Figure 1.
(A) Example of a neutrophil transmigrating at a tricellular junction of endothelial cells. The images were color-merged in ImageJ (VE-cadherin-GFP, green; phase contrast, red) to observe the location of transmigration in relation to endothelial cell borders. The white arrowhead points to the cell of interest that is transmigrating at the intersection of three endothelial cells. The time after plating neutrophils (in minutes∶seconds format) onto the endothelium is indicated in the top right corner of each image. Scale bar is 10 µm in all images. (B) Fraction of transmigrated cells (not total cells) that transmigrate via the bicellular junctions, tricellular junctions, or transcellular route, as a function of subendothelial matrix stiffness. Bars represent mean ± SEM of 3 independent experiments. * indicates P<0.05 with bicellular and tricellular junction routes on the same substrate stiffness by ANOVA, followed by Tukey's honestly significant difference criterion. There were no statistical differences between substrates for any route. (C) Time to complete transmigration as a function of route and subendothelial matrix stiffness. Bars represent mean ± SEM of pooled data from 3 independent experiments (on 0.87 kPa, N = 45, 37, 3 for bicellular, tricellular, and transcellular pathways, respectively; on 5 kPa, N = 28, 26, 5; on 280 kPa, N = 40, 21, 3). * indicates P<0.05 with 5 kPa and 280 kPa for the transcellular route by ANOVA, followed by Tukey's honestly significant difference criterion. (D) Time to complete transmigration is shown for all cells regardless of route taken, or cells taking the paracellular route (includes bicellular and tricellular junctions), as a function of subendothelial matrix stiffness. There were no statistical differences between any substrates or routes.
Figure 2.
Fraction of neutrophils that transmigrate.
Neutrophils were pretreated with DMSO (vehicle control), blebbistatin (“Bleb”; to inhibit myosin II), ML-7 (to inhibit myosin light chain kinase), nocodazole (“Noc”; to inhibit microtubule polymerization), taxol (“Tax”; to stabilize microtubules), latrunculin-A (“Lat-A”; to inhibit actin polymerization) or jasplakinolide (“Jasp”; to inhibit actin depolymerization) and plated onto a TNF-α-activated HUVEC monolayer on a 5 kPa polyacrylamide gel. Bars represent mean ± SEM of 3–4 independent experiments. * indicates P<0.05, and *** indicates P<0.001 with DMSO control using a t-test.
Figure 3.
Effects of actin stabilization (via jasplakinolide) or myosin II inhibition (via blebbistatin) on neutrophil morphology.
(A) Jasplakinolide-treated neutrophils are shown on a TNF-α-activated HUVEC monolayer on a 5 kPa polyacrylamide gel, approximately 20 minutes after plating neutrophils. White arrows point to protrusion structures that remain (and are not dynamic) throughout the entire timelapse. In subsequent panels, blebbistatin (“bleb”)-treated neutrophils were plated on (B) bare fibronectin-coated polyacrylamide gels (without an endothelium) and activated with 10 nM fMLF or (C) on TNF-α-activated HUVECs on a 5 kPa polyacrylamide gel. Arrowheads in panels B and C point to tails left behind by the neutrophils as it migrates along the gel (panel B) or as it transmigrates through the endothelium (panel C). Two examples of blebbistatin-treated neutrophils are given for panels B and C. Scale bar in panel B is 10 µm and applies to all images in panels A, B, and C.
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.
Figure 5.
Time to complete transmigration.
(A) Transmigration time (through endothelium on 5 kPa gel) for all neutrophils, from when the first protrusion is seen under the endothelium, to complete transmigration (except tails for the cases of blebbistain and ML-7), for all treatments. Bars represent mean ± SEM of pooled data from 3 independent experiments (N = 61, 103, 75, 182, 32 for DMSO, blebbistatin, ML-7, nocodazole, and taxol, respectively). ** indicates P<0.01 and *** indicates P<0.001 with DMSO vehicle control, using a t-test. (B) Transmigration times are shown separately for the “first cells” that enter the endothelium, as well as the “followers” that used the same gap to transmigrate as one of the first cells. Bars represent mean ± SEM of pooled data from 3 independent experiments (For first cells, N = 53, 77, 66, 146, 28, while for followers N = 8, 26, 9, 36, 4 for DMSO, blebbistatin, ML-7, nocodazole, and taxol, respectively). ** indicates P<0.01 and *** indicates P<0.001 with DMSO vehicle control for first cells, while ∧∧∧ indicates P<0.001 with DMSO vehicle control for followers, using a t-test. ### indicates P<0.001 between first cells and followers using a t-test. (C) Total transmigration time, including the time for the final tail to disappear beneath the endothelium. Bars represent mean ± SEM of pooled data from 3 independent experiments (N = 61,68,12 for DMSO, blebbistatin and ML-7, respectively). * and *** indicate P<0.05 or P<0.001 with the DMSO control, using a t-test. (D) Fraction of neutrophils with tails that remain above the endothelium for a prolonged length of time after transmigration of the majority of the neutrophil. Bars represent mean ± SEM of 3–4 independent experiments. * and ** indicate P<0.05 or P<0.01 with the DMSO control, using a t-test.
Figure 6.
Time to initiate transmigration.
(A) Mean time for initiation of transmigration through endothelial cells on a 5 kPa gel. Neutrophils were plated at time = 0. P>0.05 between all treatments and the DMSO vehicle control using a t-test. (B) Fraction of cells that initiated transmigration as a function of time for all treatments. Bars or data points represent mean ± SEM from 3–4 independent experiments.
Figure 7.
Schematic describing biophysical contributions of neutrophils during transmigration.
This schematic incorporates our work, as well as work of others (see text for discussion), and demonstrates the possible cross-talk between the cytoskeleton and contractility-regulating molecules during cell migration. As neutrophil LFA-1 binds to ICAM-1 on the surface of the endothelium, a signaling cascade is initiated inside the ECs, which ultimately leads to EC contraction and gap formation, as well as lateral displacement of VE-cadherin. A signaling cascade is simultaneously initiated in the neutrophils. Neutrophil actin polymerization initiates transmigration through the endothelial cell-cell junctions by protruding at the leading edge. Microtubule depolymerization activates GEF-H1, which stimulates the exchange of GDP to GTP on RhoA, and subsequent signaling to ROCK. Both ROCK and MLCK phosphorylate MLC, which induces actin-myosin contraction. MLCK- and myosin II-mediated contractile forces at the uropod promote retraction at the trailing edge, and by inhibiting these forces, the neutrophils cannot fully complete transmigration.