The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.     

Zygotic development without functional mitotic centrosomes

The centrosome is the dominant microtubule-organizing center in animal cells. At the onset of mitosis, each cell normally has two centrosomes that lie on opposite sides of the nucleus. Centrosomes nucleate the growth of microtubules and orchestrate the efficient assembly of the mitotic spindle. Recent studies in vivo and in vitro have shown that the spindle can form even in the absence of centrosomes and demonstrate that individual cells can divide without this organelle. However, since centrosomes are involved in multiple processes in vivo, including polarized cell divisions, which are an essential developmental mechanism for producing differentiated cell types, it remains to be shown whether or not a complete organism can develop without centrosomes. Here we show that in Drosophila a centrosomin (cnn) null mutant, which fails to assemble fully functional mitotic centrosomes and has few or no detectable astral microtubules, can develop into an adult fly. These results challenge long-held assumptions that the centrosome and the astral microtubules emanating from it are essential for development and are required specifically for spindle orientation during asymmetric cell divisions.     

Cell contacts orient some cell division axes in the Caenorhabditis elegans embryo

Cells of the early Caenorhabditis elegans embryo divide in an invariant pattern. Here I show that the division axes of some early cells (EMS and E) are controlled by specific cell-cell contacts (EMS-P2 or E-P3 contact). Altering the orientation of contact between these cells alters the axis along which the mitotic spindle is established, and hence the orientation of cell division. Contact-dependent mitotic spindle orientation appears to work by establishing a site of the type described by Hyman and White (1987. J. Cell Biol. 105:2123-2135) in the cortex of the responding cell: one centrosome moves toward the site of cell-cell contact during centrosome rotation in both intact embryos and reoriented cell pairs. The effect is especially apparent when two donor cells are placed on one side of the responding cell: both centrosomes are "captured," pulling the nucleus to one side of the cell. No centrosome rotation occurs in the absence of cell-cell contact, nor in nocodazole-treated cell pairs. The results suggest that some of the cortical sites described by Hyman and White are established cell autonomously (in P1, P2, and P3), and some are established by cell-cell contact (in EMS and E). Additional evidence presented here suggests that in the EMS cell, contact-dependent spindle orientation ensures a cleavage plane that will partition developmental information, received by induction, to one of EMS's daughter cells.     

Widespread RNA segregation in a spiralian embryo

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.     

The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions.     

Spindle Assembly and Mitosis without Centrosomes in Parthenogenetic Sciara Embryos

In Sciara, unfertilized embryos initiate parthenogenetic development without centrosomes. By comparing these embryos with normal fertilized embryos, spindle assembly and other microtubule-based events can be examined in the presence and absence of centrosomes. In both cases, functional mitotic spindles are formed that successfully proceed through anaphase and telophase, forming two daughter nuclei separated by a midbody. The spindles assembled without centrosomes are anastral, and it is likely that their microtubules are nucleated at or near the chromosomes. These spindles undergo anaphase B and successfully segregate sister chromosomes. However, without centrosomes the distance between the daughter nuclei in the next interphase is greatly reduced. This suggests that centrosomes are required to maintain nuclear spacing during the telophase to interphase transition. As in Drosophila, the initial embryonic divisions of Sciara are synchronous and syncytial. The nuclei in fertilized centrosome-bearing embryos maintain an even distribution as they divide and migrate to the cortex. In contrast, as division proceeds in embryos lacking centrosomes, nuclei collide and form large irregularly shaped nuclear clusters. These nuclei are not evenly distributed and never successfully migrate to the cortex. This phenotype is probably a direct result of a failure to form astral microtubules in parthenogenetic embryos lacking centrosomes. These results indicate that the primary function of centrosomes is to provide astral microtubules for proper nuclear spacing and migration during the syncytial divisions. Fertilized Sciara embryos produce a large population of centrosomes not associated with nuclei. These free centrosomes do not form spindles or migrate to the cortex and replicate at a significantly reduced rate. This suggests that the centrosome must maintain a proper association with the nucleus for migration and normal replication to occur.     

Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.     

Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.     

From stem cell to embryo without centrioles

Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.     

MSA-35: a protein identified by human autoantibodies that colocalizes with microtubules.

We have identified a putative 35-kilodalton protein that colocalizes with microtubules and displays a unique spatial and temporal distribution during the cell cycle of HeLa cells. This protein has been given the designation MSA-35. MSA-35 first appears in association with microtubules and centrosomes of interphase cells exhibiting centrosome separation as a prelude to cell division. This protein is found in conjunction with kinetochore microtubules throughout their appearance. MSA-35 transiently associates with interpolar microtubules following anaphase and the pattern of MSA-35 reactivity in telophase cells suggests that there are at least seven domains within the intercellular bridge. The distribution of MSA-35 during and following recovery from mitotic arrest with nocodazole suggest that it is also present at low levels in interphase cells, can associate with interphase centrosomes, and colocalizes with nascent microtubules. The complex spatial and temporal distribution of MSA-35 indicates that it may be necessary for a series of events in the mitotic process such as the bundling of microtubules.     

