Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes.

The ultrastructure of the Dictyostelium centrosome is markedly different from that of the well known yeast spindle pole body and vertebrate centriole-containing centrosome. It consists of a box-shaped, layered core structure surrounded by a corona with dense nodules embedded in an amorphous matrix. For further structural and biochemical analyses of this type of centrosome we used highly enriched isolated Dictyostelium centrosomes as an antigen to raise 14 new centrosomal monoclonal antibodies. Immunofluorescence microscopy and Western blot analysis revealed that at least 10 of them were directed against different antigens. Immunofluorescence microscopy also showed that the monoclonal antibodies fell into three different groups: A) antibodies localizing to the centrosome during the entire cell cycle; B) antibodies staining the centrosome mainly during mitosis; and C) antibodies labeling centrosome associated structures. All antibodies, except one, exhibited a cell cycle-dependent staining pattern underscoring the highly dynamic properties of the Dictyostelium centrosome.     

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis.     

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.     

TORC1-mediated protein synthesis regulates cilia size and function

The centrosome is the microtubule organizing center important for the establishment of the mitotic spindle in animal cells. In mitosis, cells normally contain two centrosomes, one for each pole of the bipolar spindle. If a cell acquires additional centrosomes, it has the potential to build a multi-polar spindle which could lead to catastrophic errors in chromosome segregation. Although such an event is unlikely to produce viable daughter cells, an increase in centrosome number has been shown to cause chromosome instability and produce anneuploid daughter cells ^1-3. Accordingly, supernumerary centrosomes have been found in a variety of human cancers and accumulation of additional centrosomes has been associated with the process of tumorigenesis ^{1, 4-9}. Despite the obvious importance of regulating centrosome number, relatively little is known about how centrosome duplication is regulated. Perhaps surprisingly, several recent studies, including three articles in this issue of Cell Cycle, implicate proteins involved in the regulation of chromosome cohesion in the maintenance of centrosome number during mitosis ^10-14. Here we will discuss these findings and what they may tell us about the regulation of centrosome number.     

Regulation of the paternal inheritance of centrosomes in starfish zygotes

In most animals, fertilized eggs inherit one centrosome from a meiosis-II spindle of oocytes and another centrosome from the sperm. However, since first proposed by Boveri [Sitzungsber. Ges. Morph. Phys. Münch. 3 (1887) 151-164] at the turn of the last century, it has been believed that only the paternal (sperm) centrosome provides the division poles for mitosis in animal zygotes. This uniparental (paternal) inheritance of centrosomes is logically based on the premise that the maternal (egg) centrosome is lost before the onset of the first mitosis. For the processes of the selective loss of the maternal centrosome, three models have been proposed: One stresses the intrinsic factors within the centrosome itself; the other two emphasize external factors such as cytoplasmic conditions or the sperm centrosome. In the present study, we have examined the validity of one of the models in which the sperm centrosome overwhelms the maternal centrosomes. Because centrosomes cast off into both the first and the second polar bodies (PB) are known to retain the capacity for reproduction and cell-division pole formation, we observed the behavior of those PB centrosomes with reproductive capacity and the sperm centrosome in the same zygotic cytoplasm. We prepared two kinds of fertilized eggs that contain reproductive maternal centrosomes, (1) by micromanipulative transplantation of the PB centrosomes into fertilized eggs, and (2) by suppression of the PB extrusions of fertilized eggs with cytochalasin B. In both types of eggs, the PB centrosomes could double and form cell-division poles, indicating that they are not suppressed by the sperm centrosome, which in turn indicates that selective loss of the maternal centrosome is due to intrinsic factors within the centrosomes themselves.     

Centrosome number is controlled by a centrosome-intrinsic block to reduplication

The centrosome duplicates once in S phase. To determine whether there is a block in centrosome reduplication, we used a cell fusion assay to compare the duplication potential of unduplicated G1 centrosomes and recently duplicated G2 centrosomes. By fusing cells in different cell cycle stages, we found that G2 centrosomes were unable to reduplicate in a cellular environment that supports centrosome duplication. Furthermore, G2 cytoplasm did not inhibit centrosome duplication in fused cells, indicating that the block to reduplication is intrinsic to the centrosomes rather than the cytoplasm. To test the underlying mechanism, we created mononucleate G1 cells with two centrosomes by fusing cells with enucleated cytoplasts. Both centrosomes duplicated, indicating that the block is not controlled by centrosome:nucleus ratio. We also found that human primary cells have tight control over centrosome number during prolonged S-phase arrest and that this control is partially abrogated in transformed cells. This suggests a link between the control of centrosome duplication and maintenance of genomic stability.     

Amorphous no longer: the centrosome comes into focus

Recent genetic and biochemical studies have provided new insights into the molecular basis of centrosome-mediated microtubule nucleation. In addition, molecules and mechanisms involved in microtubule severing and stabilization at the centrosome, assembly of proteins onto centrosomes and regulation of centrosome duplication and separation are being defined. Characterization of centrosome function, together with studies implicating centrosomes in tumorigenesis and demonstrating that centrosomes are highly organized, are beginning to bring into focus an organelle once viewed as an 'amorphous cloud'.     

