Biomedical subjects

L Cassimeris

Publications and source records attributed to L Cassimeris.

35 records · Page 2Linked to original sources

PubMed1995

Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.

Actin Cytoskeleton↗

PubMed1994

XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover.

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.

Animals↗

PubMed1994

Microtubule assembly and kinetochore directional instability in vertebrate monopolar spindles: implications for the mechanism of chromosome congression.

We have proposed previously a kinetochore motor-polar ejection model for chromosome congression to the metaphase plate where forces generated at the kinetochore are antagonized by away-from-the pole forces generated within each half-spindle on the chromosome arms. This model was based in large part on observations of the behavior of chromosomes on monopolar spindles. In these cells chromosomes typically become attached to the pole by only one kinetochore fiber. These mono-oriented chromosomes move to positions away from the pole even though they are pulled poleward at their kinetochores. Their arms are also ejected away from the pole when severed from the centromere. Here we have characterized further the properties of monopolar spindles in newt lung epithelial cells to determine the similarities between monopolar and bipolar spindles. We found no significant differences between monopolar and bipolar spindles over the parameters examined, which included: microtubule dynamics as measured by fluorescence redistribution after photobleaching; the ability of polar microtubule arrays to push chromosome arms away from the pole; the dependence of chromosome position relative to the pole on microtubule assembly; the number of kinetochore microtubules per kinetochore; and the directional instability of kinetochore motion during chromosome oscillations poleward and away-from-the-pole. As in bipolar spindles, kinetochore directional instability is characterized by abrupt switching between constant velocity phases of poleward and away-from-the-pole motion. From these data we conclude that the mechanism(s) responsible for chromosome positioning in monopolar spindles are fundamentally the same as those in bipolar spindles; only the geometry of the two spindle forms and the interplay between sister kinetochore directional instabilities are different. We also found no correlation in the kinetochore-to-pole distance with kinetochore microtubule number in monopolar spindles, but a strong qualitative correlation with microtubule density. This finding indicates that oscillations of mono-oriented chromosomes in both monopolar and bipolar spindles occur because chromosomes persist in poleward motion until they reach a density of polar microtubules sufficiently high to promote switching to away-from-the-pole motion. As the kinetochore and chromosome arms move away-from-the-pole, microtubule density decreases and the kinetochore switches to poleward motion, pulling the chromosome arms back into regions of higher microtubule density. The mechanism regulating kinetochore switching between poleward and away-from-the-pole motion is poorly understood, but may depend on tension at the kinetochore generated by pushing forces on the chromosome arms produced by the polar microtubule arrays.

Animals↗

PubMed1993

Regulation of microtubule dynamic instability.

Characterization of microtubule assembly in vivo and in vitro has raised questions of how cells regulate dynamic instability. While dynamic instability is an intrinsic property of the tubulin molecule, factors are required to increase the plus-end elongation rate and increase the frequencies of both catastrophe and rescue to achieve cellular tubulin turnover rates. It is likely that the activities of the transition frequency regulators are themselves regulated during the cell cycle, but the mechanisms of regulation are not known. As we identify the proteins which alter microtubule assembly, new classes of MAPs will emerge. An understanding of how these proteins function may provide further insight into how cells organize the different arrays of microtubules used for such processes as vesicle transport, polarized organization of organelles, and chromosome movement.

Animals↗

PubMed1993

Thymosin beta 4 (T beta 4) in activated platelets.

When resting human blood platelets are stimulated with thrombin, 50 to 60% of the G-actin polymerizes to F-actin within 60 seconds. The increase in F-actin is correlated with a corresponding decrease in the complex of G-actin with T beta 4. Within 5 seconds after stimulation, nucleation sites for pyrene actin polymerization increase 1.5 times in Triton-lysed supernatants. Cytochalasin D, known to inhibit the increase in F-actin after thrombin, also inhibits the fall in T beta 4-actin complex and the increase in nucleation sites. Phosphorylation of T beta 4 could not be detected in either control or activated cells. Increased T beta 4 corresponding to that lost from the T beta 4-actin complex is present in lysates from activated platelets and retains the ability to complex with actin. The data, taken together with previous estimates for the dissociation constant of the T beta 4-actin complex, show that actin polymerization following platelet activation could be controlled primarily by the increased availability of free barbed ends of actin filaments which have a higher affinity for G-actin than does T beta 4 and suggest that the increased free T beta 4 may serve to limit the degree of polymerization.

Actins↗

PubMed1992

Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin.

Fluorescent derivatives of phalloidin are widely used to measure filamentous actin (F-actin) levels and to stabilize F-actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminyl (TRITC)-phalloidin to rabbit skeletal muscle F-actin and to F-actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC-phalloidin can be used to inhibit F-actin depolymerization and to quantify F-actin without prior fixation. By equilibrium measurements, the affinity of TRITC-phalloidin binding to rabbit skeletal muscle F-actin (pyrene labeled) or to PMN lysate F-actin was 1-4 x 10(-7) M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC-phalloidin binding to PMN lysate F-actin resulted in an association rate constant of 420 +/- 120 M-1 sec-1 and a dissociation rate constant of 8.3 +/- 0.9 x 10(-5) sec-1. The affinity calculated from the kinetic measurements (2 +/- 1 x 10(-7) M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 microM TRITC-phalloidin inhibited 0.1 microM pyrenyl F-actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F-actin (less than 1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC-phalloidin can be used to measure F-actin levels in cell lysates when G-actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin-induced polymerization.

