Types and Life Cycles of Centrioles of Flagellates

SUMMARY. Two centrioles, an old one and a new one, are always present in the resting cell. From prophase onward two new ones and two old ones are present. Beginning with the resting stage, five types of centriole life cycles are described and compared with one another: In type 1, both centrioles are elongate; in type 2, the old one is long and the new one, which is short, elongates in prophase; in type 3, both are short, both elongate in prophase, and both, except for their anterior tips, degenerate in late telophase; in type 4, both are long but in prophase their distal ends become free of the rest of the centrioles, these ends migrate to center or posterior end of cell, where, after they produce the achromatic figure and it completes its function in nuclear division, they degenerate; in type 5, both are short and neither elongates at any stage of its life cycle.
New centrioles are produced by the anterior ends of old ones. In their first generation, centrioles produce only extranuclear organelles (flagella, parabasals, axostyles, etc.); in their second and later generations, they produce only the achromatic figure (gametogenesis in Trichonympha and reorganization in Barbulanympha and Rhynchonympha are exceptions to this rule).
The distal ends of centrioles in some types of cycles are surrounded by centrosomes; in others they are not. In one type of centriole life cycle a small central spindle is present in the resting cell in two genera; in the other types this is not the case.     

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.     

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally.     

Centriole number and the reproductive capacity of spindle poles

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.     

THE STRUCTURE AND FUNCTION OF CENTRIOLES AND THEIR SATELLITES IN THE JELLYFISH PHIALIDIUM GREGARIUM

Testes of jellyfish Phialidium gregarium were fixed in 2 per cent OsO₄ in Veronal-acetate buffer at pH 7.4. Thin sections showed that in young spermatids the spindle fibers of the last maturation division are attached to satellites of the filament-forming centriole. In more mature spermatids this attachment is not observed. During the developmental phase, nine satellites can be observed emanating from the interspaces between the nine tubular triplets of this centriole. A circular region on each of the enlarged distal ends of the satellites attaches them to the cell membrane. The satellites apparently provide a firm anchor for the axial filament. Each of the epithelial cells covering the testis produces a single long flagellum. On the filament-forming centriole often a satellite can be observed to which tubules are attached. These tubules are 180 A in diameter and probably represent remnants of spindle fibers. It is suggested that the distal centriole has the ability to form several satellites or appendages at appropriate times during the cell cycle. These satellites are distinct from the daughter centrioles in that they are supportive structures: in certain phases of cell life, spindle fibers may attach to them, while in other instances the distal centriole and the flagellum it is forming are anchored by them.     

Spermatogenesis of a marine snail,Littorina sitkana

Summary The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.     

Centrin-2 is required for centriole duplication in mammalian cells

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.     

In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells

Dividing cells in monolayers of the rat-kangaroo (Potorous tridactylis) cell line Pt-K₁ have large spindles and are flat, thus making possible studies of interactions between the achromatic and chromatic parts of the mitotic apparatus during the cell cycle. At prophase, asters and centrioles seem to exert pressure on the nuclear membrane leading to its rupture and penetrance of the centrioles. Apparently, the long axis of the spindle is shorter than the nuclear diameter. What appears as persistent, large portions of the nuclear membrane were observed in some metaphase and anaphase cells. Such a condition might also indicate an arrested mitosis. The midbody, which was often bipartite, was found to be of a ribonucleoprotein nature. — Three-group metaphases were of common occurrence and might represent early stages of chromosome orientation preceding the final alignment of the chromosomes on the equatorial plate. They could also be an expression of an anomalous condition as a result of mitotic arrest during prometaphase owing to spindle inactivation or breakage, errors in centromere-spindle attachments, interference with chromosome movement, or a duplicated centriolar constitution. Most of these aberrations could be attributed to the flatness of dividing cells, which might also bring about the failure of centriole separation and spindle organization in prometaphase stages, as well as multipolar mitosis.De novo organization of half spindles might take place in cells with ruptured spindles. Anaphase cells showing signs of a previous three-group orientation were rare. — Multipolar mitoses were prevalent mainly in cells with high chromosome numbers. They were often star-shaped with the chromosomes oriented between opposite and adjacent poles, and rarely as end-to-end associations of spindles. Apparently, one or more centrioles might share a common polar region. Multipolar configurations have either a mono- or multinuclear origin. Nuclei usually enter division synchronously in binucleate cells and the spindles become organized between centrioles associated with individual or different nuclei.     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.     

