Electron microscopy of the spindle in locally heated cells

Individual living cells in metaphase were exposed to a steep temperature gradient by placing a microheater near one spindle pole. The cells were then fixed and the spindle was examined by electron microscopy. The structure of the warmer half-spindle differed from the cooler half-spindle in several ways. Kinetochore microtubules were nearly parallel in the warmer half-spindle but were divergent in the cooler. The total length of microtubules in the warmer half-spindle was 52 per cent greater and the number of kinetochore microtubules per kinetochore averaged 16 per cent higher than in the cooler half-spindle. The warmer half-spindle was longer than the cooler. These observations clearly demonstrate a locally enhanced assembly of microtubules in the warmer half-spindle. The electron microscope study makes still clearer the unusual character of chromosome movement in the differentially heated cells: the structure of the warmer half-spindle is hard to distinguish from that in normal cells, yet chromosome movement there is far slower than normal (Nicklas, 1979).     

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.     

Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.     

Chromosome movement in lysed mitotic cells is inhibited by vanadate

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10-- 100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.     

Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.     

Early distant relapse after optimal local control in locally advanced rectal cancer

We present a case of locally advanced rectal cancer with initial optimal local control after neoadjuvant concurrent chemoradiotherapy followed by surgery; early liver recurrence then occurred and was treated again with curative intent with neoadjuvant combination chemotherapy followed by liver surgery. We reflect on this difficult problem and discuss relevant topics to this case report.     

Local thermal sensation and finger vasoconstriction in the locally heated hand

The effects of local heating on finger blood flow (BF) and local thermal sensation (Sensw) were studied. Finger BFs in both hands were measured simultaneously; one hand was immersed in water the temperature (Tw) of which was raised from 35 degrees C to 43 degrees C by steps of 2 degrees C every 10 min, while the other hand was kept at Tw 35 degrees C. Finger BF in the locally heated hand decreased at Tw 37 to 41 degrees C, while finger BF in the control hand did not alter. Sensw in the heated hand showed a dynamic response, initially increasing concomitantly with an increase in Tw, then gradually returning and adapting to a new level of Sensw. The dynamic response of Sensw was not perceived during mental calculation even when Tw was raised to 40 degrees C, and the reduction in finger blood flow was not observed. These results suggest that finger vasoconstriction caused by local heating closely relates to the dynamic response characteristic of local thermal sensation at Tw above core temperature, and that the perception of local thermal sensation in the central nervous system is involved in the mechanism of this vasoconstrictor response.     

Ran control of mitosis in human cells: gradients and local signals

Roles of the GTPase Ran in cell life and division rely on a largely conserved mechanism, i.e. Ran's ability to interact with transport vectors. Modes of control of downstream factors, however, are diversified at particular times of the cell cycle. Specificity and fine-tuning emerge most clearly during mitosis. In the present article, we focus on the distinction between global mitotic control by the chromosomal Ran gradient and specific spatial and temporal control operated by localized Ran network members at sites of the mitotic apparatus in human cells.     

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.     

Flattening,movement and control of division of epithelial-like cells

The kinetics of cell division and movement in four epithelial-like cell lines, grown in continuously perfused culture medium, were studied by time-lapse cinemicrography. One line exhibited “contact regulation of cell division,” so that the rate of mitosis per cell decreased steadily as population density increased. In the other three lines mitosis was not controlled as a function of population density until the cells became very crowded. An explanation for this difference was sought in terms of the hypothesis that the rate of division depends on the area of the cell membrane. Cells of the contact-regulated line flattened uniformly on the substrate. Their motility was restrained by adhesion between their borders. As they crowded together, contact inhibition of cell overlap caused a steady decrease in average surface area per cell. All three of the non-controlled lines also had contact inhibition of overlap. Cells of two of them flattened on the substrate; but these cells had little mutual adhesion and were highly motile, so that they continually changed their shapes. The areas of their cell membranes were therefore not subject to a restraint that could control the rate of division. Cells of the fourth line remained rounded or only slightly flattened during culture growth, so that no change in cell membrane area occurred that could change the rate of division.     

Mitotic spindle: laser microsurgery in yeast cells

Laser microsurgery has led to remarkable discoveries in a number of cell types. Two recent studies have shown that this classical technology can now be employed with small yeast cells. This advance will enable regional ablation to be combined with facile genetic manipulation in a eukaryotic cell.     

Chromosome aberrations induced in human bone-marrow cells by therapeutical local irradiation. Time and dose relationships

Bone-marrow cell chromosome aberrations were studied in 14 unirradiated control patients with malignant tumors and 132 comparable patients after a single localised exposure to ⁶⁰Co γ irradiation. In control patients the chromosome break frequency was 0.01 per cell and aberrant cell frequency was 1.1%.     

