Two- and three-dimensional tracking ofMFA2mRNA molecules in mating yeast

Intracellular mRNA transport contributes to the spatio-temporal regulation of mRNA function and localized translation. In the budding yeast, Saccharomyces cerevisiae , asymmetric mRNA transport localizes ∼30 specific mRNAs including those encoding polarity and secretion factors, to the bud tip. The underlying process involves RNA binding proteins (RBPs), molecular motors, processing bodies (PBs), and the actin cytoskeleton. Recently, pheromone a-factor expression in mating yeast was discovered to depend upon proper localization of its mRNA, MFA2 . MFA2 mRNAs in conjunction with PBs cluster at the shmoo tip to form “mating bodies”, from which a-factor is locally expressed. The mechanism ensuring the correct targeting of mRNA to the shmoo tip is poorly understood. Here we analyzed the kinetics and trajectories of MFA2 mRNA transport in living, alpha-factor treated yeast. Two-(2D) and three-dimensional (3D) analyses allowed us to reconstruct the granule tracks and estimate granule velocities. Tracking analysis of single MFA2 mRNA granules, labeled using a fluorescent aptamer system, demonstrated three types movement: vibrational, oscillatory and translocational. The mRNA granule transport was complex; a granule could change its movement behavior and composition during its journey to the shmoo. Processing body assembly and the actin-based motor, Myo4p, were involved in movement of MFA2 mRNA to the shmoo, but neither was required, indicating that multiple mechanisms for translocation were at play. Our visualization studies present a dynamic view of the localization mechanism in shmoo-bearing cells.