PMC

Spreading MDA-MB 231 cells depleted of myosin IIA and IIB were fixed and stained with paxillin antibodies, phalloidin, and DAPI. A) The formation of an organized actin network in the spreading cell expressing myosin IIA and IIB. Arrow-head and filled arrow indicate central fibers and transverse arcs in the scrambled siRNA received spreading cells, respectively. Focal contacts are connected to actin filaments in the lamellipodia and central part of the spreading cell. Loss of an organized actin network especially actin arcs and focal contacts are shown in the spreading cell depleted of myosin IIA. Myosin IIB-depleted cells display loss of transverse arcs and the formation of long actin fibers during spreading. In the spreading margins focal contacts are connected to these long actin filaments. The scale bars represent 20 µm.

A) Spreading HeLa-ATCC cell displaying focal contacts formation through out the membrane. Arrow and arrow-head indicate focal contacts formation in the central and cell margins of the spreading cell, respectively. B) Spreading HeLa-Clontech cells show focal contacts formation exclusively in the peripheral region. Arrow indicates loss of focal contacts formation in the central region and arrow-head shows the formation of focal contacts in the spreading margins. C) Transient expression of GFP-myosin IIB in HeLa-Clontech cells rescues the formation of focal contacts in the central part of spreading cell. The scale bars represent 20 µm. D) Graphical representation of focal contacts in spreading HeLa cells. E) Box-and-whisker plot showing the average area of focal contact. Numbers of images processed to quantify number and area of punctate structures were in between 12–16.

(A) Distributions of maximum volume loss of individual cells induced by spreading for control (n=170, N=3) and Y-27 (n=120, N=4) treated cells. (B) Average normalized volume of control HeLa Kyoto cells (gray, n=493, N=5) or treated with 100 µM Y-27632 (red, n=292, N=3) plated on PLL-PEG-coated glass. Error bars represent standard deviation. (C) Left: Average spreading area of 3T3-ATCC cells spreading on fibronectin-coated glass (n=20, N=3). Error bars represent standard deviation. Right: Average normalized volume of 3T3-ATCC cells spreading on fibronectin-coated glass (n=20, N=3). Error bars represent standard deviation. (D) Average normalized spreading area of control RPE-1 cells (gray, n=90, N=3), treated with 100 µM Y-27632 (red, n=85, N=3) or 100 µM CK-666 treated (magenta, n=48, N=3) spreading on fibronectin-coated glass. Error bars represent standard deviation. (E) Average normalized volume of control RPE-1 cells (blue, n=90, N=3) or control cells plated on PLL-PEG-coated glass (cyan, n=168, N=3). Error bars represent standard deviation. (F) Average normalized volume of control RPE-1 cells (gray, n=90, N=3), 100 µM Y-27632 treated (red, n=85, N=3) or 100 µM CK-666 treated (magenta, n=48, N=3) spreading on fibronectin-coated glass. Error bars represent standard deviation. (G) Volume flux (dV/dt) of 3T3-ATCC cells plotted versus their spreading speed (dA/dt) at the first 10 min of spreading (n=20, N=3). The data are fitted with linear regression y=−0.23x+2.51, R²=0.42. (H) Volume flux (dV/dt) of RPE-1 cells plotted versus their spreading speed (dA/dt) before (green) and after (magenta) lamellipodia formation (n=90, N=3). Error bars represent standard error. (I) Median volume flux (dV/dt) of control (blue, n=89, N=3), 100 µM Y-27632 (red, n=85, N=3) or 100 µM CK-666 (magenta, n=38, N=2) treated RPE-1 cells plotted versus their spreading speed (dA/dt) before and after lamellipodia formation. Error bars represent standard error. (J) Average spreading area (left) and average normalized volume (right) of control HeLa cells and treated with 1 mM MnCl₂ (N=3, n=57).
Figure 2—figure supplement 1—source data 1.Data tables related to quantifications in Figure 2 Supplement 1.

Cells were fixed after 60–70 min of spreading and stained with vinculin antibodies and DAPI. A) Loss of focal contacts in spreading COS-7 cells transiently expressing GFP. Arrow indicates loss of focal contact formation in the spreading cell. B) Transient expression of GFP-myosin IIA rescues formation of focal contacts. Arrow indicates focal contact formation. C) Transient expression of GFP-myosin IIB does not rescue focal contacts formation in the spreading cells. Arrow indicates loss of focal contact formation. D) Graphical representation of number of punctate structures (focal contacts) per cell. Confocal z-sections were exported into TIF images and then processed to quantify number and area of punctate structures using ImageJ program (NIH). Numbers of images processed to quantify number and area of punctate structures were in between 12–15. E) Box-and-whisker plot showing the average area of focal contact. We have observed a wide range of areas of punctate structures in pixels and therefore showed by Box-and-whisker plot.