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.     

Glycated haemoglobin and metabolic control of diabetes mellitus: external versus locally established clinical targets for primary care.

OBJECTIVES--To examine current targets for glycated haemoglobin as a marker for metabolic control in diabetes mellitus in relation to datasets from several areas, and to consider whether target setting could be improved. DESIGN--Data collected from enhanced care records of general practices for a representative community based sample of people with diabetes. SETTING AND SUBJECTS--3022 people with diabetes on the lists of 37 general practices (total list size 222,550) in South Glamorgan in 1992; samples of glycated haemoglobin had been processed at two laboratories with different methodologies and reference ranges. MAIN OUTCOME MEASURES--Last glycated haemoglobin level measured in subjects for 1992 and published data from other studies considered in relation to existing goals and standards for the metabolic control of diabetes. RESULTS--An ascertainment rate for people with diabetes of 1.36% was obtained. The rate of data capture for haemoglobin A1 was 75.7%, and the mean level for study samples was 10.5% at one laboratory and 10.0% at the other (similar values to those of comparable studies). These mean levels of haemoglobin A1 in representative populations of people with diabetes are poor or very poor according to published standards, including those of the British Diabetic Association. These findings are set in the context of the psychology of goal setting and performance in complex clinical situations. CONCLUSION--Targets for clinical care that are set in the absence of normative data and local feasibility assessments should be treated with caution. Targets are more likely to enhance health care if target setters recognise the importance of psychological aspects of goal setting and motivation.     

Variations on theme: spindle assembly in diverse cells

The mitotic spindle faithfully separates the genetic material, and has been reverently observed for well over a century. Across eukaryotes, while the mechanisms for moving chromosomes seem quite conserved, mechanisms for assembling the spindle often seem distinct. Two major pathways for spindle assembly are known, one based on centrosomes and the other based on chromatin, and these pathways are usually considered to be fundamentally different. We review observations of spindle assembly in animals, fungi, and plants, and argue that microtubule assembly at a particular location, centrosomes, or chromatin, reflects contingent, cell-type specific factors, rather than reflecting a fundamental distinction in the process of spindle building. We hypothesize that the essential process for spindle assembly is the motor-driven organization of microtubules that accumulate in the form of dense bundles at or near the chromosomes.     

Interaction between the vertical posture and movement control systems during a quick voluntary arm raise

The study was aimed at a deeper understanding of the interaction between the system of vertical posture control and the system of voluntary movement control based on the analysis of postural muscle activity components resulting from the action of the former or the latter system. For this purpose, a quick arm raise was performed in the standing and sitting positions with body fixation at different levels, when the task of maintaining a vertical posture was simplified or completely eliminated. Under these conditions, the muscle activity associated with posture control was supposed to change, while the activity of muscles raising the arm was supposed to remain invariable. The results showed that the simplification of the posture control resulted in a decrease or elimination of anticipatory changes in the activity of some muscles. However, most of the muscle activity variations were retained even in the sitting position, and these variations appeared simultaneously with the activity of muscles raising the arm. The so-called “anticipatory postural activity” during an arm raise in a normal standing position is supposed to consist of two components: an initial component reflecting the work of the posture control system and a later component reflecting the work of the movement control system. It is suggested that the planning of muscle activity and exchange of information between these two systems take place only before the beginning of the movement; after that, they act independently and in parallel.     

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.     

Chromosome displacement and spindle tubule polymerization: 1. The effect of alterations in pH on displacement frequency

In order to test whether 'chromosome displacement' (Ford and Lester, 1982) is related to spindle function, the phenomenon was examined under conditions known to alter spindle tubule polymerization, namely alterations in pH within the range 6.8-8.0. The following observations were made: (a) the frequency of cells showing chromosome displacement was not altered by variations in pH, but the number of displaced chromosomes per cell was markedly changed. Minimum numbers of chromosomes were displaced at pH 7.6. (b) For any chromosome, the extent of the response to pH change, was positively correlated with the basal displacement rate for that chromosome. (c) Chromosomes which have a 'stable' evolutionary history have a more predictable response to pH than those which have an 'unstable' history. Since displacement is significantly influenced by pH, it is concluded that the phenomenon is related to spindle structure.     

LIS1 and spindle orientation in neuroepithelial cells

Asymmetric stem cell division is thought to require precise orientation of the mitotic spindle. However, a recent study in Cell (Yingling et al., 2008) analyzes the role of LIS1 in the developing mouse brain and shows that spindle orientation is more important during early, symmetric progenitor cell divisions than for later asymmetric divisions.