Fig. 1

EPLIN is a component of actin filaments recruited to leukocyte transmigration sites and ICAM-1 clusters. (A) After irradiation of LifeAct-expressing transgenic mice followed by allogeneic transplantation of bone marrow, the mice were allowed to recover for 6 weeks. Transmigration of leukocytes was provoked in cremaster muscle by administration of 50 ng IL-1β for four hours. Mice were then euthanized, tissues was collected and laser scanning microscopy (LSM) was performed for the LifeAct-EGFP and immunolabelled EPLIN and PECAM and merged at higher magnification accompanied by Z-stacks as indicated (Z1-Z2). Lower image showes overview of LifeAct labelled inflamed cremaster muscle together with anti pan-EPLIN- and anti PECAM-1 antibodies, additionally superimposed with phase contrast images to identify the segmental nucleated neutrophils (white). Indicated line scan (X1-X2) verifies the colocalization of EPLIN and LifeAct-positive actin filaments at sites of leukocyte transmigration, whereas PECAM1 was irregularly labeled. (B) Confocal microscopy of antibody-induced ICAM-1 clusters in TNF-α-activated HUVEC. Detection of ICAM-1 (blue), EPLIN (green) by indirect immunostaining and actin filaments are labelled by phalloidin-TRITC (red) as indicated. Upper panel shows, control (no ICAM-1 Ab treatment). Middle panel shows ICAM-1 Ab treated cells. Lower panel displays the larger view of cropped areas (white boxes in the middle panel) of ICAM-1 clusters and co-labelled proteins as indicated. (C) Anti-ICAM-1-coated beads or (D) human neutrophils were added to TNF-α-activated HUVEC cultures expressing either EPLIN-α-EGFP or EPLIN-β-EGFP respectively (M/E = moderate expression). After fixation, cells were additionally labeled with phalloidin-TRITC (red) for actin filament and ICAM-1 antibody (blue) as indicated. In panel D, transmigrating neutrophils were identified by phase contrast microscopy due to their segmented polymorphic nuclei (white). The fluorescence intensity of the blue channel has been adjusted to improve the visibility of the corresponding label. The representative merged channel is presented as overview and the white boxes indicate the larger view of the cropped areas.