Unusual amoebal strains of the MyxomycetePhysarum polycephalum possessing two pro-flagellar apparatuses

Summary The microtubules structure of two stable diploid amoebal strains, each resulting from the fusion of two haploid amoebae has been studied by electron microscopy. Tridimensional reconstructions showed that these diploid amoebae-typically possessed two proflagellar apparatuses,i.e., two microtubule organizing centers 1 (mtoc 1) and two pairs of centrioles with their associated microtubular arrays. These observations account for the high frequency of biflagellated amoebae in these two strains. The presence of two mtoc 1 may account for the high percentage of mitotic abnormalities which was observed under phase contrast microscopy and electron microscopy and is in agreement with a role of the mtoc 1 as a mitotic center during mitosis. However, the presence of numerous normal mitotic apparatuses raises the question of the regulations which play a role in the mitotic process. The unusual distribution of centrioles and the unusual pro-flagellar apparatuses which were produced suggest that in interphase the anterior centriole is a necessary structure for the morphogenesis of the microtubular arrays 2 and 3 and that the posterior centriole is a necessary structure for the morphogenesis of the microtubular arrays 4 and 5.     

Variations in the number of centrioles,the number of microtubule organizing centers 1 and the percentage of mitotic abnormalities inPhysarum polycephalum amoebae

Summary Several, stable amoebal strains which differ phenotypically from the diploid parental amoebal strain have been obtained in the MyxomycetePhysarum polycephalum. They were detected using their flagellation pattern as a discriminating parameter. This approach is valid since the number of flagella by phase contrast microscopy correlates with the number of anterior centrioles obtained using three-dimensional reconstructions of the nucleo-flagellar complexes from serial thin sections. The complexity of the structures of the various nucleo-flagellar complexes suggests that in these strains the duplication time of centrioles is not strictly regulated as it is in haploid amoebae. In agreement with this hypothesis, several pro-centrioles were observed in interphase amoebae. Although the anterior centrioles are linked to the mtoc 1 during interphase, the number of mtoc 1 cannot regulate the number of centrioles since some strains possess two mtoc 1 but only one pair of centrioles. Neither the number of centrioles nor the number of mtoc 1 are related to ploidy. Stable strains with one (all haploid strains), two (some diploid strains) and three (some diploid strains) mtoc 1 have been observed. Thus each mtoc 1 is duplicated once per cell cycle implying that it must possess some information which plays a role in the morphogenesis of the new mtoc 1. Except in one case, the number of mitotic abnormalities increases exponentially with the number of mtoc 1. This observation suggests that the mtoc 1 could correspond to the interphase state of the mitotic center.     

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

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.     

Control of daughter centriole formation by the pericentriolar material

Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.     

Centriole size modifications during the cell cycle of the amoebae of the mxyomycete Physarum polycephalum

Anterior and posterior centrioles of Physarum amoebae are indistinguishable by their size during interphase but there is a correlation between the size of the two centrioles in the same amoeba. The interphase length of centrioles in diploid amoebae possessing only one pair of centrioles was 11% longer than in the case of the haploid strain. Treatment with taxol led to a 23 and 32% increase of the mean length in interphase and blocked mitosis, respectively. Conversely, during control mitosis the parental centrioles showed a 12% decrease of their mean length while the size of the daughter centrioles increased progressively. Neither nocodazole nor cold treatment induce a decrease of centriole length. The mean length of the cartwheel structure (internal proximal part) although constant during mitosis could be increased 24% in the presence of taxol. Similarly there was a correlation between the number of anterior satellites and the centriole length.     

