The influence of mechanical loading on cell structure and behavior is on the cutting edge of current cell biology research. Space flight has been postulated to alter mechanical loads on cells and it is known to alter cellular behavior. T-cell activation is known to be affected during space flight, but the association with altered mechanical environments remains unclear. Our hypothesis is that alterations in the mechanical environment to which T-cell are exposed can directly modulate the cell's ability to polarize and to integrate the signal transduction machinery. This results in a shift in the activation threshold due to altered assembly and effectiveness of cellular signaling systems. To characterize this phenomenon, primary T-cells will be activated in a cell-contact dependent manner under a variety of mechanical loading scenarios. Culture conditions include standard 1xG culture conditions, clinorotation (a ground-based culture model that provides elements of the physical environment present in cell culture during space flight) and controlled induction of mechanical loads. These experiments specifically will assess: 1) the effect of the mechanical forces on the activation response of T-cells, 2) morphological assessment of cell contact and polarization by environmental scanning electron microscopy/ESEM, 3) assessment of the integration of the mechanosensing and signaling components of the cell using confocal microscopy, and 4) proteomic assessment of early and downstream signal transduction events. This approach represents a novel application of biochemical, morphological and mechanical assessment of T-cell activation and will provide a significant insight into the behavior of T-cells under changing environmental conditions. A more fundamental understanding of how T-cells integrate the signals during activation and how the external mechanical environment affects the threshold of activation will provide a mechanistic framework for interpretation of space flight and Earth-based immunology studies.
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T cells activated under the influence of altered mechanical loads exhibit significant differences in their ability to integrate the signaling networks and commit to full activation. T cell activation experiments have shown a significant link exists between the mechanosensing of the T cell with its external environment and the integration of activating signals. Through the use of confocal microscopy, we have developed a novel method to analyze the 3D changes in the surface contact area of the T cell and an activating bead that represents an antigen presenting cell (APC). We have used post-processing algorithms to isolate microtubule structures that interact directly with the nucleus showing a direct link with how the tubulin physically communicates with the nucleus throughout T cell activation. We have tracked the posttranslational modifications in the cellular cytoskeleton that are important in controlling the trafficking of proteins to the activating site during T cell activation, as well as are responsible for regulating the re-orientation the microtubule organizing center (MTOC) to the site of TCR stimulation. Finally, we have tracked changes in the localization of proteins that play an integral role in the regulation of the mechanosensing of the T cell with their external environment which is integral to the success of T cell activation.
This culture-based system allowed us to examine the effects of altered mechanical forces on T cell polarization, cytoskeletal post-translational modifications (PTMs), spatial organization of key signaling proteins, and activation. Overall, cytoskeletal reorganization and polarization were significantly impaired during suspension culture. Examination of cell-bead constructs of T cells activated in suspension showed that although T cells contact and adhere to the beads, they remain round and do not exhibit extensive physical reorientation toward the contact site. Measurements of the cell-bead contact surface area showed a dramatic reduction of size in cells activated in suspension compared to static controls. Assessment of tubulin PTMs showed an increase in detyrosinated (Glu) microtubules in cells activated under suspension which suggests a reduction in cytoskeletal remodeling that can impact the transportation of proteins to their activating sites. T cells also required significantly more stimulation to activate in suspension culture as compared to static culture (increased activation threshold). This change in threshold involves a mechanism independent of TCR (T-cell receptor) triggering as T cells stimulated during suspension required 2- to 3-fold higher levels of TCR triggering to achieve equivalent levels of activation compared to static cells. Evaluation of the integration of the signal transduction pathways showed that ZAP 70 and Erk1/Erk2 phosphorylation was significantly inhibited in T cells activated in suspension culture. In addition, spatial reorganization and clustering of both Zap 70 and Erk1/Erk2 did not occur in the suspension activated T cells as compared to static controls. Our results suggest that mechanical forces play a primary role in the regulation and integration of signal transduction networks necessary for successful T cell activation.
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