Comparative analysis of tools for live cell imaging of actin network architecture

Abstract

Fluorescent derivatives of actin and actin-binding domains are powerful tools for studying actin filament architecture and dynamics in live cells. Growing evidence, however, indicates that these probes are biased, and their cellular distribution does not accurately reflect that of the cytoskeleton. To understand the strengths and weaknesses of commonly used live-cell probes--fluorescent protein fusions of actin, Lifeact, F-tractin, and actin-binding domains from utrophin--we compared their distributions in cells derived from various model organisms. We focused on five actin networks: the peripheral cortex, lamellipodial and lamellar networks, filopodial bundles, and stress fibers. Using phalloidin as a standard, we identified consistent biases in the distribution of each probe. The localization of F-tractin is the most similar to that of phalloidin but induces organism-specific changes in cell morphology. Both Lifeact and GFP-actin concentrate in lamellipodial actin networks but are excluded from lamellar networks and filopodia. In contrast, the full utrophin actin-binding domain (Utr261) binds filaments of the lamellum but only weakly localizes to lamellipodia, while a shorter variant (Utr230) is restricted to the most stable subpopulations of actin filaments: cortical networks and stress fibers. In some cells, Utr230 also detects Golgi-associated filaments, previously detected by immunofluorescence but not visible by phalloidin staining. Consistent with its localization, Utr230 exhibits slow rates of fluorescence recovery after photobleaching (FRAP) compared to F-tractin, Utr261 and Lifeact, suggesting that it may be more useful for FRAP- and photo-activation-based studies of actin network dynamics.

Keywords: Cytoskeleton; actin; cell architecture; fluorescent protein reporters; live cell imaging.

Figure 1.

Comparison of mCherry-actin localization with eGFP-tragged actin reporters. mCherry-actin and eGFP reporter localization in fixed S2 cells with corresponding linescans in the lamellum and lamellipod for (A–B) F-tractin-eGFP, (C–D) Lifeact-eGFP, (E–F) Utr261-eGFP, (G–H) eGFP actin (control). Scale bars indicate 5 microns.

Figure 2.

Comparison of live-cell actin probes and phalloidin in Drosophila S2 cells on ConA. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on ConA stably expressing (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP. Scale bars indicate 5 microns.

Figure 3.

Intensity plot profiles of actin probes and phalloidin in DrosophilaS2 cells on ConA Comparison of intensity profiles across lines (shown in yellow) for Alexa 564 phalloidin (top) and eGFP actin reporters (bottom) in fixed S2 cells on ConA. (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP.

Figure 4.

Comparison of live-cell actin probes and phalloidin in DrosophilaS2 cells on PDL. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed S2 cells on PDL stably expressing (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP. Scale bars indicate 5 microns.

Figure 5.

Comparison of live-cell actin probes and phalloidin in XenopusXTC cells on PLL. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed XTC cells transiently expressing (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP. Scale bars indicate 5 microns.

Figure 6.

Comparison of live-cell actin probes and phalloidin in mouse B16-F10 cells on laminin. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed B16-F10 cells stably expressing (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP.

Figure 7.

Comparison of live-cell actin probes and phalloidin in human U2-OS cells on fibronectin. Comparison of Alexa 564 phalloidin localization and eGFP actin reporters in fixed U2-OS cells stably expressing (A) eGFP, (B) eGFP-actin, (C) Utr230-eGFP, (D) Utr261-eGFP, (E) F-tractin-eGFP and (F) Lifeact-eGFP. Scale bars indicate 5 microns.

Figure 8.

FRAP of actin reporters at U2-OS cell stress fibers and B16-F10 lamellipodia. (A) Normalized FRAP recovery curves for actin reporters on U2-OS cell stress fibers. Color scheme follows: cyan, eGFP-actin; red, Utr261-eGFP; purple, Lifeact-eGFP; green, F-tractin-eGFP; orange, Utr230-eGFP. (B) Inset from (A) showing early recovery of Utr261-eGFP, F-tractin-eGFP and Lifeact-eGFP. (C) Normalized FRAP recovery curves for actin reporters in the mouse B16-F10 lamellipod. (D) Inset from (C) showing early recovery of Utr261-eGFP, F-tractin-eGFP and Lifeact-eGFP.