Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1

Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.     

Abundance and composition of the summer phytoplankton community along a transect from the Barguzin River to the central basin of Lake Baikal

The abundance and composition of phytoplankton were investigated at six stations along a transect from the Barguzin River inflow to the central basin of Lake Baikal in August 2002 to clarify the effect of the river inflow on the phytoplankton community in the lake. The water temperature in the epilimnion was high near the shore at Station 1 (17.3°C), probably due to the higher temperature of the river water, and gradually decreased offshore at Station 6 (14.5°C). Thermal stratification developed at Stations 2–6, and a thermocline was observed at a 17–22-m depth at Stations 2–4 and an 8–12-m depth at Stations 5 and 6. The concentrations of nitrogen and phosphorus nutrients in the epilimnion at all stations were <1.0 μmol N l⁻¹ and <0.16 μmol P l⁻¹, respectively. Relatively high concentrations of nutrients (0.56–7.38 μmol N l⁻¹ and 0.03–0.28 μmol P l⁻¹) were detected in the deeper parts of the euphotic zone. Silicate was not exhausted at all stations (>20 μmol Si l⁻¹). The chlorophyll a (chl. a) concentration was high (>10 μg l⁻¹) near the shore at Station 1 and low (<3 μg l⁻¹) at five other stations. The <2 μm fraction of chl. a in Stations 2–6 ranged between 0.80 and 1.85 μg l⁻¹, and its contribution to total chl. a was high (>60%). In this fraction, picocyanobacteria were abundant at all stations and ranged between 5 × 10⁴ and 5 × 10⁵ cells ml⁻¹. In contrast, chl. a in the >2 μm fraction varied significantly (0.14–11.17 μg l⁻¹), and the highest value was observed at Station 1. In this fraction, the dominant phytoplankton was Aulacoseira and centric diatoms at Station 1 and Cryptomonas, Ankistrodesmus, Asterionella, and Nitzschia at Stations 2–6. The present study demonstrated the dominance of picophytoplankton in the pelagic zone, while higher abundance of phytoplankton dominated by diatoms was observed in the shallower littoral zone. These larger phytoplankters in the littoral zone probably depend on nutrients from the Barguzin River.     

The development of cysteine proteases in freesia corms during responses to chilling

Freesia protease (FP)-A has been found in regular freesia corms (Kaneda et al., Biosci. Biotechnol. Biochem. 61 (1997) 1554). New corms were generated from original corms that were kept for several months at 4°C. In this study, two proteases (FP-B and FP-C) have been found to new corms kept for 6 months at 4°C, and have increased during new corms enlargement.

 FPs were purified from the extracts of new corms, and the M_r of those were 24k (A), 25k (B), and 24.5k (C) by SDS-PAGE, respectively.

 The N-terminal sequences of FPs were identical to those of papain with respect to the conservative residues of cysteine protease. The sequence of FP-A was identical with those of FP-B within 20 residues of its N-terminal. It may be possible that FP-B was produced by some post-translational modifications from FP-A during the chilling. On the other hand, N-terminal sequence of FP-C was different from those of FP-A and FP-B. It was explained that FP-C was a new protease of freesia corm.     

Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130^CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody.

Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell–cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell–cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129–137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.

     

Expression of nitric oxide synthase in mucosal cells of the canine colon

The expression of nitric oxide synthase (NOS) in the mucosa of the canine colon was investigated with in situ hybridzation, immunohistochemistry (using isoform specific antibodies), western analysis, and NADPH diaphorase (NADPH-d) histochemistry. In situ hybridization using a common probe for known isoforms of NOS showed that NOS mRNA was strongly expressed in mucosal cells. A gradient in the degree of hybridization was noted from the base of the crypts to the luminal surface. This gradient was also apparent using an endothelial NOS (eNOS)-specific probe. Neural NOS-like immunoreactivity (nNOS-LI) was observed in columnar epithelial cells, and the same population of cells was stained with NADPH-d. Endothelial NOS-like immunoreactivity (eNOS-LI) was also found in mucosal cells; however, this eNOS-LI was confined to mucous cells. These cells were not stained with NADPH-d. The existence of cNOS in mucosal cells was confirmed by in situ hybridization using the probe which specifically hybridized with mRNA of eNOS and by western blots which demonstrated the expression of a 135-kDa protein in mucosal homogenates. The differential expression of NOS isoforms and the gradient in expression along the length of the crypts suggest complex roles for NO in the development of colonic epithelial cells and in secretion and transport functions of the colonic mucosa.     

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial -glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.     

DNA polymerase A and its stimulative factor of baker's yeast: purification and characterization

DNA polymerase A (I or major) and its stimulative factor were purified from 15-20 kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis. The extraction was carried out in the presence of 10 or 3 mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively). These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300. Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME). After ME treatment, however, they showed two protein bands at Mr of about 145,000 and 75,000 in SDS-PAGE, except for those obtained by the procedure involving 10 mM PMSF and the batchwise adsorption-elution. All the preparations described above showed practically the same specific activity. This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145,000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75,000, which were still active and were linked to each other through a disulfide bond. Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20,000 in SDS-PAGE before and after ME treatment. The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38% with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA).oligo(dT)10, respectively. With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated. The purified factor also stimulated the activity of calf thymus DNA polymerase alpha by 150% with denatured salmon sperm DNA; Km was about 5 X 10(-10)M, practically the same as that of yeast DNA polymerase A. However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.