How ZBP1 controls beta-actin mRNA compartmentalization to implement cell directionality and its implications on development and disease

Abstract

Directed cell migration is a fundamental process in tissue patterning during development, repair after injury, and diseases like metastatic cancer. Asymmetric localization of beta-actin mRNA has been known to play an important role in guiding cell directionality, although how it instituted this control was largely unknown. We hypothesized that beta-actin mRNA targeted to developing focal adhesions at the leading edge to reinforce these adhesions and direct migration. Cytoplasmic beta-actin mRNA localization is facilitated by the zipcode binding protein, ZBP1. Binding to the cognate zipcode within the 3'UTR of beta-actin mRNA enables ZBP1 to asymmetrically distribute mRNA and control its translation, a process known to enhance directed cell migration. TIRF microscopy was used to track single particles of beta-actin mRNA in live cells and enabled us to discover a specific compartment near focal adhesions where beta-actin mRNA dwells for periods greater than one minute. In ZBP1 KO fibroblasts the probability of tracking beta-actin mRNA around the adhesion compartment was significantly reduced. Consequently, adhesion lifetimes were reduced in ZBP1 KO cells. This supported our hypothesis that ZBP1 facilitated mRNA localization to strengthen focal adhesions and therefore directed cell migration. To test whether mRNA localization to adhesions could alter adhesion dynamics and cell motility, we developed a novel mRNA tethering technique. By tethering mRNA directly to focal adhesion complexes through MS2 stem loops in the 3'UTR of beta-actin mRNA and the MS2 coat protein fused to vinculin, we were able to show increased condensation of mRNA at all adhesions. This produced a significant increase in adhesion lifetime and adhesion size in cells expressing MS2-beta-actin mRNA. The effect on adhesion stability was translation-dependent since addition of the translation inhibitor, cycloheximide, reduced adhesion lifetimes significantly in cells with beta-actin mRNA tethered to adhesions. Additionally, cell velocity was significantly reduced in cells with tethered beta-actin mRNA. These experimental results lead us to conclude that beta-actin mRNA compartmentalization at adhesions, mediated by ZBP1, supplied a novel source of actin protein to strengthen adhesions and produce an asymmetric force distribution within the cell to institute directionality.