Activating T cells reorganize their cortical actin to form a ramified transportation network beneath the immunological synapse.

Cells adjust their macroscopic mechanical properties by tuning the actin protomer concentration and activity of actin nucleators.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling.

Biological research increasingly demands the use of non-invasive and ultra-sensitive imaging techniques. The Airyscan technology was recently developed to bridge the gap between conventional confocal and super-resolution microscopy. This technique combines confocal imaging with a 0.2 Airy Unit pinhole, deconvolution and the pixel-reassignment principle in order to enhance both the spatial resolution and signal-to-noise-ratio without increasing the excitation power and acquisition time. Here, we present a detailed study evaluating the performance of Airyscan as compared to confocal microscopy by imaging a variety of reference samples and biological specimens with different acquisition and processing parameters. We found that the processed Airyscan images at default deconvolution settings have a spatial resolution similar to that of conventional confocal imaging with a pinhole setting of 0.2 Airy Units, but with a significantly improved signal-to-noise-ratio. Further gains in the spatial resolution could be achieved by the use of enhanced deconvolution filter settings, but at a steady loss in the signal-to-noise ratio, which at more extreme settings resulted in significant data loss and image distortion.