Sorting of RNAs to specific subcellular loci occurs in diverse settings from fly oocytes to mammalian neurons. Using the membrane-permeable nucleic acid stain SYTO 14, we directly visualized the translocation of endogenous RNA in living cells. Labeled RNA was distributed nonrandomly as discrete granules in neuronal processes. The labeled granules colocalized with poly(A ϩ ) mRNA, with the 60S ribosomal subunit, and with elongation factor 1␣, suggesting that granules represent a translational unit. A subset of labeled granules colocalized with ␤-actin mRNA. Correlative light and electron microscopy indicated that the fluorescent granules corresponded to clusters of ribosomes at the ultrastructural level. Poststaining of sections with heavy metals confirmed the presence of ribosomes within these granules. In living neurons, a subpopulation of RNA granules was motile during the observation period. They moved at an average rate of 0.1 m/sec. In young cultures their movements were exclusively anterograde, but after 7 d in culture, one-half of the motile granules moved in the retrograde direction. Granules in neurites were delocalized after treatment with microtubule-disrupting drugs. These results raise the possibility of a cellular trafficking system for the targeting of RNA in neurons.

No abstract

Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro.

We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (DeltaBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing DeltaBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, DeltaBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a DeltaBLCBS-myosin is similar to that in wild-type cells.

Abstract. The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues . Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment.We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation . A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin . Complex microtubule loops often formed transiently within the growth cone. Branches