Actins↗

PubMed1992

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes.

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.

Actinin↗

PubMed1992

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.

Actins↗

PubMed1991

Chromosome fiber dynamics and congression oscillations in metaphase PtK2 cells at 23 degrees C.

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 microns or greater. DTAF-tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF-tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti-beta tubulin antibody. At 2-5 minutes after injection, concentrated DTAF-tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF-tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 microns) of concentrated DTAF-tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF-containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF-containing segments: 1) a poleward flux of tubulin subunits (Mitchison, 1989) or 2) capture of DTAF-containing nonkinetochore microtubules.

Animals↗

PubMed1991

Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes.

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/- 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60-90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.

Animals↗

PubMed1990

Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities.

Chemoattractants stimulate actin polymerization in lamellipodia of polymorphonuclear leukocytes. We find that removal of chemoattractant results in rapid (within 10 s at 37 degrees C) and selective depolymerization of the F-actin located in lamellipodia. Addition of 10 microM cytochalasin B, in the presence of chemoattractant, also resulted in rapid and selective depolymerization of lamellar F-actin. The elevated F-actin level induced by chemoattractant rapidly returns to the level present in unstimulated cells after (a) a 10-fold decrease in chemoattractant concentration; (b) the addition of 10 microM cytochalasin B; or (c) cooling to 4 degrees C. The F-actin levels of unstimulated cells are only slightly affected by these treatments. Based on the similar effects of cytochalasin addition and chemoattractant dilution, it is likely that both treatments result in actin depolymerization from the pointed ends of filaments. Based on our results we propose that chemoattractant-stimulated polymorphonuclear leukocytes contain two distinct populations of actin filaments. The actin filaments within the lamellipodia are highly labile and in the continued presence of chemoattractant these filaments are rapidly turning over, continually polymerizing at their plus (barbed) ends, and depolymerizing at their minus ends. In contrast, the cortical F-actin filaments of both stimulated and unstimulated cells are differentially stable.

Actins↗

PubMed1990

Stability of microtubule attachment to metaphase kinetochores in PtK1 cells.

Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10-20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/- 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.

Animals↗

PubMed1990

Chemoattractant stimulation of polymorphonuclear leucocyte locomotion.

Chemoattractants stimulate both cell locomotion and the orientation of this locomotion (chemotaxis) in polymorphonuclear leukocytes. Cell locomotion is a complex process which includes the coordinated protrusion of cell processes, formation of attachments to the substrate and contraction of the rear of the cell. To understand how chemoattractants regulate this process, it is helpful to dissect the process into components that can be examined separately. Comparison of these components in cells before and after stimulation with chemoattractant provides information about their regulation. In this review we focus on three components: how chemoattractants induce the development of cell polarity; how chemoattractants modulate cytoskeletal components (especially actin) to cause pseudopod protrusion; and how chemoattractant modulation of cell adhesions might contribute to cell locomotion. Spatial and temporal coordination of these and other components of locomotion result in efficient and directed cell movement. Our treatment of these questions is speculative and not comprehensive. We propose simple hypothetical models which can provide the reader with a conceptual framework that integrates the information available.

Actins↗

PubMed1988

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy.

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.

Animals↗

PubMed1988

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization.

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.

Actins↗

PubMed1988

Real-time observations of microtubule dynamic instability in living cells.

Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.

Animals↗

PubMed1986

Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle.

During mitosis a monooriented chromosome oscillates toward and away from its associated spindle pole and may be positioned many micrometers from the pole at the time of anaphase. We tested the hypothesis of Pickett-Heaps et al. (Pickett-Heaps, J. D., D. H. Tippit, and K. R. Porter, 1982, Cell, 29:729-744) that this behavior is generated by the sister kinetochores of a chromosome interacting with, and moving in opposite direction along, the same set of polar microtubules. When the sister chromatids of a monooriented chromosome split at the onset of anaphase in newt lung cells, the proximal chromatid remains stationary or moves closer to the pole, with the kinetochore leading. During this time the distal chromatid moves a variable distance radially away from the pole, with one or both chromatid arms leading. Subsequent electron microscopy of these cells revealed that the kinetochore on the distal chromatid is free of microtubules. These results suggest that the distal kinetochore is not involved in the positioning of a monooriented chromosome relative to the spindle pole or in its oscillatory movements. To test this conclusion we used laser microsurgery to create monooriented chromosomes containing one kinetochore. Correlative light and electron microscopy revealed that chromosomes containing one kinetochore continue to undergo normal oscillations. Additional observations on normal and laser-irradiated monooriented chromosomes indicated that the chromosome does not change shape, and that the kinetochore region is not deformed, during movement away from the pole. Thus movement away from the pole during an oscillation does not appear to arise from a push generated by the single pole-facing kinetochore fiber, as postulated (Bajer, A. S., 1982, J. Cell Biol., 93:33-48). When the chromatid arms of a monooriented chromosome are cut free of the kinetochore, they are immediately ejected radially outward from the spindle pole at a constant velocity of 2 micron/min. This ejection velocity is similar to that of the outward movement of an oscillating chromosome. We conclude that the oscillations of a monooriented chromosome and its position relative to the spindle pole result from an imbalance between poleward pulling forces acting at the proximal kinetochore and an ejection force acting along the chromosome, which is generated within the aster and half-spindle.

Anaphase↗