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First published online 30 July 2003
doi: 10.1242/jcs.00684

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Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA

View larger version (11K): [in a new window]   Fig. 1. Under-agarose chemotactic assay. Three parallel troughs are cut into the agarose. The center trough is filled with folate and cells are placed in the peripheral troughs. Cells then migrate towards the folate, deforming the agarose upwards while simultaneously flattening themselves. Arrow indicates direction of cell movement.

View larger version (12K): [in a new window]   Fig. 2. Distance traveled by cells under 0.5% agarose. At each time point (indicated in hours) the distance that the ten front-most cells in one field of view had traveled from the trough edge was determined. AX2 (wild type), ELC- (essential light chain mutant), ELC+ (essential light chain mutant rescued by expression of essential light chain cDNA), and mhcA- (myosin heavy chain null mutants).

View larger version (93K): [in a new window]   Fig. 3. Cell morphology under 2.5 and 0.5% agarose. Images of cells under agarose were acquired 5 hours after wild-type cells were added to the trough and 2 hours after mhcA- cells were added. The trough edge interferes with the imaging, so the optical field was place about 300 µm from the edge of the trough. AX2 (wild type), ELC- (essential light chain mutant), ELC+ (essential light chain mutant rescued by expression of essential light chain cDNA), and HK321 (myosin heavy chain null mutants).

View larger version (109K): [in a new window]   Fig. 4. Fragmentation of mhcA- cells moving under agarose. Myosin mutant cells were imaged under 0.5% agarose, within 500 µm of trough edge. The origin of the gradient and direction of cell movement is indicated by the white arrow. The montage shows images of a single cell over time as it becomes stretched and fragmented. The black arrow indicates a cell fragment left behind. Numbers indicate time in seconds. A QuickTime movie showing the fragmentation events can be viewed at http://jcs.biologists.org/supplemental/.

View larger version (55K): [in a new window]   Fig. 5. GFP-ABD120 localization in mhcA- cells under agarose. Confocal images taken 100 seconds apart of GFP-ABD120 localization in mhcA- cell. The cell is moving in the direction indicated by the arrow. GFP-ABD localizes to F-actin primarily in the rear of the cell. Magnification is 63x with a z-section thickness of 0.5 µm. A QuickTime movie showing the dynamic localization can be viewed at http://jcs.biologists.org/supplemental/.

View larger version (128K): [in a new window]   Fig. 6. mhcA- cells are unable to move the cell body under the edge of a 2% agarose trough. MhcA- cells were imaged at the edge of the trough. The dotted line indicates trough edge and the numbers indicate the time in seconds. The vertical white arrow points towards the origin of the chemoattractant gradient and the direction of cell movement. The cells can be seen extending pseudopods under the agarose (arrows), but the cell body remains in the trough so the cells cannot move up the gradient. A QuickTime movie showing the transition event can be viewed at http://jcs.biologists.org/supplemental/.

View larger version (92K): [in a new window]   Fig. 7. Localization of F-actin and myosin II during under-agarose migration. Confocal images taken of cells moving in the direction of the arrow under 2.0% agarose. The images shown are at 30-40 second intervals in a focal plane a few microns above the substratum. (A) Wild-type cells expressing GFP-myosin II. (B) mlcE- cells expressing GFP-Myosin II. (C) Wild-type cells expressing GFP-ABD120 to visualize F-actin dynamics. (D) mlcE- cells expressing GFP-ABD120. The localization and dynamics of actin and myosin II are not significantly altered in mlcE- cells. QuickTime movies of the fluorescence localization can be viewed at http://jcs.biologists.org/supplemental/.

View larger version (36K): [in a new window]   Fig. 8. Three-dimensional localization of myosin II in cells moving under agarose. z-series through the cells were acquired with a confocal microscope while the cells were moving under agarose. The sections shown are spaced approximately 0.2 µm apart. (A) Wild-type cells expressing myosin-GFP. (B) mlcE- cells expressing myosin-GFP. In both cases, the edge of the cell contains myosin-GFP throughout much of the 4-5 µm thickness. This would form a vertical wall of myosin and actin at the edge. The extent of this wall from front to back of the cell varies from cell to cell and over time. In many cells, the wall is primarily in an arc around the posterior of the cell as shown in Fig. 7A,B.

View larger version (50K): [in a new window]   Fig. 9. Model of myosin II function in cortical stability. Cross hatching indicates cross-linking function of myosin II in a cortical acto-myosin complex. Arrows indicate direction of cell movement and downward pressure resultant from resistance to deformation of agarose. (A,B) Deformation of agarose and flattening of cell that occurs during wild-type cell migration under agarose. (C,D) Lack of cross-linking occurring as a result of loss of myosin heavy chain function. (C) The ability of the cell to extend a protrusion under the agarose similar to wild-type cells. In D, although the cell was able to retract its midbody and subsequently migrate; downward pressure imposed upon the cell by the agarose results in the uropods not being withdrawn.

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