Directional Transport of a Bead Bound to Lamellipodial Surface Is Driven by Actin Polymerization

Figure 6

Effect of the inhibitors of actin turnover or those of myosin II activity on the distribution of actin filaments and myosin II. Under each condition, distributions of actin and myosin II are compared in the same cell. (a) Actin filaments in the control cell; arrows, vague staining of actin filament network in the lamellipodium; arrowheads, actin bundles. (b) Myosin II in the same cell; arrowheads indicate the spot-like fluorescence attributed to myosin II mini-filaments, some of which reside along the actin bundles. Unlike actin, distribution of myosin II along the cell periphery was hardly observed. (c) Comparison of fluorescence intensity of actin filaments and that of myosin II measured along the white lines shown in (a) and (b). The fluorescence intensity was normalized as described in Section 2.9. The arrow and the dashed arrow indicate the start of the rapid rise of the profile of actin and that of myosin II, respectively. The symbol () indicates the plateau corresponding to the vague actin staining. (d) Derivative of the intensity profiles shown in (a) and (b); the arrow and the dashed arrow indicate the positions of local maxima of the derivative of the profiles of actin and that of myosin II, each of which corresponds to the rapid rise of each profile. (e) Actin filaments in the cell treated with 100 nM CytD; arrows indicate the distribution of actin bundles (i.e., stress fibers); arrowheads indicate the cell edge where rim-like distribution of actin filaments is seen. (f) The same cell stained for myosin II; there is some colocalization of myosin II with actin bundles (arrows). (g) Line profiles of fluorescence intensity for actin (black line) and myosin II (gray line) measured along the white lines shown in (d) and (e). The arrow and the dashed arrow indicate the start of the rapid rise of the profiles of actin and myosin II, respectively. The arrowhead indicates a peak of the fluorescence profile corresponding to the actin rim. ((h) and (i)) Distribution of actin filaments in the cell treated with 5 μM (−)-blebbistatin or with 5 μM Y-27632, respectively. Asterisks indicate lamellipodia; arrows in both panels indicate the vague staining of actin in lamellipodia; arrowheads in both panels indicate actin stress fibers. ((j) and (k)) Distributions of actin filaments and myosin II in the cell treated with 50 μM (−)-blebbistatin. Arrows indicate the vague staining of actin and arrowheads indicate actin bundles. (l) Line profiles of the intensity of actin filaments (black line) and that of myosin II (gray line) measured along the white lines shown in (i) and (j), respectively; the background (outside the cell) of the image field for myosin (k) was higher than that of actin probably because of the nonspecific binding of the secondary antibody to the glass surface. The wide plateau corresponding to the vague actin fluorescence is evident between ~3.5 μm and ~5.5 μm from the start of the profile plot. White thick bar in (j) indicates 10 μm.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)