Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.

We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells.

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.

Diatom gliding is the result of an actin-myosin motility system.

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.

Ultraviolet microbeam irradiations of epithelial and spermatocyte spindles suggest that forces act on the kinetochore fibre and are not generated by its disassembly.

Ultraviolet (UV) microbeam irradiations of crane-fly spermatocyte and newt epithelial spindles severed kinetochore fibres (KT-fibres), creating areas of reduced birefringence (ARBs): the remnant KT-fibre consists of two "stubs," a pole-stub attached to the pole and a KT-stub attached to the kinetochore. KT-stubs remained visible but pole-stubs soon became undetectable [Forer et al., 1996]. At metaphase, in both cell types the KT-stub often changed orientation immediately after irradiation and its tip steadily moved poleward. In spermatocytes, the chromosome attached to the KT-stub remained at the equator as the KT-stub elongated. In epithelial cells, the KT-stub sometimes elongated as the associated chromosome remained at the equator; other times the associated chromosome moved poleward together with the KT-stub, albeit only a short distance toward the pole. When an ARB was generated at anaphase, chromosome(s) with a KT-stub often continued to move poleward. In spermatocytes, this movement was accompanied by steady elongation of the KT-stub. In epithelial cells, chromosomes accelerated polewards after irradiation until the KT-stubs reached the pole, after which chromosome movement returned to normal speeds. In some epithelial cells fine birefringent fibres by chance were present along one edge of ARBs; these remnant fibres buckled and broke as the KT-stub and chromosome moved polewards. Similarly, KT-stubs that moved into pole stubs (or astral fibres) caused the pole stubs (or astral fibres) to bend sharply from the point of impact. Our results contradict models of chromosome movement that postulate that force is generated by the kinetochore disassembling the KT-fibre. Instead, these results suggest that poleward directed forces act on the KT-fibre and the KT-stub and suggest that continuity of microtubules between kinetochore and pole is not obligatory for achieving anaphase motion to the pole.

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.

The amyloid precursor protein of Alzheimer's disease is found on the surface of static but not activity motile portions of neurites.

We have previously found that the amyloid precursor protein (APP) of Alzheimer's disease is present on the surface of rat cortical neurons in culture, in a segmental pattern which first becomes evident after 24 hours and is fully developed by five days. As APP has previously been reported to have a short half-life in neuronal cell lines, and has been shown to contain binding sites for various extracellular matrix components within its extracellular domain, we hypothesized that APP would be associated with portions of neurites undergoing rapid structural change, such as growth cones. To test this hypothesis, we observed selected neurons by video time-lapse differential interference microscopy on 24-hour-old primary rat neuronal cultures for up to 45 minutes, followed by fixation and immunocytochemistry to ascertain surface APP distribution on those same neurons. In contrast to our predictions, surface APP was not found on active portions of neurites, even if the activity produced no net translational movement. This result indicates that surface APP is actually associated with stable portions of neurites, a conclusion that tallies with other recent results showing that neuronal surface APP has a longer half-life than general cellular APP, and is associated with markers of adhesion patches, which themselves are relatively stable structures.

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.

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.

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.

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.

Microtubule dynamics in the spindle. II. A thermodynamic and kinetic description.

We have previously presented a model for the assembly and disassembly of mitotic spindle microtubules (MTs) (Pickett-Heaps et al., 1986). In this paper, we describe the thermodynamics of such spindle MT assembly and present equations to describe the polymerization kinetics of different classes of spindle MTs. These equations are used to predict, in terms of kinetics parameters, the magnitude of forces extant on spindle MTs and to define the critical force needed to halt MT assembly. We calculate several of these forces for a hypothetical model cell; our predicted value for the force generated along kinetochore fibers is in close agreement with measured values taken from living cells. The model and its implications are discussed with reference to other recent models of spindle and MT dynamics.

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.

Studies on kinetochore function in mitosis. I. The effects of colchicine and cytochalasin on mitosis in the diatom Hantzschia amphioxys.

Cytochalasin does not affect mitosis (including half spindle separation and disassembly) while cleavage is partially or totally inhibited. While colchicine stops central spindle growth, it resists breakdown even after prolonged exposure. Prometaphase chromosome movement soon ceases, and some attached chromosomes slowly detach; these phenomena are correlated with a loss of the numerous microtubules (MTs) that emanate from the poles, with which chromosomes interact. "C-anaphase", often seen, is marked in vivo by spindle elongation and unequal polar distribution of those chromosomes still attached to the central spindle; this stage is characterized ultrastructurally by the accumulation of dense matrix material, probably the "collar" previously described, at the poles. Kinetochores often remain tightly associated with this matrix. We believe this result is significant, since it clearly demonstrates that the kinetochores are attached to a spindle component other than microtubules. We suspect that this matrix is contractile and part of the mitotic machinery for moving chromosomes. These colchicine effects are not reversible.