The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, ‘cadherin fingers’, which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.
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We thank G. Crabtree, M. Lin and X. Liu for providing constructs, M. Teruel for reagents to generate Giardia-diced siRNA, and the Stanford Shared FACS Facility for support. E. Wagner and M. Glotzer generously provided constructs and advice for local Rho activation. We are grateful to S. Collins, D. Garbett, A. Suvrathan, G. Dey and M. Galic for helpful discussions and comments on the manuscript. A.H. was supported by postdoctoral fellowships from the Swiss National Science Foundation and from the Human Frontiers Science Program Organization. This work was supported by NIH grants GM063702 and MH095087.
Integrated supplementary information
HUVEC plated at low and high density were stained with Hoechst and imaged using widefield fluorescence microscopy (4×, 0.2 NA) at 10 min intervals for 4 h. Streams and swirls of coordinately moving HUVEC can be seen in high-density cultures (right, 792 cells/mm2, coordination score 0.24), but not in low-density cultures (274 cells/mm2, coordination score 0.07). Scale bar, 200 μm. Displayed at 15 fps.
A monolayer of HUVEC stably expressing CDH5-mCitrine was wounded and collective migration was monitored using widefield fluorescence microscopy (20×, 0.75 NA) at 5 min intervals. Cadherin fingers extending from the rear of migrating cells are highlighted by circles. Scale bar, 20 μm. Displayed at 3 fps.
HUVEC stably expressing CDH5-mCitrine were imaged at 1 min intervals using widefield fluorescence microscopy (40×, 1.3 NA). The life-time of individual cadherin fingers ranges from minutes to hours. Scale bar, 10 μm. Displayed at 10 fps.
Loss of polarized cadherin fingers in cells treated with the Arp2/3 inhibitor CK666, the ROCK inhibitor Y27632, or Thrombin.
HUVEC stably expressing CDH5-mCitrine were subjected to live-cell imaging (40× 1.3 NA, 1 min intervals) and were either control-treated or treated with CK666 (200 μM), Y27632 (20 μM), or Thrombin (1U/ml) after the first 5 frames. In CK666 and Y27632-treated cells the number of cadherin fingers decreases after drug addition. Thrombin treatment causes serrated, symmetric cell-cell junction. Scale bar, 10 μm. Displayed at 5 fps.
In HUVEC stably expressing the reporter, and plated at low density, mTurquoise-MLC is depleted from protrusions, indicating depletion of myosin II activity from protrusive actin networks. Acquired at 5 s intervals, displayed at 8 fps. Scale bar, 10 μm.
HUVEC stably expressing Ftractin-mCherry-P2A-mTurquoise-MLC were imaged at 5 s intervals. At the interface between the leader (top) and the follower cell (bottom), mTurquoise-MLC is depleted in the front of the follower cells. Acquired at 15 s intervals, displayed at 8 fps. Scale bar, 10 μm.
HUVEC stably expressing CDH5-mCitrine were co-plated with HUVEC stably expressing Ftractin-mCherry-P2A-mTurquoise-MLC at a ratio of 10:1 and imaged at 30 s intervals. Separate channels, overlay and the ratio of mTurquoise-MLC/Ftractin are shown. A low mTurquoise-MLC/Ftractin-mCherry ratio was observed in the front near where cadherin fingers are present. Scale bar, 10 μm, displayed at 15 fps.
HUVEC stably expressing CDH5-mCitrine were imaged at 1 min intervals using widefield fluorescence microscopy (40×, 1.3 NA). Automatically detected incoming (green) and outgoing (red) cadherin fingers are shown. Scale bar, 10 μm. Displayed at 8 fps.
A monolayer of HUVEC stably expressing CDH5-mCitrine (white) was stained with Hoechst (blue) and imaged at 4 min intervals using widefield fluorescence microscopy (20×, 0.75 NA). The video focuses on a turning cell. The nuclear trajectory and incoming cadherin fingers (+) are marked. Incoming cadherin fingers accumulate laterally and predict the direction of cell turning. Scale bar, 20 μm, displayed at 8 fps.
The same turning cell as in video 9 is shown. The nuclear trajectory and outgoing cadherin fingers (+) are marked. Outgoing cadherin fingers do not predict the direction of cell turning. Scale bar, 20 μm, displayed at 8 fps.
HUVEC stably expressing CDH5-mCitrine and transiently transfected with Lyn11-FRB and mCherry-FKBP-GEF(TIAM1) were imaged live using a 40× 1.3 NA objective and at 20 s intervals. Rapamycin was added after frame 7 to synthetically activate Rac and induce protrusive actin polymerization. Increased Rac activity was insufficient to increase the number of incoming or outgoing cadherin fingers. Scale bar, 10 μm, displayed at 5 fps.
Increased contractility through synthetic Rho activation is sufficient to induce outgoing cadherin fingers.
HUVEC stably expressing CDH5-mCitrine and transiently transfected with Lyn11-FRB and mCherry-FKBP-GEF(TIAM1) were imaged live using a 40× 1.3 NA objective and at 1 min intervals. Rapamycin was added after frame 5 to synthetically activate Rho and induce actomyosin contractility. Increased Rho activity was sufficient to induce outgoing cadherin fingers, while incoming cadherin fingers were lost. Scale bar, 10 μm, displayed at 5 fps.
Increased contractility at the onset of mitotic cell rounding is sufficient to induce outgoing cadherin fingers.
HUVEC stably expressing CDH5-mCitrine were subjected to live-cell imaging, using a 40× 1.3NA objective and at 1 min intervals. The increased contractility in the cell undergoing mitotic cell rounding causes a loss of incoming cadherin fingers, but initiates formation of outgoing cadherin fingers between itself and its neighbors. Scale bar, 10 μm, displayed at 5 fps.
HUVEC stably expressing CDH5-mCitrine were transiently transfected with Stargazin-GFP-LOVpep and (PDZ)2-mCherry-GEF(LARG). Live-cell imaging was done using a 60× 1.35 NA objective for 60 frames at 30 s intervals using a 514 nm laser for illumination. Transfected cells were identified by mCherry fluorescence (not shown) and Rho activity was locally activated in circular regions using 455 nm laser at frames 11-21. Locally increased contractility induced the formation of outgoing cadherin fingers. Scale bar, 10 μm, displayed at 7 fps.
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Integrative Biology (2019)