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Proper spindle positioning and orientation are essential for asymmetric cell division and require microtubule-actin filament (F-actin) interactions in many systems. Such interactions are particularly important in meiosis, where they mediate nuclear anchoring, as well as meiotic spindle assembly and rotation, two processes required for asymmetric cell division. Myosin-10 proteins are phosphoinositide-binding, actin-based motors that contain carboxy-terminal MyTH4 and FERM domains of unknown function. Here we show that Xenopus laevis myosin-10 (Myo10) associates with microtubules in vitro and in vivo, and is concentrated at the point where the meiotic spindle contacts the F-actin-rich cortex. Microtubule association is mediated by the MyTH4-FERM domains, which bind directly to purified microtubules. Disruption of Myo10 function disrupts nuclear anchoring, spindle assembly and spindle-F-actin association. Thus, this myosin has a novel and critically important role during meiosis in integrating the F-actin and microtubule cytoskeletons.

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and ⌬fnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O 2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.

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Although the role of the actin cytoskeleton in morphogenesis of polarized epithelial sheets is generally accepted as centrally important, the regulation of actin dynamics in this process remains unclear. Here, we show that the pointed-end capping protein Tmod3 contributes to epithelial cell shape within confluent monolayers of polarized epithelial cells. Tmod3 localizes to lateral cell membranes in polarized epithelia of several cell types. Reduction of Tmod3 levels by shRNA leads to a loss of F-actin and tropomyosins from lateral cell membranes, and a decrease in epithelial cell height, without effects on localisation of tight junction or adherens junction proteins, or any apparent changes in cell-cell adhesion. Instead, distribution of αII-spectrin on lateral membranes is disrupted upon reduction of Tmod3 levels, suggesting that loss of Tmod3 function leads to destabilization and disassembly of tropomyosin-coated actin filaments followed by disorganization of the spectrin-based membrane skeleton on lateral membranes. These data demonstrate for the first time a role for pointed-end capping in morphology regulation of polarized epithelial cells through stabilization of F-actin on lateral membranes. We propose that Tmod3-capped tropomyosin-actin filaments provide crucial links in the spectrin membrane skeleton of polarized epithelial cells, enabling the membrane skeleton to maintain cell shape.

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.

The enzyme, Qi3 replicase, responsible for the replication of the RNA of Escherichia coli phage Q0, is composed of four nonidentical subunits, three of which, I, III, and IV, are coded for by the bacterial genome, while subunit II is phage-specific. Subunit IV is shown to be identical to the protein synthesis elongation factor EF Ts by the following criteria: coelectrophoresis on polyacrylamide gels in sodium dodecyl sulfate and in urea buffers, identity of the first seven amino acids at the amino-terminus, precipitation of subunit IV by anti-EF T-factor serum, and stimulation of EF Tu-GDP exchange by subunit IV. Subunit III is shown to be identical to the protein synthesis elongation factor EF Tu by the following criteria: coelectrophoresis on sodium dodecyl sulfate gels, precipitation of EF Tu by anti-QB3 replicase serum, binding of guanine nucleotides, and binding of phenylalanyl-tRNA. In addition, QB3 replicase activity can be reconstituted from subunits I and II with EF Tu and EF Ts.The RNA bacteriophage of Escherichia coli, Qu, induces an enzyme, Q0 replicase, that is responsible for replication of the phage RNA. This enzyme has been extensively characterized and purified. It will copy Q0 RNA, but not the RNA of similar E. coli RNA phages. Early in its purification, it is calpable of copying both phage RNA ("plus strands") and the RNA complement of the phage RNA ("minus strands"). The ability to copy "plus strands," however, is lost upoin further purification. The purified core enzyme can be assayed with either "minus strands" or poly(C) as template. "Plus strand" activity can be restored by addition of hostcoded factors (for a review, see Stavis and August, ref. 1).Kamen (2) and Kondo, Gallerani, and Weissmannii (3) found that the purified core enzyme consists of four nonidentical polypeptide chains of approximate molecular weights 70,000, 65,000, 45,000, and 35,000 (designated 1, 11, III, and IV in the nomenclature of Kameni). Subunit II is coded for by the phage genome, while the other three subunits are present in uninfected E. coli. The replicase of the serologically unrelated RNA phage f2 has been purified by Fedoroff and Zinder (4); it contains three host-coded polypeptides of similar, if not identical, molecular weights to those of Q,3 replicase, in addition to the phage-coded subunit.The four polypeptides of Qfl replicase can be separated into complexes of subunits I + II and subunits III + IV by incubation in a buffer of low salt concentration, followed by sedimentation on a low salt-glycerol gradient. Kamen (2) found that neither fraction alone shows activity ill the poly-(C)-dependent assay, but partial activity could be recovered after the two were mixed together.WTe report here that subunits III and IV of QO replicase are identical with EF Tu and EF Ts, respectively, two elongation factors identified as part of the mechanism of protein biosynthesis by Lucas-Lenard and Lipmanni (5,6 Abbreviations: SDS, sodium dodecyl sulfate; Phe-tRNA, phenylalanyl-charged phenylalanine-tRNA.