Mitotic forces

Li X, Nicklas RB. Mitotic forces control a cell-cycle checkpoint. Nature 1995 373 630-632. 

These considerations will center on the usual course of chromosome motion occurring when the spindle is present—prometaphase through anaphase. Three questions suggest the analytical Tormat. First, descriptive cytology What are the phenomena to be explained Second, mechanics What forces and mechanical properties are necessary to account for chromosome motion Third, preliminary attempts at molecular explanation How are mitotic forces produced and controlled This gives a progression from the best to the least understood features of chromosome motion. 

Spindle Mechanical Properties. How does the spindle respond to the mitotic forces so that force directionality is controlled and orderly chromosome motion results Artificial forces have been used to study chromosome attachment to the spindle. Especially informative are the centrifugation studies of Schrader (1934), Shimamura (1940), and Yamamoto (1964), and the micromanipulation experiments of Carlson (1952). Recent micromanipulation results (Nicklas and Staehly, 1967) add some details to the earlier observations and confirm them during normal prometaphase and anaphase motion. Artificial forces sufficient to stretch the chromosome cause little or no increase in the distance from the pole to the kinetochore, but there is much less resistance to lateral or poleward displacement. The spindle as a whole behaves in micromanipulation and during isolation as a single body it is a mechanical unit independent of the rest of the cell (reviewed by Mazia, 1961). The simplest, and the classic, interpretation attributes these mechanical properties to individual spindle fibers in order to account for the... 

Chromosomes are individually attached to the spindle by chromosomal spindle fibers rigid enough to bear any normal mitotic force. The separation of the poles is... 

Fig. 8. Diagrammatic representation of a hypothesis (McIntosh et al., 1969) of mitotic force production based upon sliding of polarized interpolar ( Ip ) and chromosomal ("Ch") microtubules caused by cross-bridges (short, diagonal arrows) between them. A., The basic motile unit. B, Metaphase, showing two sister chromatids linked together (bold horizontal bars) and a minimal number of microtubules. C, and D, Two stages in anaphase after dissolution of the chromatid linkages. Details In the text. (Adapted from McIntosh et al. 1969. Nature (London), 224 659-663.)...

Fig. 10. Diagrammatic representation of an alternative sliding hypothesis for mitotic force production. Midanaphase is depicted with two normal sister chromatids, one chromatid displaced to the right, and polarized interpolar fibers ( Mp") with attached bridges (diagonal lines or arrows) which produce force only when adjacent to appropriately oriented, polarized chromosomal fibers. The question marks denote two unspecified features of the model bridge interactions between overlapping interpolar fibers of opposite polarity (center) and possible activity of "free" bridges in regions (right and left) of force production between chromosomal and Interpolar fibers. The broken vertical line indicates the position of the equator. Details in the text.

Second, interlocked unipolar bivalents were produced by micromanipulation by Henderson and Koch (1970). Two unipolar bivalents were oriented to opposite poles, with one threaded through the other, as shown in Figure 14 (e.g., 137-minute print). The two bivalents exerted tension against each other by natural mitotic forces, recognizable from the slight elongation of each bivalent (compare the 0- and 137-minute prints). The needle was withdrawn (before 0 minutes), and the orientation of all four half-bivalents... 

In contrast, UCN-01, a staurosporine derivative, acts as a potent inhibitor of the Chkl kinase and efficiently abrogates the G2 checkpoint upon DNA damage. Die forced entry into mitosis in the presence of DNA damage results in a mitotic form of apoptosis. Several clinical trials are currently exploring a combined treatment with UCN-01 and various DNA damaging diugs. In the same vein, inhibitors of Chk2 are developed and tested in clinical trials. 

It is most unlikely that the sole functions of mysoin-Il in nonmuscle cells are to provide the contractile force to bisect cells during cytokinesis and for the contractility of stress fibers. Myosin-II is present in a variety of cell types at moderate concentrations in tissues such as brain, which are almost totally non-mitotic and do... 

Uemura T, Ohkura H, Adachi Y, Morino K, Shiozaki K, Yanagida M (1987) DNA topoisomerase 11 is required for condensation and separation of mitotic chromosomes in S. pombe. Cell 50 917-925 Ushiki T, Hoshi O, Iwai K, Kimura E, Shigeno M (2002) The structure of human metaphase chromosomes its histological perspective and new horizons by atomic force microscopy. Arch Histol Cytol 65 377-390... 

Fetner, R. H. Mitotic Inhibition Induced in Grasshopper Neuroblasts by Exposure to Ozone. Technical Documentary Report SAM-TDR 63 39. Brodcs Air Force Base, Texas USAF School of Aerospace Medicine, Aerospace Medical Diviskm (AFSa, June 1%3. 

The elastic stress may be external or internal. External stresses are exerted on the chromatin during the cell cycle when the mitotic spindle separates chromosome pairs. The 30-nm fiber should be both highly flexible and extensible to survive these stresses. The in vitro experiments by Cui and Bustamante demonstrated that the 30-nm fiber is indeed very soft [66]. The 30-nm fiber is also exposed to internal stresses. Attractive or repulsive forces between the nucleosomes will deform the linkers connecting the nucleosomes. For instance, electrostatic interactions, either repulsive (due to the net charge of the nucleosome core particles) or attractive (bridging via the lysine-rich core histone tails [49]) could lead to considerable structural rearrangements. 

