Yamashiro S et al. (2019), Convection-Induced Biased Distribution of Actin...

XB-ART-56612

Biophys J 2019 Jan 08;1161:142-150. doi: 10.1016/j.bpj.2018.11.022.

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Convection-Induced Biased Distribution of Actin Probes in Live Cells.

Yamashiro S , Taniguchi D , Tanaka S , Kiuchi T , Vavylonis D , Watanabe N .

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Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics. Because of its fast kinetics, Lifeact is generally believed to distribute evenly throughout cellular actin structures. In this study, however, we demonstrate misdistribution of Lifeact toward the rear of lamellipodia where actin filaments continuously move inward along the retrograde flow. Similarly, phalloidin showed biased misdistribution toward the rear of lamellipodia in live cells. We show evidence of convection-induced misdistribution of actin probes by both experimental data and physical models. Our findings warn about the potential error arising from the use of target-binding probes in quantitative live imaging.

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Genes referenced: actb
GO keywords: lamellipodium

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	Figure 1. (A) Images of Lifeact-mCherry (LA-mCherry, left) and EGFP-actin (middle) in live Xenopus XTC cells. After fixation, F-actin was stained with Alexa 647 phalloidin (A647-phalloidin, right). Representative data are shown (n = 6 cells). (B) Merged images of (A) showing rear-biased distribution of LA-mCherry in lamellipodia are given. (C) The average fluorescence intensity of the images in (A) along the yellow lines in the inset is shown. (D and E) Similar distribution of Atto-550 Lifeact (Atto-550-LA) and rhodamine-phalloidin (Rh-phalloidin) in fixed XTC cells is shown. Staining with Rh-phalloidin was carried out after washing out Atto-550 Lifeact, which is an exchangeable probe (6). Representative data are shown (n = 3 cells, see Fig. S1).
	Figure 2. (A) Fluorescent speckle images of Alexa 546 phalloidin (A546-phalloidin, left) and CF680R-actin (middle) in a live fish keratocyte. The probes were delivered into keratocytes by electroporation (15). F-actin was stained with Oregon Green phalloidin (OG-phalloidin) after fixation (right). (B) The average fluorescence intensity of the images in (A) along the white lines in the inset is shown. Representative data are shown (n = 4 cells). The region where the lamellipodium detached from the glass was excluded from analysis (the blue arrow in (A)).
	Figure 3. (A) Convection-induced biased distribution model. Because of fast reassociation and the retrograde actin flow, the probes are biased toward back of lamellipodia. (B) Measurement of Atto-488 Lifeact binding to cytoplasmic (upper) or muscle (lower) G-actin (blue dots) and F-actin (red dots) by fluorescence anisotropy is shown. Atto-488 Lifeact binds to F-actin with the lines showing the best fit to Kd = 3.4 μM for cytoplasmic actin and Kd = 2.3 μM for muscle actin. (C) Calculated distribution of LA-mCherry (red) in a model lamellipodium with a linear decrease in F-actin concentration (green) is shown. (D) Calculated distribution of Alexa phalloidin (red) in a model lamellipodium with a uniform F-actin distribution (green) is shown.
	Figure 4. Simulated concentration profile of free (A) and total (B) actin probes with various Kd. The other parameters, except for kon, are the same as those shown in Fig. 3 C and Table S1. F-actin distribution is depicted by a green dotted line. The amount of probe is measured in the same units on both panels.
	Figure 5. Simulated concentration profile of total LA-mCherry (A) and Alexa phalloidin (B) with various retrograde flow speeds. The other parameters, except for retrograde flow speed, are the same as those adopted in Fig. 3 C and Table S1. The linear F-actin distribution is depicted by a green dotted line.
	Figure 6. (A, C, E, and G) Images of Lifeact-EGFP (LA-EGFP) and Alexa 546 phalloidin (A546-phalloidin) in four live Xenopus XTC cells with the retrograde flow speeds indicated. The distributions of LA-EGFP and A546-phalloidin are compared to the F-actin distribution visualized by miRFP703-actin in live cells (A, E, and G) or staining with A546-phalloidin after fixation (C). (B, D, F, and H) The average fluorescence intensity of the images in (A, C, and E) or (G) along the yellow lines in the inset is shown. Representative data are shown (n = 11 cells). Bars, 5 μm.

References [+] :

Abraham, The actin-based nanomachine at the leading edge of migrating cells. 1999, Pubmed