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J D Pickett-Heaps

Publications and source records attributed to J D Pickett-Heaps.

At least 19 recordsLinked to original sources
PubMed2001

Pac-Man does not resolve the enduring problem of anaphase chromosome movement.

The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates. We summarise these anomalies as follows: poleward movement of chromosomes occurring without insertion of any MTs at the kinetochore; "anaphase" shortening of kinetochore fibres in spindles entirely devoid of chromosomes and, presumably, kinetochores; continued movement of chromosomes while their severed kinetochore stub elongated poleward after treatment with UV microbeams; and fluxing of tubulin subunits through kinetochore MTs during anaphase A, indicating that during anaphase, kinetochore MTs disassemble partly or solely at the poles.

Anaphase↗
PubMed2001

Phallacidin stains the kinetochore region in the mitotic spindle of the green algae Oedogonium spp.

We found previously that in living cells of Oedogonium cardiacum and O. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (< 2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore of Oedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.

Actins↗
PubMed1998

Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.

Living crane-fly spermatocytes were treated with 10-20 microg/ml cytochalasin D (CD) or 0.3 microg/ml latrunculin (LAT) at various stages of meiosis I. The drugs had the same effects on chromosome behaviour, but CD effects were reversible and LAT effects generally were not. When applied in mid-prometaphase to metaphase, both drugs altered subsequent anaphase poleward movements: half-bivalents either moved more slowly than normal, or moved more slowly after a brief period of movement at normal rate or stalled for 10 min or more immediately after disjunction. CD effects were reversible: within 1 min after washing out the CD, stopped chromosomes started moving and slowed chromosomes sped up. When applied in anaphase, both drugs stopped or slowed poleward chromosome movements, usually reversibly. When applied near the end of prophase, both drugs often prevented one or more bivalents in the cell from attaching to the spindle. Attached bivalents behaved as in cells treated with drugs at later stages, as described above. Unattached bivalents in the same cells moved to poles or cytoplasm in early prometaphase, where they remained motionless; at anaphase they sometimes did not disjoin, but when they did disjoin the half-bivalents did not move, either in the continued presence of the drug or when CD was washed out, confirming that they were not atttached. When CD or LAT prevented all bivalents in the cell from attaching, spindles kept in the drug were invaded by granules at about the time of normal anaphase. Conversely, when CD was washed out during late prometaphase, chromosomes often attached to spindle fibres and later entered anaphase. As CD and LAT are different antiactin drugs, but have the same effect on chromosome behaviour, the results implicate actin in early interactions of chromosomes with spindle fibres and in anaphase chromosome movements.

Actins↗
PubMed1997

Traction fibre: toward a "tensegral" model of the spindle.

Most current hypotheses of mitotic mechanisms are based on the "PAC-MAN" paradigm in which chromosome movement is generated and powered by disassembly of kinetochore microtubules (k-MTs) by the kinetochore. Recent experiments demonstrate that this model cannot explain force generation for anaphase chromosome movement [Pickett-Heaps et al., 1996: Protoplasma 192:1-10]. Another such experiment is described here: a UV-microbeam cut several kinetochore fibres (k-fibres) in newt epithelial cells at metaphase and the half-spindle immediately shortened: in several cells, the remaining intact spindle fibres bowed outwards as they came under increased compression. Thus, severing of k-MTs can lead to increased tension between chromosomes and poles. This observation cannot be explained by models in which force is produced by motor molecules at the kinetochore actively disassembling k-MTs. Rather, we argue that tensile forces act along the whole k-fibre, which, therefore, can be considered as a classic "traction fibre." We suggest that anaphase polewards force is generated by MTs interacting with the spindle matrix and when k-MTs are severed, polewards force continues to act on the remaining kMT-stub; spindle MTs act as rigid struts concurrently resisting and being controlled by these forces. We suggest that the principles of "cellular tensegrity" [Ingber, 1993: J. Cell Sci. 104:613-627] derived from the behaviour and organization of the interphase cell apply to the spindle. In an evolutionary context, this argument further suggests that the spindle might originally have evolved as the mechanism by which a single tensegral unit (cytoplast) is divided into two cytoplasts; use of the spindle for segregating chromosomes might represent a secondary, more recent development of this primary function. If valid, this concept has implications for the way the spindle functions and for the spindle's relationship to cytokinesis.

Animals↗
PubMed1994

The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta.

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage. These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpublished), its mode of action is different to that of most anti-MT agents.

Cell Polarity↗
PubMed1993

An extended corona attached to metaphase kinetochores of the green alga Oedogonium.

Mitotic cells of the green alga Oedogonium were treated with the anti-microtubule agent oryzalin (1.0-0.1 microM) for 5 to 10 min. Within 5 min treatment of living cells, metaphase spindles became spherical with disorganized chromosomes, and anaphase spindles collapsed. At lower concentrations, the effects were slower, and partial recovery was observed about 10 to 20 min after the drug was washed out. Following breakdown of the spindle, considerable disorganized activity detected by time-lapse continued within the nucleus, isolated from the cytoplasm by its intact nuclear membrane. Under the electron microscope, spindle microtubules (MTs) were absent in oryzalin-treated cells. Paired metaphase kinetochores displayed an array of fine filamentous material extended, usually straight, about 3 microns into the nucleoplasm. In cells recovering from oryzalin treatment, MTs became associated with kinetochores in the usual manner. However, this filamentous array, the "extended corona" (EC), was almost undetectable, even when the MTs were short and poorly organized. The EC is appreciably larger by metaphase than the corona of prophase chromosomes and so it may assemble during early mitosis. Fine filaments interspersed with kinetochore MTs have been described in carefully fixed cells of this alga (M.J. Schibler, J.D. Pickett-Heaps, Eur. J. Cell Biol. 22, 687-698 (1980)). The EC apparently represents a less organized form of this material remaining after its scaffold of MTs has been removed. These fibers appear involved in MT capture upon spindle recovery from anti-MT drugs. They could function during prometaphase and even anaphase movement along spindle MTs.

