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PubMed2003

Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment.

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.

Actins↗
PubMed2006

Peripheral and posterior pole retinal lesions in association with high myopia: a cross-sectional community-based study in Hong Kong.

PURPOSE: To evaluate the prevalence and factors associated with posterior pole and peripheral retinal lesions in Chinese subjects with high myopia. METHODS: Three hundred and thirty-seven asymptomatic adults with high myopia of refractive error <or=-6 D were examined in a cross-sectional community-based study. All subjects underwent cycloplegic refraction, ultrasound biometry and dilated fundal examination. Statistical analysis was performed to assess factors associated with the presence of posterior pole and peripheral retinal lesions. RESULTS: The mean age of the 337 subjects was 36.0 years and the mean spherical equivalent refractive error was -10.2 D. Thirty-eight eyes (11.3%, 95% CI=8.1-15.2%) were found to have one or more posterior pole lesions and subjects with posterior pole lesion had significantly older age, longer axial length and higher degree of myopia (all P<0.001) compared with subjects without posterior pole lesion. After controlling for axial length, both the severity of refractive error and older age were significantly associated with the presence of posterior pole lesion (both P<0.001). For peripheral retinal lesions, 189 eyes (56.1%, 95% CI=50.6-61.5%) were found to have one or more peripheral retinal lesions. The presence of peripheral retinal lesion was associated with younger age and higher degree of refractive error (P=0.046 and 0.002, respectively). CONCLUSION: Posterior pole and peripheral retinal degenerative lesions were found in a considerable proportion of subjects with high myopia. As some of these retinal lesions might predispose to visual impairment, highly myopic individuals should be educated on the symptoms of various eye conditions and seek care immediately if symptoms arise.

Adolescent↗
PubMed2000

Load carriage energy expenditure with and without hiking poles during inclined walking.

The purpose of this study was to compare load carriage energy expenditure with and without using hiking poles. Twenty male volunteers aged 20-48yr (Mean=29.8yr) completed two randomly ordered submaximal treadmill trials with poles (E) and without poles (C). Poles and load (15 kg backpack) were fitted for each subject according to the manufacturers' suggestions. Heart rates (HR), minute ventilation (V(E)), oxygen consumption (O2), caloric expenditure (Kcal), and rating of perceived exertion (RPE) were recorded at the end of each minute. Two trials separated by one week consisted of a constant treadmill speed of 1.5 mph and 1 min at 10% grade, 2 min at 15% grade, 2 min at 20% grade, and 10 min. at 25% grade. Mean HR (E = 144.8 +/- 24.4 b x min(-1); C = 144.0 +/- 25.7 b x min(-1)) and mean V(E) (E=51.4 +/- 15.8L x min(-1); C=50.8 +/- 17.0L x min(-1)), VO2 (E = 26.9 +/- 6.1 ml x kg(-1) x min(-1); C = 27.4 +/- 6.6 ml x kg(-1) x min(-1)), and Kcal (E = 10.6 +/- 2.9 Kcal x min(-1); C = 10.8 +/- 3.1 Kcal x min(-1)) were not significantly different between the two conditions. RPE (E = 13.28 +/- 1.2; C = 14.56 +/- 1.2) was significantly lower (P < 0.05) with hiking poles. Analysis of paired time points yielded no significant differences in HR, VO2, V(E), and Kcal, however, RPE means were significantly lower for 5 of the last 7 trial minutes with the use of poles. These results suggest that during load carriage on moderate grade, the weight and use of hiking poles does not increase energy expenditure but may provide reduced perceptions of physical exertion.

Adult↗
PubMed1985

Centriole number and the reproductive capacity of spindle poles.

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.

Animals↗
PubMed2000

TPX2, A novel xenopus MAP involved in spindle pole organization.

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

Amino Acid Sequence↗
PubMed2001

Chromosome movement in mitosis requires microtubule anchorage at spindle poles.

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

Antibodies↗
PubMed2004

Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel.

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

Absorbable Implants↗
PubMed2001

ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae.

ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.

Cell Membrane↗
PubMed2004

Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other.

To test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.

Animals↗
PubMed2005

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans.

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.

Anaphase↗
PubMed1998

Poling forces during roller skiing: effects of technique and speed.

