Figure 4. Incomplete transmigration upon inhibition of contractility.

(A) A representative phase contrast sequence of a DMSO-treated neutrophil transmigrating through the endothelium on a 5 kPa gel. The neutrophil on top of the endothelium appears bright white in phase contrast microscopy, while the portion that has already transmigrated through the endothelium appears dark (white arrow). (B) A representative phase contrast sequence of blebbistatin-treated neutrophils transmigrating through the endothelium. Black arrowhead points to the first cell of interest. White arrows point to the darkened portion of the neutrophil under the endothelium. White arrowheads point to the portion of the tail that remains above the endothelium for several minutes after the rest of the neutrophil has transmigrated. Gray arrowheads point to another neutrophil that attempts to transmigrate, but does not ever fully succeed. For panels A and B, time = 0:00 (in minutes∶seconds format) in the upper right corner of the first image in each sequence indicates the time of the frame just before transmigration begins. (C) An example of an ML-7-treated neutrophil (indicated by black arrowhead) that makes two attempts at transmigration. A protrusion is sent beneath the endothelium (designated by white arrow at 0:30), but the neutrophil does not transmigrate on this attempt. Later, at time = 2:50 the neutrophil begins to transmigrate again (designated by white arrows in images 2:50–3:10). Similar to blebbistatin, a tail is left behind (white arrowhead at 3:10), but full transmigration, including the tail, occurs by time = 4:30. Scale bar for the first image in each sequence is 20 µm. (D) A time sequence is shown for a blebbistatin-treated neutrophil that transmigrates, leaves a tail behind, and later detaches from the tail. Time after plating neutrophils is shown in the upper right corner of each image in minutes∶seconds fomat. Scale bar in panel D is 20 µm and applies to all images in panel D.