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First published online 4 March 2003
doi: 10.1242/jcs.00340

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Mechanics of cell spreading: role of myosin II

Tetsuro Wakatsuki1, Robert B. Wysolmerski2 and Elliot L. Elson1,*

1 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA
2 Department of Pathology and Anesthesiology, St Louis University School of Medicine, St Louis, MO 63104, USA



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  		Fig. 3. Effect of CD concentration on STA- and KT-treated cells. The dependence of cell area on CD concentration was observed for STA or KT-treated cells spreading for 30 minutes (error bars are standard errors of at least 20 measurements). The sigmoidal curve was well fit to the data plotted on the semi-logarithmic scale and yielded the effective half-maximum inhibitory dose (IC50) of CD on STA- and KT-pretreated cells of 3.5 nM and 20 nM, respectively.

 


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  		Fig. 1. Myosin activity during cell spreading with and without STA and KT. During cell spreading, 30-40% of the myosin RLC was phosphorylated at a steady level for 1 hour and the RLC was further phosphorylated at 2 hours after spreading even without serum (•). The specific RLC kinase inhibitor KT (1 µM) reduced the amount of the phosphorylated RLC to less than 10% of the total RLC for 30 minutes after re-plating ({circ}). The relatively nonspecific kinase inhibitor STA (100 nM) ({triangleup}) suppressed the RLC phosphorylation to less than 5% for a much longer period (2 hours) compared with KT. The quantitative level of RLC phosphorylation (A) was detected by modified western blots (B). Data represent the averaged value of at least three independent experiments (A). U, 1P and 2P indicate unphosphorylated, mono-phosphorylated and di-phosphorylated myosin light chains respectively (B).

 


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  		Fig. 2. Quantitative analysis of cell spreading with and without STA and KT. Cells treated with KT and STA started to spread much earlier than did cells without these compounds (A). Quantitative measurements of cell areas clearly showed an acceleration of the rate of cell spreading by STA and KT (error bars are standard deviations) (B). The difference in the area of cell spreading with and without STA and KT at 30 minutes is statistically significant (P<0.05).

 


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  		Fig. 4. Localization of actin and myosin II during cell spreading. Actin was stained with rhodamine-labeled phalloidin, and myosin II was immunolocalized using myosin II antibodies stained with Alexa-488-labeled secondary antibody. The cells shown in the images in the first (A,D,G,J,M), second (B,E,H,K,N) and third (C,F,I,L,O) column from the left are treated with DMSO, KT and STA, respectively. The cells shown in the first/second rows (A-F) and third/fourth rows (G-L) and the last row (M-O) of the images are fixed 10, 30 and 120 minutes after seeding the cells on the dishes, respectively. At 10 minutes after beginning cell spreading, myosin II was localized diffusely with or without STA and KT, yet the samples treated with STA and KT showed many F-actin spikes (A-C). Cross-sectional views of the cells in A-C are shown in D-F, whose cell heights were significantly reduced by myosin II inhibition. At 30 minutes, control cells started to spread asymmetrically, but cells treated with STA and KT were spread into almost circular shapes (G-I). This suggests that myosin might be required for polarization of spreading cells. Localization of myosin II at this stage was not different with or without the KT and STA. Cross-sectional views of cells showed significant differences in flattening of cells due to the increased rate of cell spreading in KT- and STA-treated cells (J-L). The myosin II strongly co-localized with actin filaments at 120 minutes without STA and KT (M). Cells treated with KT also started to show co-localization with actin filaments, which is consistent with the level of myosin RLC phosphorylation shown in Fig. 2 (N). Many cells treated with STA started to break into fragments (O).

 


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  		Fig. 5. Mechanical properties of cells with and without KT at early stages of cell spreading. The indentation measurements were performed on cells spreading 10-30 minutes after they settled on the substratum. The first point of contact on the cell made by a vertical cylindrical tip as well as the initial distance of the tip from the substratum were observed by indentation on the cell and the substrate next to the cell. The difference between them is the cell height shown in A. As we observed fixed cells by microscopy (Fig. 4), the heights of live cells treated with KT are significantly reduced (P<0.05) compared with those of control cells (A). Force responses to indentation by the cylindrical tip were plotted against % indentation (B). The data represent averaged values and standard errors of 25 and 15 cells with and without KT, respectively. The curve has an indentation phase (loading) indicated by a solid arrow and a retraction phase (unloading) indicated by a dotted arrow. The indentation phase force responses showed a significant reduction of stiffness in KT-treated cells compared with the control. The retraction phase for control cells showed no significant change in KT-treated cells (B).

 


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  		Fig. 6. Forces involved in cell spreading. Myosin II maintains the integrity of the actin cytoskeleton (small white arrows) as well as generating the traction force on the substratum (large black arrows). In the presence of serum, myosin II is fully activated. Hence the traction force is detectable (A). Without serum, the traction force is not detectable; either with or without serum, myosin II activity lasts briefly and counterbalances the protruding force (small gray arrows) due to actin polymerization (B). Hence, the traction force is not detectable. When myosin II activity is inhibited, the cell can spread without the counterbalancing force (C). Therefore, inhibition of myosin II activity increases the rate of cell spreading.

 

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© The Company of Biologists Ltd 2003