Molecular phylogeny of Cercomonadidae and kinetid patterns of Cercomonas and Eocercomonas gen. nov. (Cercomonadida, Cercozoa)

Cercomonads are among the most abundant and widespread zooflagellates in soil and freshwater. We cultured 22 strains and report their complete 18S rRNA sequences and light microscopic morphology. Phylogenetic analysis of 51 Cercomonas rRNA genes shows in each previously identified major clade (A, B) two very robust, highly divergent, multi-species subclades (A1, A2; B1, B2). We studied kinetid ultrastructure of five clade A representatives by serial sections. All have two closely associated left ventral posterior microtubular roots, an anterior dorsal root, a microtubule-nucleating left anterior root, and a cone of microtubules passing to the nucleus. Anterior centrioles (=basal bodies, kinetosomes) of A1 have cartwheels; the posterior centriole does not, suggesting it is older, and implying flagellar transformation similar to other bikonts. Strain C-80 (subclade A2) differs greatly, having a dorsal posterior microtubule band, but lacking the A1-specific fibrillar striated root, nuclear extension to the centrioles, centriolar diaphragm, extrusomes; both mature centrioles lack cartwheels. For clade A2 we establish Eocercomonas gen. n., with type Eocercomonas ramosa sp. n., and for clade B1 Paracercomonas gen. n. (type Paracercomonas marina sp. n.). We establish Paracercomonas ekelundi sp. n. for culture SCCAP C1 and propose a Cercomonas longicauda neotype and Cercomonas (=Neocercomonas) jutlandica comb. n. and Paracercomonas (=Cercomonas) metabolica comb. n.     

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE V. BLEPHAROPLAST DEVELOPMENT IN MARCHANTIA POLYMORPHA

Coaxial centrioles and a microtubule organizing center (MTOC) constitute each centrosome in spermatid mother cells of Marchantia polymorpha. During cell division the centrosome separates at its midregion and the two centrioles undergo a planar rotation that brings them to lie somewhat staggered and nearly parallel with their proximal ends embedded in osmiophilic granular material similar in appearance to that of the MTOC. Microtubules of the multilayered structure (MLS) arise in this material below the posterior centriole and parallel to its long axis. The rotation of centrioles and the initiation of S₁ tubules below the posterior centriole determine polarity of the incipient blepharoplast. Lower MLS strata are formed under the anterior centriole by the compaction of granular, osmiophilic matrix. Formation and growth of S₂ vertical lamellae occur at the left front edge of the MLS in association with MTOC-like matrix localized near the cell membrane. The MLS enlarges to about 0.4 μm wide by 0.6 μm long and is ovoid in outline except for a short distal projection underlying the posterior centriole. Subsequently the lamellae are transformed into homogenous, osmiophilic matrix that contributes directly to the expansion of all MLS strata including microtubules. The stratum of lamellae is interpreted as a planar MTOC subject to morphogenetic control. Each of the four strata grows proximally while the tapering distal projection lengthens beneath the posterior basal body. Dense matrix above the MLS, apparently elaborated by the S₂ layer, is organized into cartwheel and triplet components of the basal bodies’ proximal extensions. Organization of triplet tubules proceeds from proximal to distal toward preexisting triplets. Osmiophilic matrix contributes to the formation of microtubule keels and osmiophilic crests and may serve as a cementing material that stabilizes the spatial relationships of blepharoplast components. After full expansion of the MLS’ lower strata, the S₂ layer is reorganized into lamellae. Flagellar growth in Marchantia is postulated to involve a process whereby subunits or their precursors are elaborated by the MLS, translocated to the distal end of the flagellum and incorporated into the axonemal tubules. When MLS microtubules elongate to form a long, narrow band, the distal half of the S₂ layer is again in the osmiophilic matrix state.     

Spatial relationships between centrioles and the centrosphere in monoasters induced by taxol inPhysarum polycephalum amoebae

Summary Monoasters induced by taxol in the amoebae of the MyxomycetePhysarum polycephalum show an unusual tridimensional location of the centrioles. Tridimensional reconstructions of individual monoasters with either two or four centrioles show that the position of centrioles is not random. The characteristics of these monoasters suggest that the centrioles are not linked to the mitotic center in the monoaster since mitotic centers completely devoid of centrioles in adjacent or central location are observed. However, the preferential centrifugal orientation of the centrioles in the centrosphere induced by taxol suggests that centrioles are initially located in the mitotic center in agreement with the attachment of the centrioles to the mitotic center during interphase.     

