The physiological significance of beta -actin mRNA localization in determining cell polarity and directional motility

Abstract

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Figure 1

Movement of CEFs in the presence of (Upper) zip code sense (C⁺) and (Lower) antisense oligonucleotides (C⁻). Pictures depict frames 5 min apart from the video analysis of two fields of cells. Arrows indicate direction of movement of each cell over the subsequent frames. Note that cells move in the presence of sense, but not antisense, oligonucleotides.

Figure 2

β-actin mRNA localization in the presence of zip code sense oligonucleotides (C⁺) or antisense oligonucleotides (C⁻). Perimeter and centroid (dots) plots are from ODN-treated cells over the time frames of analysis (1 min). These results are representative of the analysis of populations of cells depicted in Fig. 1. (A) Sense-treated cell. (B) Antisense-treated cell. Note that the antisense oligonucleotides cause loss of polarized cell movement defined as a linear centroid track (arrow in A).

Figure 3

Sites of rhodamine actin incorporation in zip code sense-treated (C⁺) and antisense treated (C⁻) cells. (A–C) Sense treatment. (D–F) Antisense treatment. (A and D) Rhodamine actin incorporation showing the location of free barbed ends on actin filaments. (B and E) FITC-phalloidin-labeling of all actin filaments. (C and F) Phase-contrast image. (Bar, 10 μm.) Note that rhodamine actin incorporation sites are unpolarized in antisense-treated cells.

Figure 4

Localization of β-actin protein in zip code antisense (A and B) compared with sense-treated cells (C and D). (A and C) Staining with anti-β-actin antibodies. (B and D) Nomarski optics. (Bar, 10 μm.) Note that the β-actin staining is not as prominently localized to the leading edge in antisense-treated cells.