Close association of centrosomes to the distal ends of the microbody during its growth, division and partitioning in the green alga Klebsormidium flaccidum

Summary. Division and partitioning of microbodies (peroxisomes) of the green alga Klebsormidium flaccidum, whose cells contain a single microbody, were investigated by electron microscopy. In interphase, the rod-shaped microbody is present between the nucleus and the single chloroplast, oriented perpendicular to the pole-to-pole direction of the future spindle. A centriole pair associates with one distal end of the microbody. In prophase, the microbody changes not only in shape, from a rodlike to a branched form, but also in orientation, from perpendicular to parallel to the future pole-to-pole direction. Duplicated centriole pairs are localized in close proximity to both distal ends of the microbody. In metaphase, the elongated microbody flanks the open spindle, with both distal ends close to the centriole pair at either spindle pole. The microbody further elongates in telophase and divides after septum formation (cytokinesis) has started. The association between the centrioles and both distal ends of the microbody is maintained throughout mitosis, resulting in the distal ends of the elongated microbody being fixed at the cellular poles. This configuration of the microbody may be favorable for faithful transmission of the organelle during cell division. After cytokinesis is completed, the microbody reverts to the perpendicular orientation by changing its shape. Microtubules radiating from the centrosomes flank the side of the microbody throughout mitosis. The close association of centrosomes and microtubules with the microbody is discussed in respect to the partitioning of the microbody in this alga. Correspondence: H. Hashimoto, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan. Present address: M. Honda, Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

What is the function of centrioles?

The function of centrioles has been controversial and remains incompletely resolved. This is because centrioles, in and of themselves, do not directly perform any physiological activity. Instead, their role is only to act as a jig or breadboard onto which other functional structures can be built. Centrioles are primarily involved in forming two structures-centrosomes and cilia. Centrioles bias the position of spindle pole formation, but because spindle poles can self-organize, the function of the centriole in mitosis is not obligatory. Consequently, lack of centrioles does not generally prevent mitosis, although recent experiments suggest acentriolar spindles have reduced fidelity of chromosome segregation. In contrast, centrioles are absolutely required for the assembly of cilia, including primary cilia that act as cellular antennae. Consistent with this requirement, it is now becoming clear that many ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel Syndrome, and Oral-Facial-Digital syndrome, are caused by defects in centriole-associated proteins.     

OBSERVATIONS ON THE CENTRIOLES OF THE SEA URCHIN SPERMATOZOON

The spermatozoon of Lytechinus variegatus has two parallel centrioles. The basal body of the flagellum consists of the proximal centriole (a short cylinder of nine tubule-triplets) and its distal extension of nine tubule-doublets. The distal centriole lies near the distal end of the basal body, between the nucleus and the mitochondrion. The observations suggest that both the proximal and the distal centrioles are polarized structures, their tubule-triplets pitched in the same direction and their distal ends associated with the flagellar axoneme and with the mitochondrion, respectively. The distal centriole in different spermatozoa occupies different positions around the basal body-flagellum complex.     

Male gametogenesis without centrioles

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis.     

Cell center of macrophages, granulocytes and lymphocytes during in vitro cell spreading, polarization and movement

Different motile blood cells behave in a different way upon spreading on the glass surface. Macrophages pass through all the stages of spreading described for fibroblasts (Vasiliev, Gelfand, 1976); granulocytes are polarized after a short staying in badly spread conditions, lymphocytes are polarized immediately after setting of the glass surface. In relation to the leading edge and the cell nucleus, centrioles in the described cell types are located differently. In macrophages they are mainly in the front or on one side of the nucleus, in granulocytes they lie within the ring-like nucleus, in lymphocytes they are strictly located behind the nucleus in the uropode. In all the cases, however, centrioles are localized in the central region of the cytoplasm. Their location does not appear to be connected with the movement direction of blood cells. The distal ends of the active centrioles are faced to the upper cell surface in the examined cells. It is suggested that the centriole can distinguish the free cell surface and the surface associated with the substrate.     