Dinitrobenzenes↗
PubMed1991

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.

Anaphase↗
PubMed1990

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

Anaphase↗
PubMed1988

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella.

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.6 micron/min, the rate of anaphase B is 2.3 +/- 0.3 micron/min, and the extent of anaphase B is 26.7 +/- 9.7% of metaphase spindle length. Colchicine was added to metaphase cells in order to inhibit any further addition of microtubule (MT) subunits onto the spindle. Colchicine, which does not break down the well-ordered Surirella central spindle, caused no significant change in the rate of anaphase A (1.3 +/- 0.3 micron/min) while it significantly decreased both the rate of anaphase B (1.2 +/- 0.4 micron/min) and the extent of anaphase B (14.8 +/- 8.3% of metaphase spindle length). Surirella cells were also treated with the metabolic inhibitor 2-4-dinitrophenol (DNP) in order to test the effects of energy depletion on anaphase. When DNP was added early in anaphase A, prior to the completion of sister chromosome separation, anaphase A was inhibited. When DNP was added after initiation of sister chromosome separation, anaphase A continued to completion, although at a lower rate than control cells (0.5 +/- 0.2 micron/min). Anaphase B was completely inhibited by DNP, but upon recovery from DNP resumed at a normal rate (2.2 +/- 0.5 micron/min) and progressed to a slightly larger than normal extent (44.0 +/- 13.0% of metaphase length).(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphase↗
PubMed1987

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.

Adenosine Triphosphate↗
PubMed1986

Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassembly reactions in vivo.

The mitotic spindle contains several classes of microtubules (MTs) whose lengths change independently during mitosis. Precise control over MT polymerization and depolymerization during spindle formation, anaphase chromosome movements, and spindle breakdown is necessary for successful cell division. This model proposes the site of addition and removal of MT subunits in each of four classes of spindle MTs at different stages of mitosis, and suggests how this addition and removal is controlled. We propose that spindle poles and kinetochores significantly alter the assembly-disassembly kinetics of associated MT ends. Control of MT length is further modulated by localized forces affecting assembly and disassembly kinetics of individual sets of MTs.

Adenosine Triphosphate↗
PubMed1984

Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms.

Lesions ("ARBs") generated in metaphase and anaphase central spindles of Hantzschia by an ultraviolet microbeam are devoid of microtubules previously present. In vivo, the poleward transverse edge of the lesion invariably loses birefringence poleward, until this segment has vanished; the loss is slow during metaphase and faster at anaphase. The other transverse edge, proximal to the overlap, remains stable until disassembly of the whole spindle. We conclude that the central spindle microtubules are not in flux during metaphase to telophase, and that depolymerization of these microtubules takes place only from the end distal to the pole, as during normal spindle disassembly. Microtubule polarity and the creation of free ends may determine which microtubules are disassembled during later mitosis and how disassembly proceeds.

Anaphase↗
PubMed1984

The organization of microtubules during anaphase and telophase spindle elongation in the rust fungus Puccinia.

The entire framework of microtubules (MTs) in the meiotic spindle of the rust fungus Puccinia has been reconstructed during the later stages of meiosis I, by tracking MTs through transverse serial sections. This spindle is of special interest because it elongates considerably during anaphase spindle elongation, from 5 microns at metaphase to 15 microns at telophase. The spindle is composed mainly of MTs from opposite poles which interdigitate or overlap in the middle of the spindle. In the overlap region, MTs from one pole seek out as near neighbors, MTs from the opposite pole at a preferred spacing of 43 to 55 nm. During anaphase elongation three changes in spindle structure occur: 1) the region of overlap decreases, but this reduction in overlap cannot account for all the increase in spindle length; 2) interdigitated MTs (MTs from one pole that are within 80 nm of a MT from the opposite pole) dramatically increase in length by MT polymerization and; 3) kinetochore MTs, free MTs (those unattached to the poles) and non-interdigitated polar MTs shorten and disappear. The mechanism of anaphase elongation and the control over MT polymerization and depolymerization during anaphase are discussed.

Anaphase↗
PubMed1983

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation.

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.

Anaphase↗
PubMed1983

A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy.

A simple method is described to embed material in resin, in the form of microscope slides, to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block-holder for ultramicrotome are the specific tools of this method.

Microbiological Techniques↗
PubMed1983

Near-neighbor analysis of spindle microtubules in the alga Ochromonas.

The near-neighbor spacing of microtubules (MTs) in the spindle of the alga Ochromonas is analyzed. The technique of near-neighbor analysis of MTs (as developed by McDonald et al. [9]) in the mid-region of the Ochromonas spindle (overlap) shows that MTs from one pole preferentially associate with MTs from the opposite pole at a center-to-center distance of 35 to 43 nm. However, in the half spindle between the chromosomes and the poles, kinetochore MTs (kMTs) do not preferentially associate with other MTs in the half spindle but instead are arranged essentially at random. Individual polar MTs (MTs attached to one pole), kMTs and free MTs (MTs unattached to the poles) were selected for near-neighbor analysis over their entire lengths. The spacing of MTs in the overlap is compatible with those models for mitosis which propose that separation of the poles is accomplished by sliding between closely spaced MTs of opposite polarity. In contrast to the overlap, the arrangement of MTs in the half spindle is not compatible with MT2MT sliding theories that propose that chromosome movement is accomplished by sliding between kMTs and polar MTs.

Anaphase↗