Spatial mapping of affinity changes for the integrin LFA-1 during cell migration using clusters identified based on local density.

Localization microscopy methods like Stochastic Optical Reconstruction Microscopy (STORM) are very well suited for exploring clustering of proteins, as the data inherently provide a list of molecular coordinates. Here we use state-of-art cluster analysis algorithms (DBSCAN) to explore the clustering behaviour of different affinity forms of the integrin LFA-1. It has been suggested that LFA-1 may form clusters, in order to increase the avidity to ICAM-1. However, this hypothesis still seems to be controversial. In this study, we found, variations in clustering behaviour among the different affinity forms of LFA-1 in migrating T-cells. We found that panLFA-1 is located in clusters throughout the polarised cell on ICAM-1, with an increased density of molecules and clusters in the mid area and rear of the cell, whereas the intermediate and high affinity form of LFA-1 showed an increased number in the mid area of a migrating cell and the high affinity form of LFA-1 in the front and rear. Together, these data suggest that, in addition to LFA-1 conformation, protein clustering might play a role in controlling cell-substrate adhesion on ICAM-1.By applying the cluster analysis algorithm DBSCAN to localization microscopy data, integrin clusters could be identified and different cluster parameters could be quantified.