lament network by dynamic cotranslation

Intermediate fi lament (IF) proteins are assembled into either homopolymer or heteropolymer 10-nm-diam cytoskeletal fi laments in a complex multistep process (Strelkov et al., 2003). Pairs of protein chains interact in parallel and in register to form an α-helical coiled–coil dimer, which is the basic building block of IF. Little is known about the mechanisms responsible for dimer formation. However, it is known that in vitro dimers assemble into antiparallel tetramers that associate laterally to form unit-length fi laments (ULFs). The ULFs are 60-nm-long and contain 32 protein chains. These anneal end-to-end to form 10-nm-diam IFs (Strelkov et al., 2003). Of these various IF structures, only small amounts of tetramer have been detected in soluble fractions of lysed cells (Soellner et al., 1985; Eriksson et al., 2004). Type III IF proteins such as vimentin and peripherin exist in several states within cells, including nonfi lamentous particles (Prahlad et al., 1998). Particles form short IF, or squiggles, which form the long IF that comprise the cytoskeletal networks of interphase cells. Although the composition of particles is unknown, they most likely contain essential IF building blocks such as dimers or ULFs. The assembly of particles into IF networks has been studied in spreading fi broblasts and differentiating nerve cells (Helfand et al., 2003a). During spreading, 70% of the particles move rapidly along MT in a kinesinand dynein-dependent manner (Helfand et al., 2004). As spreading progresses, many particles are converted into polymerized IF. Similar particles are seen in other IF systems, including type IV neurofi laments (Prahlad et al., 1998) and the types I and II keratins in epithelial cells (Liovic et al., 2003). Although it is obvious that nonfi lamentous particles are precursors in the assembly pathway of cytoskeletal IF, little is known about their formation in cells. We show that in rat pheochromocytoma cells (PC12), a signifi cant fraction of peripherin particles are assembled cotranslationally in a process that we have termed dynamic cotranslation. Evidence for dynamic cotranslation is derived from RNA FISH and the simultaneous live imaging of both peripherin mRNA and its protein product. Individual peripherin mRNA particles (messenger RNPs [mRNPs]) possess numerous copies of peripherin mRNA, suggesting a mechanism involving the coordinated synthesis of coiled–coil dimers, the building blocks of IF. The results provide important and novel insights into the linkages among the motile properties and targeting of mRNAs, their translational control, and the dynamic properties and assembly of IF proteins. 10.1083/jcb.200511033200511033Assembling an intermediate fi lament network by dynamic cotranslation