In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.
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This work was initiated as a collaborative effort between R.S.F. and G.D. (NIH R21 CA124990). H.E. and G.D. are supported by NIH R01 GM090317. R.S.F., R.S.A., K.A.M. and C.M.W. are supported by the NHLBI Division of Intramural Research.
Integrated supplementary information
3D z-stacks of AECs expressing TdTm-CAAX were acquired at 10 minute intervals using a spinning disk confocal microscope. Shown is maximum intensity projection reconstruction. Grid scale: Major tick marks, 10 μm. Total elapsed time shown at lower right in min.
First frame: rendering of the segmented surface. Subsequent frames show successive rounds of computational thinning to achieve the morphological skeleton. Frame size is 130 μm lateral × 65 μm axial.
Curvature category mapping on the surface of a cell treated with 20 μM blebbistatin, with curvature categories as described in Fig. 1 and Supplementary Fig. 2.
Rotation is about an axis 70 degrees off of the z-axis, to show sides of thin branches. Total frame size is 95 μm × 60 μm.
Maximum intensity reconstruction of fluorescence (red) of an AEC expressing TdTm-CAAX migrating in a 3D collagen gel. Time-lapse 3D image sequence was collected by spinning disk confocal microscopy at 10 min intervals (elapsed time shown in lower right as hours:min:sec). In each frame, the position of the cell morphological skeleton is shown in green (branches) and blue (body segments), and branch tips and vertices are highlighted with large and small red spheres, respectively. Morphological skeleton evolution over time is shown in colour scale from purple (T = 0) to white (T = 300 min) show in white. Scale grid = 5 μm, minor tick marks = 1 μm. Total elapsed time shown at lower right in minutes.
Maximum intensity reconstruction of fluorescence (red) of an AEC expressing TdTm-CAAX migrating in a 3D collagen gel collected by 3D spinning disk confocal microscopy imaging at 10 min intervals. The blue sphere shows the centermost point, the coloured trace shows path over time, with red indicating the starting frame and white indicating final position. Grid scale = major tick marks, 5 μm, minor tick marks = 1 μm. Total elapsed time shown at lower right in minutes.
Red values indicate highest maximal local curvature, blue values indicate lowest values. Scale grid is in microns. Time interval between frames is 300 s.
Example of a segmented region of interest fused for analysis of local curvature and cortical GFP-myosin IIA intensity over time.
Maximum intensity reconstruction of fluorescence (red) of time-lapse 3D imaging of an AEC expressing GFP-myosin-IIA collected by spinning disk confocal microscopy at 10 minute intervals during migration in a 3D collagen gel. Grey area indicates region segmented and cropped for surface curvature measurement and cortical GFP-myosin-IIA intensity. Time stamp shown in lower left in min, total time 60 min. Grid spacing = 10 μm.
Maximum intensity reconstruction of fluorescence (red) of time-lapse 3D imaging of an AEC expressing GFP-myosin-IIA collected by spinning disk confocal microscopy at 3 minute intervals during migration in a 3D collagen gel. Frame playback is paused during movie presentation to illustrate regions of cortical GFP-myosin IIA accumulation at the base of branches oriented normal or non-normal to the major axis of the cell body. Grid spacing = 10 μm. Total elapsed time shown at lower left in min.
About this article
Acta Mechanica Solida Sinica (2019)