PURPOSE: Although it has been reported that the majority of propulsive forces are generated through the poles with ski skating, no study has systematically examined poling forces among different skating techniques. The objective of the present study was to examine poling forces and timing during roller skiing on a 2.1% uphill. METHODS: Nine highly skilled cross-country skiers roller skied at three paced speeds and maximal speed using the V1 skate (V1), V2-alternate (V2A), V2 skate (V2), and double pole (DP) techniques while poling forces and timing were measured with piezoelectric transducers. RESULTS: Peak force (PF) values with the skating techniques were significantly lower than with DP and ranged from 18.9 +/- 3.1% of body weight (BW) to 31.5 +/- 5.6% BW across the speeds of the study. Average force over the entire cycle (ACF) increased with speed with DP, V2A and V1 (P < 0.01) but not with V2. PF and ACF were higher (P < 0.01) with V2 than V1 and V2A. Poling time was longer (P < 0.01) with V2A compared with V1 and V2. CONCLUSIONS: The results of this study suggest that 1) the use of the upper body is greater with V2 than with other skating techniques while there is a relatively greater reliance on the lower body for generation of the additional propulsive forces required to increase velocity, and (2) poling forces do not appear to be as effectively applied with V2 as with V2A.

Acceleration↗
PubMed2003

Prediction of lower pole stone clearance after shock wave lithotripsy using an artificial neural network.

PURPOSE: We performed this study as a comprehensive evaluation of variables reported to affect lower pole stone clearance after shock wave lithotripsy using artificial neural network analysis. MATERIALS AND METHODS: The radiographic images and treatment records of 680 patients with lower pole renal calculi treated with primary shock wave lithotripsy using the Wolf Piezolith 2500 (Wolf, Knittlingen, Germany) lithotriptor were retrospectively evaluated by applying artificial neural network analysis. Successful stone clearance was defined as absent fragments of any size detected on plain x-ray with tomography and/or excretory pyelography performed 6 months after treatment. Prognostic variables included patient characteristics, laboratory values, stone characteristics and the spatial anatomy of the lower pole, as defined by infundibular length, diameter, caliceal pelvic height, 2 measurements of the lower infundibulopelvic and infundibuloureteropelvic angles as well as the pattern of dynamic urinary transport. RESULTS: Artificial neural network analysis had 92% accuracy for correctly predicting lower pole stone clearance. The pattern of dynamic urinary transport represented the most influential predictor of stone clearance, followed by a measure of the infundibuloureteropelvic angle, body mass index, caliceal pelvic height and stone size. Anatomical measurements of lower pole anatomy and classification of the type of urinary transport were well reproducible with low intra-observer and interobserver variability (correlation coefficient alpha >0.8). CONCLUSIONS: In a comprehensive analysis of variables reported to influence lower pole stone clearance artificial neural network analysis predicted stone clearance with a high degree of accuracy. The relative importance of dynamic urinary transport in lower pole stones and the usefulness of artificial neural network analysis to predict shock wave lithotripsy outcomes in individuals must be confirmed in a prospective trial.

Adolescent↗
PubMed1998

Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p.

Schizosaccharomyces pombe divides by means of a centrally placed division septum. The initiation of septation must be tightly coordinated with events in mitosis, as premature formation of the septum can lethally cut the undivided nucleus. The Spg1p GTPase and the Cdc7p kinase, with which it interacts, play a central role in signaling the initiation of septum formation. Loss-of-function mutations in either gene prevent septation, whereas inappropriate activation of Spg1p can induce septum formation from G1 or G2 interphase cells. Increased expression of either gene leads to multiple rounds of septation without cell cleavage, emphasizing the need for precise cell cycle regulation of their activity. To understand the mechanisms underlying this regulation, we have investigated whether these key initiators of septum formation are controlled by changes in their activity and/or location during mitosis and cytokinesis. We demonstrate that Spg1p localizes to the spindle pole body in interphase and to both spindle poles during mitosis. In contrast, Cdc7p shows no discrete localization during interphase, but early in mitosis it associates with both spindle pole bodies and, as the spindle extends, is seen on only one pole of the spindle during anaphase B. Spg1p activity is required for localization of Cdc7p in vivo but not for its kinase activity in vitro. Staining with an antiserum that recognizes preferentially GDP-Spg1p indicates that activated GTP-Spg1p predominates during mitosis when Cdc7p is associated with the spindle pole body. Furthermore, staining with this antibody shows that asymmetric distribution of Cdc7p may be mediated by inactivation of Spg1p on one spindle pole. Deregulated septation in mutant cells correlates with segregation of Cdc7p to both spindle poles.

Cell Cycle Proteins↗
PubMed1994

A sigma 54 transcriptional activator also functions as a pole-specific repressor in Caulobacter.

