Imaging native beta-actin mRNA in motile fibroblasts

Abstract

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.

FIGURE 1

Sequestration of molecular beacons in the nuclei of chicken embryonic fibroblasts. (A) Changes in the intracellular distribution of a molecular beacon analog that lacks a quencher, and is designed to not have a target in the cell, as a function of time. (B) Identification of the sites within the nucleus where molecular beacons preferentially localize. TMR-labeled molecular beacon analog was injected into the cytoplasm of a fibroblast. Ten minutes after injection, the cells were fixed, permeabilized, and stained with fibrillarin-specific antibodies. Two bean-shaped regions within the nucleus in the diffraction interference contrast (DIC) image are the nucleoli, which acquire molecular beacon analog (TMR) and are stained by fibrillarin-specific antibodies (Cascade blue). (C) Kinetics of nuclear sequestration of the molecular beacon analog under different conditions. Solid symbols show the increase in nuclear fluorescence and open symbols show the decrease in cytoplasmic fluorescence. Square symbols represent free molecular beacon analog at 37°C, triangular symbols represent free molecular beacon analog at 25°C, and circular symbols represent molecular beacon analog complexed with streptavidin at 37°C. (D) Effect of prior cytoplasmic injection of wheat germ agglutinin (green) on the distribution of the molecular beacon analog (red).

FIGURE 2

Intracellular distribution of molecular beacon analogs complexed with streptavidin, as a function of time after cytoplasmic injection. The measurements of nuclear and cytoplasmic fractions as a function of time are presented in Fig. 1 C (solid and open circles).

FIGURE 3

Kinetics of hybridization of molecular beacons to chicken embryonic fibroblast β-actin mRNA. (A) Comparison of the kinetics of in vitro hybridization of free molecular beacons (continuous line) with molecular beacons linked to streptavidin (broken line). Fluorescence intensity is plotted as a function of time after the addition of a synthetic β-actin mRNA transcript. (B) Changes in total cellular fluorescence after injection of a mixture of streptavidin and biotinylated molecular beacons. Three of the molecular beacons in the mixture were labeled with TMR and were specific for β-actin mRNA (solid circles), and one was labeled with Texas red and had no target mRNA in the cells (open circles).

FIGURE 4

Imaging the distribution of β-actin mRNA in living fibroblasts. A mixture of three TMR-labeled β-actin mRNA-specific molecular beacons and a Texas red-labeled control molecular beacon, each complexed with streptavidin, was microinjected into each cell. After 1 h of incubation, a typical cell was imaged in quick succession with respect to (A) TMR and (B) Texas red. A ratio image (C) was obtained by dividing the fluorescence intensity of TMR by the fluorescence intensity of Texas red at every pixel in the image. The color of each pixel reflects the value of the ratio, with warmer colors representing higher ratios and cooler colors representing lower ratios. (D and E) Two ratio images of a typical cell that was grown in the absence of serum. Image D shows the steady state in the absence of serum, and image E shows the changes that occurred 2 min after the addition of fresh serum to the medium. (F) Table of colors used to represent the ratio at each pixel in images C, D, and E.

FIGURE 5

Translation of GFP mRNA hybridized to molecular beacons. A synthetic GFP mRNA hybridized within its coding region to three molecular beacons was injected into the cytoplasm of Chinese hamster ovary cells, and the cells were imaged for GFP and molecular beacons (TMR) after an overnight incubation. Injected cells are identified by the red fluorescence of the excess molecular beacons that accumulated in their nuclei. Two of the four injected cells show production of green fluorescent protein.