Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the?daughter centriole, rather than an "immortal" mother centriole, is ultimately retained in these stem cells.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Outer Dense Fiber 2 Is a Widespread Centrosome Scaffold Component Preferentially Associated with Mother Centrioles: Its Identification from Isolated Centrosomes

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.     

Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase

The Cyclin-Dependent Kinase (CDK)-activating phosphatase CDC25B, localises to the centrosomes where its activity is both positively and negatively regulated by several kinases including Aurora A and CHK1. Our recent data also demonstrate a role for CDC25B in the centrosome duplication cycle and microtubule nucleation in interphase that appears to involve the recruitment of γ-tubulin to the centrosomes. In the present study, we report that CDC25B, along with CHK1, CDK1 and WEE1, localise asymmetrically around the mother centrosome from S to G2-phases, and gradually become evenly distributed to the two centrosomes by late G2 phase, concomitant with centrosome maturation. We further demonstrate that siRNA inhibition of CDC25B results in an accumulation of cells in G2 phase with two separated centrosomes, each containing only a single centriole, suggesting a requirement for CDC25B in centriole duplication. We propose that the localisation of key cell cycle regulators to the mother centrosome ensures synchrony between the centrosome duplication and cell division cycles.     

The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm

Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle.     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.     

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.     

Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction

During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1-Mst2-Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the kinesin-5 Eg5. We propose that hSav1-Mst2-Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction.     

The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.     

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility.     

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.     

Loading and Unloading: Orchestrating Centrosome Duplication and Spindle Assembly by Ran/Crm1

The cell cycle is an intricate process of DNA replication and cell division thatconcludes with the formation of two genetically equivalent daughter cells. In thisprogression, the centrosome is duplicated once and only once during the G1/S transitionto produce the bipolar spindle and ensure proper chromosome segregation. The presenceof multiple centrosomes in cancer cells suggests that this process is mis-regulated duringcarcinogenesis. This suggests that certain factors exist that license the progression ofcentrosome duplication and serve to inhibit further duplications during a single cell cycle.Recent studies suggest that the Ran/Crm1 complex not only regulates nucleocytoplasmictransport but is also independently involved in mitotic spindle assembly. Factors that arecapable of interacting with Ran/Crm1 through their nuclear export sequences, such ascyclins/cdks, p53 and Brca1/2, may potentially function as centrosome checkpoints akinto the G1/S and G2/M checkpoints of the cell cycle. Our recent findings indicate thatnucleophosmin, a protein whose trafficking is mediated by the Ran/Crm1 network, is oneof such checkpoint factors for maintaining proper centrosome duplication. We proposethat Ran/Crm1 may act as a ‘loading dock’ to coordinate various checkpoint factors inregulating the fidelity of centrosome duplication during cell cycle progression, and thedisruption of these processes may lead to genomic instability and an acceleration ofoncogenesis.     

Plk2 regulated centriole duplication is dependent on its localization to the centrosome and a functional polo-box domain

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell-cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G1/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G1 phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated down-regulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.     

HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.     

Centriole duplication: analogue control in a digital age

In preparation for mitosis, the centrosome doubles once and only once to provide the two poles of the mitotic spindle. The presence of more than two centrosomes increases the chances that mitosis will be multipolar, and chromosomes will be distributed unequally. Since the number of mother-daughter centriole pairs determines the number of centrosomes, it is important that only one daughter centriole is assembled at, but slightly separated from, the proximal end of each mother centriole. This numerical and spatial specificity has led to the belief that a 'template' on the mother centriole provides a unique site for procentriole assembly. We review observations that are leading to the demise of this intuitively attractive idea. In its place, we are left with the notion that pericentriolar material at the wall of the mother centriole provides a local environment that promotes the assembly of a macromolecular complex that seeds the daughter centriole. Even though the system normally behaves in a digital fashion to go from zero to just one daughter centriole per mother, this behaviour appears to be based in the precise analogue control of multiple proteins, their activities, and the structure provided by the mother centriole.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells

Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function.     

Centrobin/Nip2 expression in vivo suggests its involvement in cell proliferation

Centrobin/Nip2 was initially identified as a centrosome protein that is critical for centrosome duplication and spindle assembly. In the present study, we determined the expression and subcellular localization of centrobin in selected mouse tissues. Immunoblot analysis revealed that the centrobin-specific band of 100 kDa was detected in all tissues tested but most abundantly in the thymus, spleen and testis. In the testis, centrobin was localized at the centrosomes of spermatocytes and early round spermatids, but no specific signal was detected in late round spermatids and elongated spermatids. Our results also revealed that the centrosome duplication occurs at interphase of the second meiotic division of the mouse male germ cells. The centrobin protein was more abundant in the mitotically active ovarian follicular cells and thymic cortex cells than in non-proliferating corpus luteal cells and thymic medullary cells. The expression pattern of centrobin suggests that the biological functions of centrobin are related to cell proliferation. Consistent with the proposal, we observed reduction of the centrobin levels when NIH3T3 became quiescent in the serum-starved culture conditions. However, a residual amount of centrobin was also detected at the centrosomes of the resting cells, suggesting its role for maintaining integrity of the centrosome, especially of the daughter centriole in the cells.