Fig. 2
From: A specific role for endothelial EPLIN-isoform-regulated actin dynamics in neutrophil transmigration
Dynamics of fluorescently labeled EPLIN-isoform in endothelial cells during neutrophil transmigration. (A) Fluorescence and phase contrast live cell images of transmigrating neutrophils through TNF-α-activated HUVEC monolayers moderately expressing EPLIN-α-EGFP. After leukocyte attachment (t = 00:00), EPLIN-α-EGFP is rapidly recruited to the gap where neutrophils adhere (dotted lines). Transmigration was nearly complete after t = 02:00. As expected EPLIN-α-EGFP also appears as a component of linear actin filaments (red arrows). Subsequently, extended EPLIN-α-EGFP structures appeared in the time range between 02:00–07:10, indicating membrane protrusions (white arrows) followed by gap closure (t = 02:00—07:10, mm:ss). (B) Illustrations of the dynamic process shown in A. (B) The indicated line scans show extended gap formation (black arrows) at time 01:30. Subsequently, from time 02:40 to 05:20, the intensity of EPLIN-α-EGFP increases, followed by upregulated dynamics leading to gap closure. (C) Illustrations of the dynamic process shown in A. EPLIN-α positive protrusions can occur at the onset of transmigration as part of docking structures, but consistently appear during pore closure. (D) Simultaneous expression of EPLIN-α-mCherry together with LifeAct-EGFP in HUVEC show colocalization of EPLIN-α and actin filaments during leukocyte transmigration. Arrow heads indicate colocalization of actin filament and EPLIN-α-mCherry at membrane protrusions (white arrowheads). The red cross indicates another leukocyte entering the image. White dashed area shows yellow spots indicating colocalization, but also show separations of EPLIN-α-mCherry and actin around the gap, characteristic for EPLIN-α dynamics as it moves behind branched actin filaments to terminate protrusions by binding to the Arp2/3 complex. (E) Pearson correlation coefficient analysis of three neutrophil transmigration sites shows transient increased colocalisation of EPLIN-α-mCherry and LifeAct-EGFP at 05:25, coinciding with pore closure followed by decrease. Unpaired t-test, p = 0.0409.