Multipolar mitosis in procaine-treated polyspermic sea urchin eggs and in eggs fertilized with UV-irradiated spermatozoa with a computer model to simulate the positioning of centrosomes

Procaine-treated eggs can be penetrated by more than one spermatozoon. Supernumerary male pronuclei can fuse with the female one giving raise to multipolar spindles or remain isolated within the egg's cytoplasm forming their own spindle. In all types of multiple mitotic figures (asters and spindles) the distribution of asters is equidistant either uniplanar or at maximum distance like at the apices of a polyhedron. Astral rays are not different from spindle fibers: they can attach to and attract chromosomes of "foreign" mitotic figures. When several mitotic figures are present in one egg, the partner asters are always of the same size, and microtubules of one aster never interdigitate with those of others. The hypothesis that positioning of centrosomes is brought about by spreading of a centrosome organizer in the form of an expanding calotte on the surface of the nucleus (Mazia, D., Int. Rev. Cytol. 100, 49-92 (1987)) is supported by a computer model.     

Neck region of gerbil spermatozoa

In the course of the reorganization and degeneration of the proximal centriole in the mature acentriolate spermatozoon of the Mongolian gerbil, both the proximal and distal centrioles appear in the early cap phase of spermatid development. During the acrosome phase, both distal and proximal centrioles become highly active in the formation of a segmented column. The proximal centriole becomes actively involved in the formation of the capitulum, while the distal centriole forms the axonemal complex and dense fibers. During the maturation phase of spermatid development, the “pinwheel” arrangement of the proximal centriole becomes an “S”-shaped structure, turned 90° on its vertical axis. The few “doublet” microtubules that can be detected later in that stage completely disappear during spermiation. The distal centriolar area develops a single central pair of microtubules and membranous elements. Another prominent feature in the neck region of the gerbil spermatozoa is the presence of two dense rudimentary columns in association with the mitochondria. Although their density is similar to that of the other columns, these two columns have no connection with the dense fibers; in fact, they are closely associated with the mitochondria.     

Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies

It is believed that in most animals only the paternal centrosome provides the division poles for mitosis in zygotes. This paternal inheritance of the centrosomes depends on the selective loss of the maternal centrosome. In order to understand the mechanism of centrosome inheritance, the behavior of all maternal centrosomes/centrioles was investigated throughout the meiotic and mitotic cycles by using starfish eggs that had polar body (PB) formation suppressed. In starfish oocytes, the centrioles do not duplicate during meiosis II. Hence, each centrosome of the meiosis II spindle has only one centriole, whereas in meiosis I, each has a pair of centrioles. When two pairs of meiosis I centrioles were retained in the cytoplasm of oocytes by complete suppression of PB extrusion, they separated into four single centrioles in meiosis II. However, after completion of the meiotic process, only two of the four single centrioles were found in addition to the pronucleus. When the two single centrioles of a meiosis II spindle were retained in the oocyte cytoplasm by suppressing the extrusion of the second PB, only one centriole was found with the pronucleus after the completion of the meiotic process. When these PB-suppressed eggs were artificially activated to drive the mitotic cycles, all the surviving single centrioles duplicated repeatedly to form pairs of centrioles, which could organize mitotic spindles. These results indicate that the maternal centrioles are not equivalent in their intrinsic stability and reproductive capacity. The centrosomes with the reproductive centrioles are selectively cast off into the PBs, resulting in the mature egg inheriting a nonreproductive centriole, which would degrade shortly after the completion of meiosis.     

The centrosome and bipolar spindle assembly: Does one have anything to do with the other?

In vertebrate somatic cells, the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles, such as plant cells and many oocytes, bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization, both before and after nuclear envelope breakdown (NEB), is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.Key words: centrosome, centriole, mitosis, spindle, cell cycle, meiosis, plant cell, microsurgery