TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation

Abstract

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dualcolor fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.A principal function of most T cells is to recognize foreign antigens on other cell surfaces, with the specificity determined by the T cell antigen receptor (TCR). Once the TCR has engaged its ligand and the signaling cascade has been initiated through the associated cluster of differentiation 3 (CD3) subunits, the kinase Zap70 transmits the signal to the adaptor Lat (linker for activation of T cells; A001392). Within seconds, actin-dependent signaling microclusters are formed in the periphery of the contact site between the T cell and antigenpresenting cell, and these microclusters then migrate to the center of the interface and form the immunological synapse [1][2][3][4][5][6] . Many signaling molecules, including the TCR and Lat, colocalize in the microclusters during early T cell activation [3][4][5][6] . On the basis of electron-microscopy studies, varying degrees of preclustering of plasma membrane molecules, including the TCR and Lat, have been proposed 7-9 . However, there are concerns about potential artifacts with this approach (for example, from fixation, gold particle staining and/or drying). Additionally, fluorescence microscopy and biochemical technologies suggest the coexistence of monomeric and multivalent TCR-CD3 complexes 9,10 . It is not clear to what extent these multivalent complexes exist or if they form through protein-protein interactions or localization in membrane domains. In the case of biochemical approaches, cell lysis itself could introduce additional artifacts. To overcome such limitations and to gain insight into the organization of plasma membrane proteins in situ, we analyzed the organization of the TCR and Lat in quiescent and activated native T cell plasma membrane sheets and live T cells. We achieved this with a combination of three techniques: a modified high-speed version of photoactivated localization microscopy (hsPALM) 11-13 , dual-color fluorescence cross-correlation spectroscopy (dcFCCS) [14][15][16] and transmission electron microscopy (TEM) 7,8,17,18 . NIH Public AccessWe show that both the TCR and Lat were preclustered i...

Bayesian cluster identification in single-molecule localization microscopy data

“…T-tests were used and their p-values were computed by permutation (see Supplementary Materials and Methods). In good agreement with previous reports 21,32 , CD3ζ was clustered in stimulated and non-stimulated cells, and cluster parameters were significantly altered. Despite no large scale changes in the percentage of localisations found in clusters (29 ± 2% in PLL to 33 ± 1% in activated cells, P > 0.05), a significant increase in the number of clusters and a significant decrease in the size of clusters was observed from resting to activated cells (8 ± 1 clusters per region on PLL versus 20 ± 3 in activated cells; P ≤ 0.005 and 82 ± 4 nm radius versus 48 ± 2 nm after activation; P ≤ 0.0005; Fig.…”

“…Each localisation is then disturbed by Gaussian noise with variance drawn from a Gamma distribution with mean 30 nm and standard deviation 13 nm (emulating the localisation error of the microscope). These parameters were chosen to approximately reflect typical clustering behaviour of proteins at the immunological synapse 14,15,20,21 . The three remaining scenarios have the same parameters as the Standard Conditions except where stated otherwise.…”

“…Here, it is accepted that proteins, including the CD3ζ subunit, arrange into microclusters upon synapse formation. While many other biological processes involve the clustering of proteins at the cell surface, this application is especially informative because both the K-function strategy and PC have been applied previously 14,15,20,21 . For experimental data, it is important that artefacts caused by multiple blinking of individual fluorophores and overlapped PSFs, inherent to the methodology of SMLM, are removed (or accounted for).…”

Fluorescence nanoscopy. Methods and applications

“…Curiously, the individual islands within the microclusters remained intact, and intermingling of TCR and Lat molecules did not occur [196]. An explanation to this result also came from PALM experiments: The Gaus group visualised Lat clusters growing in fluorescence intensity and then fading, which is characteristic of species localised in vesicles.…”

Beyond ‘furballs’ and ‘dumpling soups’ – towards a molecular architecture of signaling complexes and networks

“…The complex role of cellular membranes in signaling is also well illustrated in the case of the T-cell receptor (TCR) signalosome [10,11]. Before TCR activation, the TCR subunits and a fraction of the membrane-associated scaffold protein LAT are separated into different clusters in the plasma membrane [75]. Another fraction of LAT is simultaneously stored in small vesicles, which can interact with components at the membrane [16].…”