Rac1-Dependent Lamellipodial Motility in Prostate Cancer PC-3 Cells Revealed by Optogenetic Control of Rac1 Activity

<div><p>The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P₃ and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.</p></div

Local and reversible control of lamellipodial dynamics by photomanipulation of PA-Rac1 activity.

<p>Time-lapse images of a PC-3 cell expressing mCherry-PA-Rac1 were acquired during PA-Rac1 photo-manipulation by local laser irradiation of different areas. Selected phase-contrast and mCherry fluorescence images are shown. First, region 1 was irradiated for 10 min. The irradiation was then moved to region 2. At 25 min, the irradiation was turned off. Selected time-lapse images of phase-contrast and mCherry fluorescence are shown. The extending and retracting lamellipodia are outlined in red and yellow, respectively. Scale bar, 10 µm.</p

Effect of LY294002 on the extended lamellipodial motility in PC-3 cells expressing constitutively active Rac1Q61L.

<p>PC-3 cells were transfected with pmCitrine-Rac1Q61L. The time-lapse images of phase-contrast and mCitrine-Rac1Q61L fluorescence were captured before (left) and after (right) the addition of LY294002. Kymographs were created to show the changes in length of a lamellipodium at the position of the two-headed line. The mCitrine-Rac1Q61L-expressing cell had an extended lamellipodium around its entire circumference. After the addition of 50 µM LY294002, the extended lamellipodium had shrunk only slightly, but the peripheral ruffling was pronounced. The kymographs show dynamic changes in length due to enhanced ruffling after the addition of LY294002. Scale bars, 10 µm.</p

PI3K-dependent WAVE2 recruitments to the leading edge of the extending lamellipodium during PA-Rac1 activation.

<p>PC-3 cells were co-transfected with pTriEx/mCherry-PA-Rac1 and pEGFP-N1-WAVE2. Phase-contrast, mCherry-PA-Rac1 (red fluorescence), and EGFP-WAVE2 (green fluorescence) images were acquired before and after PA-Rac1 photoactivation. PA-Rac1 photoactivation was repeated in the same cell region in the absence (control) or presence of 50 µM LY294002. The yellow arrowheads indicate that WAVE2 was recruited to the leading edge of the extending lamellipodium. In the presence of LY294002, WAVE2 was not recruited to the periphery of the cells where PA-Rac1 was photoactivated. The blue-dotted rectangle indicates the photoactivation area. Scale bar, 10 µm.</p

PI3K is required for lamellipodial extension but not for peripheral ruffling.

<p>(A) PC-3 cells were transiently transfected with pTriEx/mCherry-PA-Rac1. The cells were subjected to repeated photoactivation in the absence (control) or presence of 50 µM LY294002. The leading edge of the extending lamellipodium is outlined in red. Scale bar, 10 µm. (B) The increased cell area due to lamellipodial extension was quantified by subtracting the area of the cell before photoactivation from the area of the cell 7 min after the beginning of PA-Rac1 activation. This increase was verified in 22 PC-3 cells. The data plot shows the increased area due to lamellipodial expansion that was induced by PA-Rac1 photoactivation in the absence [LY(−)] or presence of LY294002 [LY(+)]. The significance of the differences between LY(−) and LY(+) was confirmed with the Wilcoxon t-test (right). The increase in the lamellipodial area in the presence of LY294002 was significantly lower than that of the control cells (p<0.01). (C) Kymographic analysis was performed at a two-headed arrow placed across the lamellipodium of a PC-3 cell expressing mCherry-PA-Rac1 before and after the addition of LY294002. The laser-irradiated area is indicated with a blue rectangle. The white line outlines the extending lamellipodium, and the dotted line outlines the original cell shape. The lower panel shows the kymograph of a lamellipodium undergoing changes in length. The kymograph demonstrates the extension and retraction of a lamellipodium during PA-Rac1 activation (blue 1) and deactivation (black 1), respectively. The green arrow indicates the addition of LY294002. The PI3K inhibitor had less of an inhibitory effect on lamellipodial extension and ruffling (blue 2). However, the initiation of lamellipodial extension was drastically inhibited (black 2–blue 3). Scale bars, 10 µm.</p

Development of an Artificial Calcium-Dependent Transcription Factor To Detect Sustained Intracellular Calcium Elevation

The development of a synthetic transcription factor that responds to intracellular calcium signals enables analyzing cellular events at the single-cell level or “rewiring” the intracellular information networks. In this study, we developed the calcium-dependent transcription factor (CaTF), which was cleaved by calpain and then translocated to the nuclei where it induced reporter expression. Our results demonstrated that CaTF-mediated reporter expression was stable and responded to the intracellular calcium level and calpain activity. In addition, CaTF detected the sustained calcium increase that was induced by physiological stimulation with epidermal growth factor (EGF). These results suggest that CaTF could be a useful tool to analyze intracellular calcium signals and be an interface between an endogenous signal network and synthetic gene network