Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)-fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis.     

Repeated cycles of chilling and warming effectively terminate prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis (Coleoptera: Curculionidae) requires one or more years to complete its life cycle, owing to prolonged larval diapause. To compare the effects of temperature cycles and total periods of chilling on the termination of prolonged diapause, larvae were subjected to different chilling (5 degrees C) and warming (20 degrees C) cycles ranging from 30 to 720 days, and all cycles were repeated until the sum of chilling and warming periods reached 720 days. The prolonged diapause of C. sikkimensis was more effectively terminated by repeated cycles of chilling and warming than by prolonging the continuous chilling period. However, extremely short temperature cycles were not highly effective in enhancing diapause termination, even when such cycles were repeated many times. To examine the role of warming periods on diapause termination, diapause larvae were subjected to a sequence of chilling (120 days at 5 degrees C) and warming (240 days at 20 degrees C) with a warming period (0-120 days at 20 degrees C) inserted in the chilling period. Diapause larvae that were not reactivated in the first chilling period required exposure to a certain period of warming before they were able to complete diapause development in the subsequent chilling. Thus, C. sikkimensis appears to spread its reactivation times over several years in response to seasonal temperature cycles.     

Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.     

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Sucrose transporter NtSUT4 from tobacco BY-2 involved in plant cell shape during miniprotoplast culture

Sucrose plays an important role in several cellular processes since it is a general source of metabolic energy, serves as a precursor for starch and cellulose synthesis, and is a metabolic starting point for carboxylate- and amino acid synthesis. While plant vacuole is the main cellular storage pool, where sucrose accumulates to high concentrations, only a small number of vacuolar sugar transporters have been identified and characterized to date. We initially identified a vacuolar sucrose transporter (NtSUT4) from tobacco BY-2 cells and established transgenic tobacco BY-2 cell lines that overexpress NtSUT4-GFP (BY-SUTG cells). Using a model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that NtSUT4-GFP overexpression inhibited cell growth towards the cell major axis. Moreover, under the same conditions, we found that the cell walls were well stained by calcofluor in BY-SUTG cells than in wild type BY-2 cells. These results suggest that NtSUT4 is involved in cell shape via sucrose homeostasis in plant cells.     

Effects of abnormal ovarian cycles during pre-service period postpartum on subsequent reproductive performance of high-producing Holstein cows

The primary objective of this study was to investigate the effects of abnormal ovarian cycles during the pre-service postpartum period on subsequent reproductive performance of high-producing Holstein cows. The study was conducted in a commercial dairy farm with approximately 150 lactating cows, in a subtropical region of Japan. Animals were kept in free-stall barn, and fed a total mixed ration. Cows that calved from June 2001 to July 2002 were included in the study. Milk samples were collected twice weekly from 2 to 11 weeks postpartum, and progesterone concentrations in skim milk were determined by ELISA. After a voluntary waiting period of 40 days, cows detected in estrus were bred by artificial insemination (AI). Pregnancy was confirmed by palpation per rectum 40-70 days after AI. Out of a total of 91 cases, 39 (42.9%) had normal ovarian cycles (ovulation within 45 days after calving, followed by normal ovarian cycles), 32 (35.2%) had prolonged luteal phase (i.e. luteal activity for >20 days), and 12 (13.2%) had anovulation or delayed first ovulation (i.e. first ovulation did not occur until >45 days after calving). The remaining (8.8%) had other types of abnormalities. When compared with cows with a normal ovarian cycle, prolonged luteal phase cows had a lower 100 days AI submission rate, conception rate and pregnancy rate (84.2% versus 56.3%; P<0.05, 50% versus 16.7%; P<0.05 and 42.1% versus 9.4%; P<0.01, respectively), and longer intervals to first AI and to conception ( 67+/-6 days versus 98+/-7 days and 95+/-9 days versus 136+/-11 days; P<0.01 for each). Similarly, when compared with cows with normal ovarian cycles, those with anovulation had lower 100 days conception rate and pregnancy rate (50% versus 0%; P<0.05 and 42.1% versus 0%; P<0.01, respectively), and longer intervals to first AI and to conception ( 67+/-6 days versus 93+/-12 days; P<0.05 and 95+/-9 days versus 155+/-14 days; P<0.01, respectively). Survival analysis of the data for calving to conception interval showed that cows with prolonged luteal phase or anovulation were getting pregnant at a slower rate, and took longer to get pregnant than the cows with normal resumption of ovarian cyclicity postpartum. In conclusion, abnormal ovarian cycles during the pre-service period postpartum adversely affected reproductive performance, including AI submission rate, pregnancy rate, interval to first AI, and calving to conception interval in high-producing Holstein cows.     

Identification of coenzyme aldimine proton in 1H NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes

The pyridoxal form of the alpha subform of cytosolic aspartate aminotransferase (EC 2.6.1.1) is fully active and binds pyridoxal 5'-phosphate via an aldimine formation with Lys-258 whereas the gamma subform is virtually inactive and lacks the aldimine linkage. Comparison of 1H NMR spectra between the alpha and gamma subforms suggested that peak 1 of the alpha subform at 8.89 ppm contains a resonance assignable to the internal aldimine 4'-H. Reaction with a reagent that cleaves or modifies the internal aldimine bond [(amino-oxy)acetate, L-cysteinesulfinate, NH2OH, NaBH4, or NaCNBH3] caused the disappearance of a resonance line at 8.89 ppm that possessed a broad line width and corresponded in intensity to a single proton. These reagents were also used successfully for the identification of the aldimine 4'-H resonance in the mitochondrial isoenzyme. In contrast to the cytosolic isoenzyme whose resonance for the 4'-H did not show any detectable change in chemical shift with pH, the corresponding resonance in the mitochondrial isoenzyme exhibited pH-dependent chemical shift change (8.84 ppm at pH 5 and 8.67 ppm at pH 8) with a pK value of 6.3, reflecting the interisozymic difference in the microenvironment provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: the resonance assigned to the 4'-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and the resonance appeared upon reconstitution of the apoenzyme with [4'-1H]pyridoxal phosphate but not with [4'-2H]pyridoxal phosphate.     

Actin microfilament dynamics and actin side-binding proteins in plants

Actin microfilaments are highly organized and essential intracellular components of organelle movement and cell morphogenesis in plants. The organization of these microfilaments undergoes dynamic changes during cell division, elongation, and differentiation. Recent live-cell imaging of plant actin microfilaments has revealed their native organization and remarkable dynamics. In addition, characterization of plant actin side-binding proteins has progressed rapidly by genetic, biochemical, and bioinformatic approaches. The gathering and integration of microscopy-based information from actin microfilament dynamics and the molecular identification of actin side-binding proteins have provided considerable insights into actin microfilament-dependent events and actin microfilament organization in plants.