Central Spindles Research Articles

AIM-1: a mammalian midbody-associated protein required for cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain.

Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles.

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.

Comparison Of Spindle Elongation In Vivo And In Vitro In Stephanopyxis Turris

The spindle in dividing cells of the diatom Stephanopyxis turris contains three distinct classes of microtubules: central spindle microtubules, which slide over each other and grow during anaphase spindle elongation; kinetochore-attached microtubules, which are located on the outer surface of the central spindle; and peripheral microtubules, which fan out from the spindle poles in astral-like arrays. The poles are multilayered structures, which remain attached to the spindle after isolation. In vitro, after addition of ATP, central spindles elongate and the two half-spindles slide completely apart with a concurrent decrease in the extent and magnitude of the zone of microtubule overlap. Spindle elongation takes place in spindles whose chromatin has been removed by enzymic digestion and the extent of elongation in vitro is increased by the addition of neurotubulin. After ATP addition the arrays of interdigitating microtubules in the zone of overlap become disordered and selectively depolymerize from the overlap zone polewards. In some reactivated spindles an unusual structure, a striated fibre, can be seen running from the pole plates part of the way towards the spindle midzone. The fibre has no precedent in mitotic ultrastructure and its function is unclear. These results demonstrate that we can duplicate the essential elements of anaphase B in vitro and that this system will be useful for further studies of the molecular basis of spindle elongation.

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.

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare.

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three-dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.

Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.

On the mechanism of anaphase spindle elongation in Diatoma vulgare.

Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.

Reproduction by Binary and Multiple Fission in Gigantomonas*

SYNOPSIS. Gigantomonas usually exists in the multinucleate stage. Hence, multiple fission is more common than binary fission. In its flagellate as well as its aflagellate state it is always an amoeboid cell which possesses from one to several large, clear pseudopodia. Fairly often in the multinucleate, as well as the uninucleate, stage no extranuclear organelles are present except large plain centrioles, two of which are always closely associated with each nucleus. During nuclear reproduction four centrioles, two old ones and two new ones, are always present with each nucleus. During all nuclear reproductions, regardless of the number of nuclei present, extranuclear organelles, such as flagella, axostyle, undulating membrane, and costa when present are discarded. If they are renewed, it is by the new centrioles at the same time that the old ones produce new central spindles, two always cooperating in the process. Thus, Gigantomonas, like other genera of the Devescovinidae, the family to which it belongs, never has for each nucleus present more than one set of extranuclear organelles, a characteristic which Devescovinidae and Lophomonadidae have in common. It is the only genus of Devescovinidae without a parabasal body.Owing to the liquidness of the cytoplasm, the central spindle, which becomes very long indeed, often extends beyond the cytoplasm and thus pushes the nucleus fastened to this end of it completely out of the cell. Mainly because of this situation, multinucleate forms with an odd number of nuclei occur often; otherwise the nuclear numbers would be 2, 4, 8, 16, 32, etc., because, no matter how many nuclei are present, they all reproduce simultaneously or nearly so.An unusual situation occurs in which Gigantomonas ingests and digests a small species of Holomastigotoides, while several hundred individuals of the latter become attached to and destroy Gigantomonas.

Induced Rocking

Circular objects such as disks fitted with central spindles rest on a horizontal table in inclined positions which are in neutral equilibrium. If the disk is loaded eccentrically with a small body the disk will rest in stable equilibrium when that body is in its lowest position. If the whole is displaced to one side and released it will rock to and fro about that position. Theory and experiment are applied to find how the period of the rocking motion depends on the dimensions of the disk, spindle, and affixed body, the inclination of the spindle to the horizontal, and the masses and moments of inertia of disk, spindle, and affixed body. The expression found for the length L of the equivalent simple pendulum is L = I cosρ/ma, where I is the total moment of inertia of disk, spindle, and affixed body about the line joining the points of contact of the spindle and the disk with the table, ρ is the angle of inclination of the spindle to the horizontal, m is the mass of small affixed body, and a is the distance of the center of this body from the axis of the disk and spindle.

A Factual Analysis of Chromosomal Movement in Barbulanympha

SYNOPSIS. The role of eleven different types of achromatic figures in chromosomal movement of Barbulanympha is analyzed. When only one pole is present, no chromosomes are ever connected with it, and hence they do not move. The chromosomes go through their usual life cycle including pairing, but remain in the parent nucleus, which, of course, does not divide. When two poles are present with only one pole near the nucleus, the poles, which are the distal ends of the elongate centrioles, do not cooperate in the formation of a central spindle and the chromosomal behavior is just as if there were only one pole—no movement. The same is true when more than two poles are present with only one near the nucleus.Unless a central spindle is present, movement of chromosomes never occurs. However, when many central spindles are present, sister chromosomes may separate and move to poles which are not directly connected by a central spindle. In other words, sisters may separate without moving along a central spindle.In binucleate cells with one central spindle the chromosomes of one nucleus move to the poles, but those of the other do not. Movement always occurs in the nucleus that has its nuclear membrane depressed by the central spindle. When two or more central spindles are present, the chromosomes of both nuclei may move to the poles.

Achromatic Figure Formation by Multiple Centrioles of Barbulanympha

SUMMARY. The role of centrioles in achromatic figure production is considered when the number present varies from 1–8. Each centriole after it becomes elongate produces astral rays from its distal end. Some of these rays remain free; some, by joining centromeres, become chromosomal fibers; and some, by joining and growing along those produced by one or more other centrioles, produce the central spindle portion of the achromatic figures. Thus, one centriole may function cooperatively with one to several others in the production of central spindles. But at least two centrioles must be present, and in the proper spacial relation to each other, to form a central spindle; one by itself can form only free astral rays, no central spindle or chromosomal fibers.The flagellated areas (to which the centrioles are anchored anteriorly) play an important role in determining the position of the distal ends of the centrioles with respect to one another, and the position of these ends, in turn, in a large measure, determines the types of achromatic figures produced, particularly the number of central spindles.