SummaryNoncentrosomal microtubules play an important role in polarizing differentiated cells, but little is known about how these microtubules are organized. Here we identify the spectraplakin, Short stop (Shot), as the cortical anchor for noncentrosomal microtubule organizing centers (ncMTOCs) in the Drosophila oocyte. Shot interacts with the cortex through its actin-binding domain and recruits the microtubule minus-end-binding protein, Patronin, to form cortical ncMTOCs. Shot/Patronin foci do not co-localize with γ-tubulin, suggesting that they do not nucleate new microtubules. Instead, they capture and stabilize existing microtubule minus ends, which then template new microtubule growth. Shot/Patronin foci are excluded from the oocyte posterior by the Par-1 polarity kinase to generate the polarized microtubule network that localizes axis determinants. Both proteins also accumulate apically in epithelial cells, where they are required for the formation of apical-basal microtubule arrays. Thus, Shot/Patronin ncMTOCs may provide a general mechanism for organizing noncentrosomal microtubules in differentiated cells.

Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA-protein and protein-protein cross-linking experiments showed that DBP40 interacts with poly(A)(+) RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.

The major core protein of cytoplasmic messenger ribonucleoprotein particles (p50) has been shown previously to inhibit protein synthesis in vitro and in vivo. Furthermore, p50 is highly homologous to the Y-boxbinding transcription factor family of proteins, binds DNA containing the Y-box motif, and thus may have a dual function in cells as a regulator of both transcription and translation. Here we show that binding or removal of p50 from rabbit reticulocyte lysate by monospecific antibodies to p50 strongly inhibits translation of endogenous and exogenous globin mRNAs as well as prokaryotic ␤-galactosidase mRNA in a rabbit reticulocyte cell-free system. Thus, depending on the conditions, p50 not only may act as a translational repressor, but may also be required for protein synthesis. Translation inhibition with anti-p50 antibodies is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it does not affect elongation/termination of polypeptide chains. The inhibition with anti-p50 antibodies is followed by a decay of polysomes and accumulation of the 48 S preinitiation complex. These results suggest that p50 participates in initiation of protein biosynthesis. Although uninvolved in the formation of the 48 S preinitiation complex, p50 is necessary either for attachment of the 60 S ribosomal subunit or for previous 5 -untranslated region scanning by the 43 S preinitiation complex.

Specifying one oocyte from many In many animals, only one cell from a cyst of germ cells is selected to become the oocyte. Using fruit flies as a model, Nashchekin et al . identified the microtubule minus end-binding protein Patronin/CAMSAP as a key factor for specifying oocyte fate. Patronin amplifies an initial asymmetry provided by the fusome to form a noncentrosomal microtubule network focused on one cell, along which dynein transports oocyte fate determinants. This mechanism for selecting a single oocyte may be shared in other organisms. —BAP