Mitotic cells may be collected by selection of the correct shearing forces (Crespi and Thilly, 1982 Crespi et al., 1981 - see Chapter 11). 

This involves the use of a special rotor and the adaptation of the centrifuge for constant flow as for the zonal rotor. This is expensive, but once installed, reproducible separations of 108-109 cells can be achieved in under an hour and cells of all sizes (rather than just the smallest) are obtained. It is particularly suited to the isolation of Gl-phase cells from suspension cultures for which mitotic detachment procedures are not applicable. The separation chamber is kite-shaped with the buffer solution entering at the acute point on the rim of the centrifuge and leaving at the obtuse point towards the centre of rotation (Fig. 11.2). Thus the centrifugal forces on particles within the chamber are countered by the centripetal flow of buffer and particles come to equilibrium within the chamber. When equilibrium has been reached, the samples are pumped out, by increasing the rate of buffer flow, and collected. Meistrich et al. (1977) obtained 3 fractions of L-P59 mouse fibroblasts which were over 90% Gl-phase, 70% S-phase cells and 60% G2 + M-phase cells, respectively. 

The G2-phase of the cell cycle is perhaps the most difficult to study as it is the most difficult phase in which to obtain a synchronised cell population. This is because, if cells are synchronised by selection at mitosis or accumulation at the Gl/S boundary, by the time they reach G2 much of the synchrony has been lost. This is because of the dispersion forces arising from the different rates at which individual cells in a population traverse the cycle. G2 populations are always contaminated with cells in other phases of the cycle and the maximum fractions of Chinese hamster (CHO) cells obtainable in G2 are 0.7 by double thymidine block and 0.4 by mitotic selection (Enger et al., 1968). 

Equation 13 correlates a mixed set of compounds which produce an abnormal type of mitosis resembling that caused by colchicine. The role of hydrophobic forces causing this kind of mitotic activity is closely related to that causing hemolysis (Equation 18), inhibiting quinea pig ileum (Equation 17), I50 red blood cell oxygen consumption, etc. The results of Table II indicate that Equation 18 can be used as a model for nonspecific membrane perturbation in various systems. 

Inoue, S. 1996. Mitotic organization and force generation by as-sembly/disassembly of microtubules. Cell Struc. Funct. 21 375-379. 

The first evidence that diffusible factors regulate the cell cycle came from cell-fusion experiments with cultured mammalian cells. When interphase cells in the Gi, S, or G2 phase of the cell cycle were fused to cells in mitosis, their nuclear envelopes retracted and their chromosomes condensed (Figure 21-3). This finding indicates that some diffusible component or components in the cytoplasm of the mitotic cells forced interphase nuclei to undergo many of the processes associated with early mitosis. We now know that these factors are the mitotic cyclin-CDK complexes. 

Catastrophic genetic damage can occur If cells progress to the next phase of the cell cycle before the previous phase is properly completed. For example, when S-phase cells are induced to enter mitosis by fusion to a cell In mitosis, the MPF present In the mitotic cell forces the chromosomes of the S-phase cell to condense. However, since the replicating chromosomes are fragmented by the condensation process, such premature entry into mitosis is disastrous for a cell. 

During mitosis, mitotic spindles attach to centromeres and provide the critical force needed to separate chromosomes prior to segregation into the two daughter cells. Human centromeres have also been shown to contain long stretches of AT rich sequences, including a highly conserved repetitive sequence (GGAAT)n. 

X values (see fig. 10.17). However, irregular oscillations of cdc2 kinase have been observed in the model, at least in the transient phase. The possibility of chaotic dynamics resulting from the periodic stimulation of the mitotic oscillator by pulses of growth factor remains very hypothetical. The occurrence of chaos in relation to the cell cycle has been discussed by Mackey (1985), and by Lloyd, Lloyd Olsen (1992), who used an abstract model of the mitotic oscillator subjected to forcing by a sinusoidal input with much shorter period. 

Lasers and cancer So what use do scientists have for these tiny tweezers One group of scientists is using them to study cell organelles. They are studying the forces exerted by mitotic spindles— the grouping of microtubules that coordinates cell division. The spindles guide replicated chromosomes to opposite sides of the cell—a key role in cell division. However, scientists do not know exactly how the spindles perform this function. 

SLIDING. Shear forces between filaments were suggested as a possible mitotic motile mechanism by BHaf (1929) even before the advent of the highly successful sUding filament model of force production in muscle. Since then such speculations have increased (Mazia, 1961 Bajer, 1968a,c Subirana, 1968 McIntosh et al., 1969). The commendably specific and testable model of McIntosh et al. will be considered first, followed by a more general discussion. 

Wherever reorientation is an essential feature of mitosis, it probably has the same cause as in meiosis, although direct evidence is lacking (Nicklas and Koch, 1969). Thus, appropriate bipolar orientation subjects the mitotic chromosome to oppositely directed forces (cf. the deformations observed by Bajer, 1958a, Fig. 6). By analogy with meiosis, the resultant tension directly or indirectly stabilizes these orientations, and reorientation occurs until this state is reached. 

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