The differential localization of proteins in the Caulobacter predivisional cell leads to the formation of two distinct progeny cells: a motile swarmer cell and a sessile stalked cell. Pole-specific transcription in the predivisional cell is one mechanism responsible for protein localization. Here we show that the sigma 54 transcriptional activator FlbD, which activates swarmer pole-specific transcription of a subset of late flagellar genes, is also capable of functioning as a pole-specific repressor of the early flagellar fliF operon. DNase I footprinting and methylation interference assays indicate that FlbD binds to regions of the fliF promoter at regions that would be likely to interfere with the binding of RNA polymerase. A mutation that abolishes FlbD binding results in up to a fourfold increase in fliF promoter expression. This mutation alters both the spatial and temporal pattern of fliF expression resulting in the inappropriate expression of the fliF operon in the swarmer pole of the predivisional cell. These results demonstrate that FlbD represses early flagellar gene expression in the swarmer pole of the Caulobacter predivisional cell. This is the first instance in which a protein specifically involved in pole-specific repression has been identified in Caulobacter. The restriction of FlbD activity to the swarmer pole accomplishes two regulatory missions by simultaneously activating late flagellar gene expression and repressing early flagellar genes.

Bacterial Proteins↗
PubMed1993

Two types of pole cells are present in the Drosophila embryo, one with and one without splicing activity for the third P-element intron.

In Drosophila, it has been postulated that the third intron of the P-element is spliced only in germ-line cells. To test whether this postulate is applicable to pole cells, the progenitor cells of germ line, we carried out a histochemical assay to detect the splicing activity in embryos. The splicing activity was detected in pole cells and primordial germ cells. The activity increased to reach a maximum at 5-6 hours AEL (after egg laying), then decreased to an undetectable level by 8-9 hours AEL. The splicing activity showed a small second peak at 12-15 hours AEL. It was rather unexpected that not all pole cells were capable of splicing the third intron. Almost all pole cells that had the splicing activity at 5-6 hours AEL penetrated the embryonic gonads and differentiated into primordial germ cells. Our findings suggest that pole cells are selected to penetrate the gonads while they are migrating from the proctodeal cavity to the gonads. Furthermore, these results suggest that the machinery to splice the P-element is active in some pole cells, and that this activity is used for processing transcripts of genes that play important roles in the differentiation of pole cells into primordial germ cells.

Animals↗
PubMed1995

Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

Animals↗
PubMed1991

Pole-to-chromosome movements induced at metaphase: sites of microtubule disassembly.

Metaphase spindles can be induced to shrink by treating cells with microtubule-depolymerizing agents. During treatment, the paired sister chromatids remain at the metaphase plate and the poles move toward them. The question we asked is whether this pole-to-chromosome movement was accompanied by a loss of subunits from the kinetochore ends of the microtubules, the polar ends, or both ends. LLC-PK cells were injected at late prometaphase with Xrhodamine tubulin and at metaphase the fluorescent spindles were marked by photobleaching a bar between one pole and the chromosomes. Nocodazole at low concentrations was briefly applied to the cells to induce the shortening of the spindle and movement of the poles inward toward the chromosomes. In the induced shortening, the distance between the photobleached bar and the chromosomes decreased substantially while the distance between the bar and the pole showed a smaller change. Upon reversal from nocodazole, new polymer was added to the spindle as determined by recovery of fluorescence, and the cells progressed through mitosis and cytokinesis. We conclude that the movement of the poles to the chromosomes induced by nocodazole treatment during metaphase is similar to the chromosome-to-pole movement occurring during anaphase in that under both conditions the primary site for kinetochore microtubule disassembly is at the kinetochore.

Anaphase↗
PubMedDate not supplied

Copper, zinc, and arsenic in soil surrounding Douglas-fir poles treated with ammoniacal copper zinc arsenate (ACZA).

The levels of copper, zinc, and arsenic in soil surrounding Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] utility poles treated with ammoniacal copper zinc arsenate (ACZA) were investigated at sites in Florida, Virginia, and New York. Copper levels were elevated near the poles and declined with both horizontal distance away from the pole and depth beneath the soil surface. Zinc levels were also elevated next to the poles, but the levels declined more slowly than did those of copper. Arsenic levels were elevated in soil immediately next to the poles but declined to near background levels farther away. The results indicate that metals can leach from ACZA-treated poles, but do not migrate far in the soil, and thus the levels decline sharply with distance from the poles.

Arsenates↗