Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a "headless" AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation.     

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.     

Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC

We determined the stoichiometry of KaiA-KaiC interactions. Using immunoblotting and two-dimensional Native- and SDS-PAGE (2DNS-PAGE) analysis, we demonstrated that the reaction products of KaiA-KaiC interactions in the presence of ATP consisted of only phosphorylated KaiC whereas in the presence of the unhydrolyzable analogue 5'-adenylylimidodiphosphate (AMPPNP) they consisted of KaiA and KaiC. In the presence of ATP, the KE (molar ratio of KaiA dimer to KaiC hexamer giving half saturation in the enhancement of KaiC phosphorylation) was 0.25, and IAsys affinity biosensor analysis demonstrated that 1 molecule of KaiA dimer interacted with 1 molecule of KaiC hexamer. In the presence of AMPPNP, the ratio of KaiA dimer to KaiC hexamer in KaiA-KaiC complexes was determined to be 2 by 2DNS-PAGE, Native-PAGE/Scatchard plot, and IAsys analyses. These results suggest that 2 molecules of KaiA dimer can interact with 1 molecule of KaiC hexamer, and that interactions of at least 1 molecule of KaiA dimer with 1 molecule of KaiC hexamer are enough to enhance the phosphorylation of KaiC by KaiA at an almost saturated level.     

Oxygen affinity of hemoglobin regulates O2 consumption,metabolism, and physical activity

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O(2) delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the alpha-globin gene or Presbyterian mutation in the beta-globin gene. The mutant mice showed increased O(2) consumption and CO(2) production in tissue metabolism, suggesting enhanced O(2) delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O(2) consumption, physical activity, and behavior in mice.     

Pathophysiology of orthostatic hypotension after bed rest: paradoxical sympathetic withdrawal

Although orthostatic hypotension is a common clinical syndrome after spaceflight and its ground-based simulation model, 6 degrees head-down bed rest (HDBR), the pathophysiology remains unclear. The authors' hypothesis that a decrease in sympathetic nerve activity is the major pathophysiology underlying orthostatic hypotension after HDBR was tested in a study involving 14-day HDBR in 22 healthy subjects who showed no orthostatic hypotension during 15-min 60 degrees head-up tilt test (HUT) at baseline. After HDBR, 10 of 22 subjects demonstrated orthostatic hypotension during 60 degrees HUT. In subjects with orthostatic hypotension, total activity of muscle sympathetic nerve activity (MSNA) increased less during the first minute of 60 degrees HUT after HDBR (314% of resting supine activity) than before HDBR (523% of resting supine activity, P < 0.05) despite HDBR-induced reduction in plasma volume (13% of plasma volume before HDBR). The postural increase in total MSNA continued during several more minutes of 60 degrees HUT while arterial pressure was maintained. Thereafter, however, total MSNA was paradoxically suppressed by 104% of the resting supine level at the last minute of HUT (P < 0.05 vs. earlier 60 degrees HUT periods). The suppression of total MSNA was accompanied by a 22 +/- 4-mmHg decrease in mean blood pressure (systolic blood pressure <80 mmHg). In contrast, orthostatic activation of total MSNA was preserved throughout 60 degrees HUT in subjects who did not develop orthostatic hypotension. These data support the hypothesis that a decrease in sympathetic nerve activity is the major pathophysiological factor underlying orthostatic hypotension after HDBR. It appears that the diminished sympathetic activity, in combination with other factors associated with HDBR (e.g., hypovolemia), may predispose some individuals to postural hypotension.     

Alpha-adrenergic vascular responsiveness to sympathetic nerve activity is intact after head-down bed rest in humans

Space-flight and its ground-based simulation model, 6 degrees head-down bed rest (HDBR), cause cardiovascular deconditioning in humans. Because sympathetic vasoconstriction plays a very important role in circulation, we examined whether HDBR impairs alpha-adrenergic vascular responsiveness to sympathetic nerve activity. We subjected eight healthy volunteers to 14 days of HDBR and before and after HDBR measured calf muscle sympathetic nerve activity (MSNA; microneurography) and calf blood flow (venous occlusion plethysmography) during sympathoexcitatory stimulation (rhythmic handgrip exercise). HDBR did not change the increase in total MSNA (P = 0.97) or the decrease in calf vascular conductance (P = 0.32) during exercise, but it did augment the increase in calf vascular resistance (P = 0.0011). HDBR augmented the transduction gain from total MSNA into calf vascular resistance, assessed as the least squares linear regression slope of vascular resistance on total MSNA (0.05 +/- 0.02 before HDBR, 0.20 +/- 0.06 U.min-1.burst-1 after HDBR, P = 0.0075), but did not change the transduction gain into calf vascular conductance (P = 0.41). Our data indicate that alpha-adrenergic vascular responsiveness to sympathetic nerve activity is preserved in the supine position after HDBR in humans.     

Formation of Keto and Hydroxy Compounds of Linoleic Acid in Submitochondrial Particles of Bovine Heart

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A₂, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and -tocopherol. The cardiolipin–cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid–hemoprotein lipid peroxidation systems such as the cardiolipin–cytochrome c system.     

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.