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...