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.

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.

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.

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.

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.

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.

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.

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.