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R B Nicklas

Publications and source records attributed to R B Nicklas.

At least 19 recordsLinked to original sources
PubMed1992

Orientation and segregation of a micromanipulated multivalent: familiar principles, divergent outcomes.

We studied the orientation and segregation of a particular quadrivalent in living grasshopper spermatocytes. Quadrivalents were detached from the spindle by micromanipulation, then placed and bent as desired. The detached quadrivalents reattach and orient on the spindle. Their orientation is determined by the same principles that apply to ordinary chromosomes in mitosis and meiosis, but the outcome is different. Certain characteristics of the quadrivalent lead to a variety of orientations rather than the single one typical of ordinary chromosomes. Two kinetochores in the quadrivalent are linked to the others by unusually long, flexible chromosome arms. These kinetochores may face either the same pole or opposite poles and tend to orient initially to the pole toward which they face. Consequently, the initial orientation of the flexibly linked kinetochores is variable, and, moreover, they frequently reorient. In contrast, the other two kinetochores are as rigidly connected as those in a small bivalent and so display the typical back-to-back arrangement. Usually, this arrangement leads quickly to a stable orientation of the two kinetochores to opposite poles. Sometimes, however, the back-to-back arrangement changes to a side-by-side arrangement so that the orientation of both kinetochores to the same pole is favored. The combined effect of this diverse behavior is that the quadrivalent has four stable orientations, each leading to a different distribution of chromosomes in anaphase. The result is genetic chaos. Ironically, this chaos is produced by the same mechanisms that, in ordinary bivalents and mitotic chromosomes, produce a single stable orientation and genetically appropriate chromosome distribution.

Aneuploidy↗
PubMed1992

Evolution and the meaning of metaphase.

We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.

Animals↗
PubMed1989

Tension, microtubule rearrangements, and the proper distribution of chromosomes in mitosis.

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such mal-orientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterized mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.

Animals↗
PubMed1989

The motor for poleward chromosome movement in anaphase is in or near the kinetochore.

I have tested two contending views of chromosome-to-pole movement in anaphase. Chromosomes might be pulled poleward by a traction fiber consisting of the kinetochore microtubules and associated motors, or they might propel themselves by a motor in the kinetochore. I cut through the spindle of demembranated grasshopper spermatocytes between the chromosomes and one pole and swept the polar region away, removing a portion of the would-be traction fiber. Chromosome movement continued, and in the best examples, chromosomes moved to within 1 micron of the cut edge. There is nothing beyond the edge to support movement, and a push from the rear is unlikely because cuts in the interzone behind the separating chromosomes did not stop movement. Therefore, I conclude that the motor must be in the kinetochore or within 1 micron of it. Less conclusive evidence points to the kinetochore itself as the motor. The alternative is an external motor pulling on the kinetochore microtubules or directly on the kinetochore. A pulling motor would move kinetochore microtubules along with the chromosome, so that in a cut half-spindle, the microtubules should protrude from the cut edge as chromosomes move toward it. No protrusion was seen; however, the possibility that microtubules depolymerize as they are extruded, though unlikely, is not ruled out. What is certain is that the motor for poleward chromosome movement in anaphase must be in the kinetochore or very close to it.

Anaphase↗
PubMed1989

Mechanically cut mitotic spindles: clean cuts and stable microtubules.

We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.

Animals↗
PubMed1986

Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.

Animals↗
PubMed1985

Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation.

The relationship between chromosome movement and microtubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.

Animals↗
PubMed1985

The total length of spindle microtubules depends on the number of chromosomes present.

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.

Animals↗
PubMed1984

A quantitative comparison of cellular motile systems.

Cellular motile systems as diverse as muscle and the mitotic spindle have been compared by their specific power output: the maximum power they develop per unit of engine volume. Striated muscles and flagella have high specific output; their performance is comparable to that of typical automobile engines. The cytokinetic furrow and the mitotic spindle have very much lower specific power output. The furrow's output is 7,000 times lower than muscle and the spindle's is 300,000 times lower. Different macromolecules have been used to generate power in systems with similar output (muscles and flagella) and, conversely, the same macromolecular motor has been used in systems with very different output (muscles and cytokinetic furrows). The common feature amid this diversity is adaptation to a particular biological role, which specific power output reflects very well. High values are found where a powerful, compact engine should be advantageous, while low values are found where precision, not power, matters most.

Actomyosin↗
PubMed1983

Measurements of the force produced by the mitotic spindle in anaphase.

The force the spindle exerts on a single moving chromosome in anaphase was measured with a flexible glass needle calibrated in dynes per micron of tip deflection. The needle was used to produce a force on the chromosome, which opposed that produced by the spindle and was measurable from needle tip deflection. The measurements were made in intact grasshopper spermatocytes after proving that the presence of materials such as the cell surface did not interfere. The results from 12 experiments in seven cells are as follows: Chromosome velocity was not affected until the opposing force reached approximately 10(-5) dyn, and then fell rapidly with increasing force. The opposing force that caused chromosome velocity to fall to zero--the force that matched the maximum force the spindle could produce--was of order 7 X 10(-5) dyn. This directly measured maximum force potential is nearly 10,000 times greater than the calculated value of 10(-8) dyn for normal chromosome movement, in which only viscous resistance to movement must be overcome. The spindle's unexpectedly large force potential prompts a fresh look at molecular models for the mitotic motor, at velocity-limiting governors, and at the possibility that force may sometimes affect microtubule length and stability.

Anaphase↗
PubMed1982

Spindle microtubules and their mechanical associations after micromanipulation in anaphase.

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.

Anaphase↗
PubMed1979

Chromosome movement and spindle birefringence in locally heated cells: interaction versus local control.

A microheater was used to produce a temperature gradient within the mitotic spindle of living cells. The slope of the temperature gradient was estimated from thermal conductivity calculations and confirmed by measurements of spindle birefringence and by experiments on striated muscle. When the microheater was placed at one spindle pole or at one group of kinetochores, the gradient was steep enough to cause a large difference in birefringence between the two half-spindles, but the velocity of chromosome movement in anaphase was nearly the same in the warmer and cooler half-spindles. When the heater was shifted from the pole toward the interzone, the average velocity of chromosome movement increased approximately two-fold but was, again, nearly uniform in the two half-spindles. The rate of spindle elongation was especially sensitive to the site of heating, increasing ten-fold when the heater was shifted from the pole to the interzone. Regardless of heater position, the rate of chromosome movement was determined largely by the temperature of the coolest spindle region--chromosomes in the warmer half-spindle moved more slowly than expected from estimates of the temperature in that region. Since the microheater produces a substantial temperature gradient within the spindle, the near uniformity of chromosome velocity in both half-spindles must be due to some biological property of the spindle. Two very different explanations for the results are considered the most likely. According to one explanation, the near uniformity of velocity in both half-spindles is determined by the structure of the interpolar spindle, while changes in velocity involve force producers located both in the half-spindles and in the interzone. On the other explanation, the velocity is nearly the same in both half-spindles because the force producers are located exclusively in the interzone (Margolis et al., 1978).

Anaphase↗
PubMed1979

Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes.

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.

Animals↗
PubMed1977

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.

Animals↗