Gradient Distribution of LFA-1 cluster Size in the Immunological Synapse

Abstract

Coordinated rearrangement of membrane receptors into clusters upon ligand binding has emerged as an important regulator of cellular signaling. However, due to cell membrane complexity and constant change in membrane morphology, probing protein clusters that are below optical resolution in live cells has long been a challenging task. Here, we use a hybrid live cell-supported membrane interface to enable high resolution fluorescence fluctuation measurements. We explore the cluster distribution of LFA-1:ICAM-1 complexes that form a ring that spans ∼10µm in diameter in a mature immunological synapse(IS). IS formation is triggered by exposing primary T cells to a supported membrane that is functionalized with activating peptide-MHC and ICAM-1-YFP. We scan across the IS with a focused laser beam to monitor the photobleaching and fluorescence fluctuations of LFA-1 bound ICAM-1-YFP molecules within the optical area. By changing the excitation laser from continuous to a pulsed form and applying photon counting histogram analysis, we are able to distinguish between the fluorescent signals of large clusters from those of small clusters. Instead of an even distribution, our results indicate that LFA-1:ICAM-1 complexes at the IS exhibit two differently clustered populations. One population shows a strong association to the T cell cytoskeleton and thus displays low lateral mobility. Interestingly, the fraction and the size of clusters of the cytoskeleton-associated complexes increase toward the center of the IS. The second population of LFA-1:ICAM-1 complexes exhibits random diffusion and a small cluster number that is no more than 3 on average. Therefore, more compartmentalized domains exist beyond the classical picture of three concentric zones at the IS. This supports the frictional force coupling model as the mechanism of IS formation, i.e. larger clusters experience larger transport force toward the center of the IS.