Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.
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Comparison of GFP-UtrCH and fluorescent phalloidin in wounded, fixed oocytes. (
a) Low magnification images showing that in samples fixed with an aldehyde-based fixative, fluorescent phalloidin (AX-568-PH) and GFP-UtrCH colocalize, and reveal a striking concentration of F-actin around wounds (arrow). Immunolocalization of tubulin reveals modest preservation of microtubules in the interior of the wound (arrowhead). ( b) In samples extracted with methanol after aldehyde fixation, phalloidin staining is completely eliminated, while staining with GFP-UtrCH is preserved. Microtubule preservation is superior under these conditions. ( c) High magnification view showing colocalization of fluorescent phalloidin and GFP-UtrCH on cables of F-actin extending away from the wound. ( d) High magnification view showing colocalization of fluorescent phalloidin and GFP-UtrCH on actin “fingers” that extend into the wound. ( e) Images showing that in cells treated with latrunculin to disrupt F-actin, phalloidin and GFP-UtrCH colocalize in large, unorganized patches around wounds (W). Scale bars is 20 μm.
The UtrCH probes do not perturb the F-actin cytoskeleton. (
a) Microinjection of 40 or 160 ng of GFP-UtrCH mRNA into Xenopus oocytes does not increase the amount of pelletable (P) F-actin compared to uninjected (Unj) controls as indicated by Western blotting. ( b) Oocytes microinjected with GFP-UtrCH appear to heal normally after wounding. Comparisons of several timepoints from 4D movies showed no physiological differences in overall appearance or structure, healing rate, or myosin-based cortical flow between GFP-UtrCH or AX-568 actin injected oocytes. ( c) Kymographs of oocytes injected with GFP-UtrCH, RFP-UtrCH or AX-568 actin reveal similar rates of F-actin movement toward the wound site (W). Actin flow rates were determined by measuring the distance travelled by individual fluorescent points (arrowheads) over time (T). ( d) Quantification of actin flow rates in cells injected with 40 or 160 ng GFP-UtrCH mRNA, 40 ng RFP-UtrCH mRNA, or AX-568 actin. There are no significant differences in the measured flow rates ( P > 0.05).
GFP-UtrCH labels F-actin structures in living echinoderm embryos and oocytes. (
a) Frames from a 4D sequence of a 16-cell purple urchin embryo expressing GFP-UtrCH from injected mRNA; vegetal view. The four small cells in the center are the micromeres; their sisters, the macromeres, divide in this sequence. Each image is a brightest-point projection of ten 1-μm sections. Probe accumulation (arrowhead) in the equatorial cortex is approximately coincident with the onset of furrowing. ( b) Frames from a time-lapse sequence at a single focal plane through the blastula epithelium in a sand dollar embryo. GFP-UtrCH accumulates on the nuclear membrane in inter-phase, brightening just before nuclear envelope breakdown (see cells labeled 1–4; the pair of cells labeled “3” has just divided at the beginning of the sequence, and by 3 min. have accumulated F-actin on the nuclear membrane). ( c) Germinal vesicle (GV) breakdown in a sea star oocyte. Actin assembly proceeds in a wave starting at the interior side of the GV. Each timepoint is a projection of eight 1-μm sections. Scale bar is 25 μm.
Labeling and distinguishing between relatively more dynamic and stable actin structures in
Xenopus oocytes. ( a) Frames from a 4D movie show that GFP-UtrCH more effectively labels the stable actin that resides on the interior of contractile ring (arrowhead) than AX-568-actin. ( b) Comparisons of GFP-UtrCH to total actin distributions by antibody staining in fixed oocytes show few differences at the leading or on the contractile ring but do exhibit differences at the trailing edge. ( c) Quantifications of fluorescent intensities at the leading edge (LE), on the contractile ring (CR), and locations away from the contractile structure (BG) showed no differences ( P > 0.05, n = 24) between antibody staining and the UtrCH probe. Significant differences in fluorescent intensities were observed at the trailing edge (TE, P < 0.05, n = 24). ( d) Quantifications of actin comets from oocytes injected with AX-488 actin and RFP-UtrCH revealed that the ratio of AX-488 actin to RFP-UtrCH is greater at the newly assembled head of the comet than the older tail regions (Asterisks indicate P < 0.05, n = 24). ( e) High magnification images showing the growing actin comet head (arrowhead) is labelled more intensely with AX-488 actin than RFP-UtrCH.
Using photobleaching and photoactivation with the UtrCH probes to monitor the contributions of the stable and dynamic F-actin populations. (
a) Frames from a 4D sequence comparing the simultaneous fluorescence recovery of AX-568 actin and fluorescence loss of PaGFP-UtrCH in the same region of the oocyte cortex. ( b) Measurements of fluorescence recovery of AX-568 actin parallels the inverse of fluorescence loss of PaGFP-UtrCH. ( c) Frames from a 4D sequence contrasting the fluorescence loss of PaGFP-UtrCH in control and jasplakinolide treated oocytes. ( d) Quantification of fluorescence loss of PaGFP-UtrCH in control and jasplakinolide treated cells reveals that fluorescence loss is reduced in jasplakinolide treated oocytes. ( e) Measurements of PaGFP-UtrCH fluorescence loss in point-activated control and jasplakinolide treated oocytes also exhibited a reduced rate of fluorescence loss in jasplakinolide treated oocytes. ( f) Fluorescence decay half-times of point-activated PaGFP-UtrCH in jasplakinolide-treated and untreated controls. Asterisks indicate P < 0.05. ( g) Combined use of RFP-UtrCH and PaGFP-UtrCH reveals a clear subdivision in F-actin populations around wounds. The oldest (i.e. most stable; green around the wound) F-actin becomes enriched on the interior of the actin array. This population of F-actin becomes surrounded by more dynamic F-actin (red). Scale bars = 30 μm.
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