has been done with direct activation of skeletal muscle. Researchers therefore aimed to determine the feasibility of using transdermal light to directly induce isometric contractions of the triceps surae in-vivo, measured with the 1300A. The authors targeted expression of ChR2 in the skeletal muscle of mice and showed that optogenetic stimulation bypassed the nerve and resulted in similar torque production to that of electrical stimulation at a frequency of 10Hz. Higher frequencies led to a larger decay in the muscle contractions, shedding light on the significance of membrane repolarization in this process. These results showed the potential for a non-invasive alternative to electrical stimulation of muscle.