Analysis of LFA-1 distribution with respect to the actomyosin network. (A) Time-lapse series of human T lymphoblasts expressing Lifeact-GFP and spreading on planar bilayers coated with anti-CD3 linked to Alexa Fluor 647–labeled streptavidin and Alexa Fluor 594–labeled ICAM-1. Imaging was initiated after full spreading was reached. Radial profiles were generated across the indicated dotted line. Representative of 10 cells analyzed. (B) Kymographs generated from the sequence shown in A. (C) Human primary CD4⁺ T cells were conjugated to SEE-pulsed Raji B cells (dashed outlines and asterisks) for 25 min and labeled with antibodies to the CD3ε chain of TCR and to the β2 chain in the extended and open conformations. Z stacks of whole conjugates were collected and rendered in 3D in the IS plane (arrowhead). Representative rendered images are shown. (D) T lymphoblasts spreading on bilayers for 30 min were labeled as in Fig. 1. Shown are single cell images and population average projections. (E) Ex vivo T cells spreading on bilayers for 30 min were labeled with conformation-specific LFA-1 and anti-myosin IIA antibodies. Bars, 5 µm.