Centrosome movement in the early divisions of Caenorhabditis elegans: a cortical site determining centrosome position

In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus (Hyman, A. A., and J. G. White. 1987. J. Cell Biol. 105:2123-2135). A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.     

Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules

Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species.     

Molecular and cellular dynamics of early embryonic cell divisions in Volvox carteri

Cell division is fundamental to all organisms and the green alga used here exhibits both key animal and plant functions. Specifically, we analyzed the molecular and cellular dynamics of early embryonic divisions of the multicellular green alga Volvox carteri (Chlamydomonadales). Relevant proteins related to mitosis and cytokinesis were identified in silico, the corresponding genes were cloned, fused to yfp, and stably expressed in Volvox, and the tagged proteins were studied by live-cell imaging. We reveal rearrangements of the microtubule cytoskeleton during centrosome separation, spindle formation, establishment of the phycoplast, and generation of previously unknown structures. The centrosomes participate in initiation of spindle formation and determination of spindle orientation. Although the nuclear envelope does not break down during early mitosis, intermixing of cytoplasm and nucleoplasm results in loss of nuclear identity. Finally, we present a model for mitosis in Volvox. Our study reveals enormous dynamics, clarifies spatio-temporal relationships of subcellular structures, and provides insight into the evolution of cell division.

Analysis of cell divisions of the microalga Volvox reveals enormous dynamics of cytoskeletal and membranous structures with coordination of intranuclear spindle formation by cytosolic centrosomes.

IN A NUTSHELLBackground: Mitosis, a type of cell division, is fundamental to all eukaryotic life and must be carried out very accurately. Even though the process of mitosis itself is highly conserved among eukaryotes, there are significant differences between animals, fungi, plants, and algae. From an evolutionary point of view, the green alga Volvox carteri used here possesses both key animal and plant functions and it exhibits important features of the last common eukaryotic ancestor that have been lost in other lineages. Prior to our work, a comprehensive in vivo analysis of the entire process of cell division in green algae was lacking.Question: How exactly does cell division work in green algae? How do the cytosolic centrosomes deal with the persistent nuclear envelope in this process? What is the relationship between different microtubular structures?Findings: Our study reveals enormous dynamics during mitosis, clarifies spatio-temporal relationships of subcellular structures, and provides insights into evolution of cell division. Although the nuclear envelope does not break down during early mitosis of Volvox, it becomes permeable and the nucleus temporarily loses its identity. Two microtubule-organizing centers, the centrosomes, located immediately outside the nuclear envelope participate in initiation of the mitotic spindle formation inside the nuclear envelope. This process also defines the orientation of the mitotic spindle. In cytokinesis, an algae-specific microtubule structure, the phycoplast, replaces the spindle. The microtubules of the phycoplast may play a direct role in promoting the cell membrane invagination of the cleavage furrow.Next steps: How are the massive rearrangements of subcellular structures regulated? What happens at the nuclear pores when the nuclear envelope becomes permeable at the onset of mitosis? What determines in later embryogenesis which cells then divide asymmetrically rather than symmetrically?     

QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.     

Spatial patterns of cells in dividing epithelia.

The spatial patterns of cell boundaries in a view of the apical surface of a dividing epithelium are explored by constructing a hypothetical cell pattern of an epithelium of dividing cells. The two elements specified in the hypothetical pattern are the orientation of division planes and the separation between the division planes in neighbouring cells. The orientations of division planes in one generation are all the same but are orthogonal to those in the preceding generation. The division-plane orientations follow in an orthogonal succession, as happens in early embryos. The division planes in neighbouring cells are offset. The contractions of division planes that would occur during cytokinesis distort existing boundaries creating various types of cell shapes. The patterns generated resemble cell patterns found in life. The hypothetical pattern is regenerative and shows how epithelial cell patterns where cells divide might arise. It has enabled the putative identification of sister cells and first cousins in the embryonic chick chorion.     

Asymmetric cell division: recent developments and their implications for tumour biology

The ability of cells to divide asymmetrically is essential for generating diverse cell types during development. The past 10 years have seen tremendous progress in our understanding of this important biological process. We have learned that localized phosphorylation events are responsible for the asymmetric segregation of cell fate determinants in mitosis and that centrosomes and microtubules play important parts in this process. The relevance of asymmetric cell division for stem cell biology has added a new dimension to the field, and exciting connections between asymmetric cell division and tumorigenesis have begun to emerge.     

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.