@article {48621, title = {3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse.}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017 Jun 22}, pages = {4077}, abstract = {

Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell{\textquoteright}s nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM). Also, existing methods that could be extended to 3D SMLM are usually subject to user defined analysis parameters, which remains a major drawback. Here, we present a new open source cluster analysis method for 3D SMLM data, free of user definable parameters, relying on a model-based Bayesian approach which takes full account of the individual localisation precisions in all three dimensions. The accuracy and reliability of the method is validated using simulated data sets. This tool is then deployed on novel experimental data as a proof of concept, illustrating the recruitment of LAT to the T-cell immunological synapse in data acquired by iPALM providing ~10 nm isotropic resolution.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-04450-w}, author = {Griffi{\'e}, Juliette and Shlomovich, Leigh and Williamson, David J and Shannon, Michael and Aaron, Jesse and Khuon, Satya and L Burn, Garth and Boelen, Lies and Peters, Ruby and Cope, Andrew P and Cohen, Edward A K and Rubin-Delanchy, Patrick and Owen, Dylan M} } @article {48434, title = {Live-cell super-resolution reveals F-Actin and plasma membrane dynamics at the T Cell synapse.}, journal = {Biophysical Journal}, volume = {112}, year = {2017}, month = {2017 Apr 25}, pages = {1703-13}, abstract = {

The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T\ cells. Building on previous studies at the T\ cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component,\ α-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.

}, doi = {10.1016/j.bpj.2017.01.038}, author = {Ashdown, George W and Burn, Garth L and Williamson, David J and Pandzic, Elvis and Peters, Ruby and Holden, Michael and Ewers, Helge and Shao, Lin and Wiseman, Paul W and Owen, Dylan M} }