Antizyme Restrains Centrosome Amplification by Regulating the Accumulation of Mps1 at Centrosomes

Extra centrosomes are found in many tumors, and their appearance is an early event that can generate aberrant mitotic spindles and aneuploidy. Because the failure to appropriately degrade the Mps1 protein kinase correlates with centrosome overproduction in tumor-derived cells, defects in the factors that promote Mps1 degradation may contribute to extra centrosomes in tumors. However, while we have recently characterized an Mps1 degradation signal, the factors that regulate Mps1 centrosomal Mps1 are unknown. Antizyme (OAZ), a mediator of ubiquitin-independent degradation and a suspected tumor suppressor, was recently shown to localize to centrosomes and modulate centrosome overproduction, but the known OAZ substrates were not responsible for its effect on centrosomes. We have found that OAZ exerts its effect on centrosomes via Mps1. OAZ promotes the removal of Mps1 from centrosomes, and centrosome overproduction caused by reducing OAZ activity requires Mps1. OAZ binds to Mps1 via the Mps1 degradation signal and modulates the function of Mps1 in centrosome overproduction. Moreover, OAZ regulates the canonical centrosome duplication cycle, and reveals a function for Mps1 in procentriole assembly. Together, our data suggest that OAZ restrains the assembly of centrioles by controlling the levels of centrosomal Mps1 through the Cdk2-regulated Mps1 degradation signal.     

Centrosomes and tumour suppressors

Centrosomes are microtubule organising centres that act as spindle poles during mitosis. Recent work implicates centrosomes in many other processes, and shows that centrosome defects can cause genetic instability. Many regulators of mammalian centrosome function were predicted from studies of model systems. Surprisingly, some well-known tumour suppressors have recently been found at centrosomes, where they influence centrosome duplication and function, suggesting that control of centrosome function is central to genetic stability.     

Centrosome-independent mitotic spindle formation in vertebrates

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.     

The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis

Centrosomes are the major microtubule-organizing centers of mammalian cells. They are composed of a centriole pair and surrounding microtubule-nucleating material termed pericentriolar material (PCM). Bipolar mitotic spindle assembly relies on two intertwined processes: centriole duplication and centrosome maturation. In the first process, the single interphase centrosome duplicates in a tightly regulated manner so that two centrosomes are present in mitosis. In the second process, the two centrosomes increase in size and microtubule nucleation capacity through PCM recruitment, a process referred to as centrosome maturation. Failure to properly orchestrate centrosome duplication and maturation is inevitably linked to spindle defects, which can result in aneuploidy and promote cancer progression. It has been proposed that centriole assembly during duplication relies on both PCM and centriole proteins, raising the possibility that centriole duplication depends on PCM recruitment. In support of this model, C. elegans SPD-2 and mammalian NEDD-1 (GCP-WD) are key regulators of both these processes. SPD-2 protein sequence homologs have been identified in flies, mice, and humans, but their roles in centrosome biogenesis until now have remained unclear. Here, we show that Cep192, the human homolog of C. elegans and D. melanogaster SPD-2, is a major regulator of PCM recruitment, centrosome maturation, and centriole duplication in mammalian cells. We propose a model in which Cep192 and Pericentrin are mutually dependent for their localization to mitotic centrosomes during centrosome maturation. Both proteins are then required for NEDD-1 recruitment and the subsequent assembly of gamma-TuRCs and other factors into fully functional centrosomes.     

Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos

In a number of embryonic systems, centrosomes that have lost their association with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules. To identify additional activities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently labeled tubulin. Our recordings follow multiple rounds of free centrosome duplication and separation during the cortical division. The rate and distance of free sister centrosome separation corresponds well with the initial phase of associated centrosome separation. However, the later phase of separation observed for centrosomes associated with a spindle (anaphase B) does not occur. Free centrosome separation regularly occurs on a plane parallel to the plasma membrane. While previous work demonstrated that centrosomes influence cytoskeletal dynamics, this observation suggests that the cortical cytoskeleton regulates the orientation of centrosome separation. Although free centrosomes do not form spindles, they display relatively normal cell cycle-dependent modulations of their astral microtubules. In addition, free centrosome duplication, separation, and modulation of microtubule dynamics often occur in synchrony with neighboring associated centrosomes. These observations suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with aphidicolin supports this conclusion; large numbers of abnormal nuclei recede into the interior while their centrosomes remain on the cortex. Following individual free centrosomes through multiple focal planes for 45 min after the injection of aphidicolin reveals that they do not undergo normal modulation of their astral dynamics nor do they undergo multiple rounds of duplication and separation. We conclude that in the absence of normally dividing cortical nuclei many centrosome activities are disrupted and centrosome duplication is extensively delayed. This indicates the presence of a feedback mechanism that creates a dependency relationship between the cortical nuclear cycles and the centrosome cycles.     

Spermatozoa of the loach Misgurnus fossilis as a test system for identification of new centromere proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. for identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein gamma-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes has been obtained from the loach spermatozoa. These centrosomes contained gamma-tubulin although they lost their ability to induce polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and on then microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising object for identification of new proteins of the mammalian centrosome.     

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.     

Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination

In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.     

The mammalian centrosome and its functional significance

Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome's functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome's role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome's significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease.     

The centrosome in normal and transformed cells

The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.     

The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.     

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     

Centrosome separation: respective role of microtubules and actin filaments

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 microm), and the elongation stage (from 1 to 7 microm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.