Morphology and phylogeny of Sainouron acronematica sp. n. and the ultrastructural unity of Cercozoa

Sainouron are soil zooflagellates of obscure taxonomy. We studied the ultrastructure of S. acronematica sp. n. and sequenced its extremely divergent 18S rDNA and that of Cholamonas cyrtodiopsidis (here grouped as new family Sainouridae) to clarify their phylogeny. Ultrastructurally similar, they weakly group together, deeply within Monadofilosa. Sainouron has three cytoplasmic microtubules; all organelles specifically link to them or the nucleus. Mature centrioles have fibrous rhizoplasts. The posterior centriole bearing the motile cilium (with cortical filaments) has a transitional hub-lattice; a dense spiral fibre links its thicker rhizoplast and triplets; its ciliary root has two microtubules: mt1, underlying the plasma membrane, initiates at the spiral fibre; mt2, laterally attached to mt1 and nucleus, initiates in the amorphous centrosomal region. The anterior younger cilium, an immotile stub with submembrane skeleton as in Cholamonas, lacks axoneme, microtubular root, rhizoplasts and spiral fibre, but becomes the posterior one every cell cycle. The nuclear envelope donates coated vesicles directly to the Golgi, which makes kinetocyst-type extrusomes, concentrated at the cell anterior for extrusion into phagosomes. Ciliary transition region proximal hub-lattices (postulated to contain centrin) and distal nonagonal fibres are cercozoan synapomorphies, found with slight structural variation in all flagellate Cercozoa, but not in outgroups.     

A molecular marker for centriole maturation in the mammalian cell cycle

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.     

Centrioles: some self-assembly required

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.     

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.     

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.     

Reproductive maternal centrosomes are cast off into polar bodies during maturation division in starfish oocytes

Animal egg inherits a maternal centrosome from the meiosis-II spindle and sperm can introduce another centrosome at fertilization. It has been believed that in most animals only the sperm centrosome provides the division poles for mitosis in zygotes. This uniparental (paternal) inheritance of the centrosome must depend on the loss of the maternal centrosome. In starfish, suppression of polar body (PB) extrusion is a prerequisite for induction of parthenogenesis (Washitani-Nemoto et al. (1994) Dev. Biol. 163, 293-301), suggesting that the centrosomes cast off into PBs have reproducing capacity. Due to the absence of centriole duplication in meiosis II of starfish oocytes, each centrosome of a meiosis-II spindle has only one single centriole, whereas in meiosis I each has a pair of centrioles (Sluder et al. (1989) Dev. Biol. 131, 567-579; Kato et al. (1990) Dev. Growth Differ. 32, 41-49). Hence, the first PB (PB1) has two centrioles, whereas the second PB (PB2) and the mature egg have only one centriole, respectively. The present study examined the reproductive capacity of PB centrosomes by transplanting them into artificially activated eggs, and then the recipient egg nucleus with the surrounding cytoplasm was removed. A transplanted PB2 centrosome with a single centriole formed a monopolar spindle at the first mitosis, followed by formation of a bipolar spindle in the next mitosis, leading to actual cleavage and subsequent development. This proves the reproducing capacity of the single centriole in the PB2 centrosome. The behavior of the transplanted PB1 centrosome was exactly the same as in the PB2 centrosome, in spite of the difference in the number of centrioles. These results clearly show that four maternal centrioles are heterogeneous in duplicating capacity, during meiosis only one centriole in each of the centrosomes of a meiosis-I spindle pole retains duplicating capacity, the reproductive centrioles are successively cast off into PBs, and finally a mature egg inheriting a nonreproductive centriole alone is formed, and the presence of a single reproductive centriole is sufficient condition for embryonic development in starfish.     

Ultrastructural Studies on the Behavior of Centrioles during Meiosis of Starfish Oocytes

The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis . Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of Pisaster ocraceus and Asterias forbesi . The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte.     

Mode of centriole duplication and distribution

Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling of biotinylated and total tubulin permitted us to distinguish newly formed from preexisting centrioles. Daughter centrioles incorporated biotinylated tubulin, and at mitosis each cell received a centrosome containing one new and one old centriole. We conclude that in each cell cycle tubulin incorporation into centrioles is conservative, and centriole distribution is semiconservative.     

Daughter Centriole Elongation Is Controlled by Proteolysis

The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L₃VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L₃VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L₃VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.