The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.     

Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation

Oocyte maturation is an important process required to achieve optimal oocyte quality, and later affects fertilization potential and subsequent embryo development. The maturation process includes synchronized nuclear and cytoplasmic remodeling, in which cytoskeletal and centrosome dynamics play an important role and significantly participate in cellular signaling. Centrosome remodeling within the maturing oocyte is essential for accurate meioisis I and II spindle formation, specifically to separate chromosomes accurately during the two successive, highly asymmetric meiotic cell divisions. Centrosomal abnormalities result in inaccurate microtubule organization and inaccurate chromosome alignment, with failures in chromosome segregation leading to aneuploidy and chromosomal abnormalities. The present review is focused on cytoskeletal and centrosome remodeling during oocyte maturation, with specific attention to γ-tubulin, pericentrin, the Nuclear Mitotic Apparatus (NuMA) protein, and microtubule organization. Species-specific differences will be discussed for rodent (mouse) and non-rodent (bovine, porcine) species, and for human oocytes.     

Orbit/CLASP Is Required for Germline Cyst Formation through Its Developmental Control of Fusomes and Ring Canals in Drosophila Males

Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.     

XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes

During the maturation of Xenopus oocytes, a transient microtubule array (TMA) is nucleated from a novel MTOC near the base of the germinal vesicle. The MTOC-TMA transports the meiotic chromosomes to the animal cortex, where it serves as the precursor to the first meiotic spindle. To understand more fully the assembly of the MTOC-TMA, we used confocal immunofluorescence microscopy to examine the localization and function of XMAP215, XKCM1, NuMA, and cytoplasmic dynein during oocyte maturation. XMAP215, XKCM1, and NuMA were all localized to the base of the MTOC-TMA and the meiotic spindle. Microinjection of anti-XMAP215 inhibited microtubule (MT) assembly during oocyte maturation, disrupting assembly of the MTOC-TMA and subsequent assembly of the first meiotic spindle. In contrast, microinjection of anti-XKCM1 promoted MT assembly throughout the cytoplasm, disrupting organization of the MTOC-TMA and meiotic spindle. Finally, microinjection of anti-dynein or anti-NuMA disrupted the organization of the MTOC-TMA and subsequent assembly of the meiotic spindles. These results suggest that XMAP215 and XKCM1 act antagonistically to regulate MT assembly and organization during maturation of Xenopus oocytes, and that dynein and NuMA are required for organization of the MTOC-TMA.     

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.     

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.     

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.     

Acentrosomal spindle assembly and maintenance in Caenorhabditis elegans oocytes requires a kinesin-12 nonmotor microtubule interaction domain

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in Caenorhabditis elegans oocytes, where kinesin-5 is not required to generate outward force and the kinesin-12 family motor KLP-18 instead performs this function. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which KLP-18 promotes acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and its adaptor protein MESP-1 activates nonmotor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.     

Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.     

Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification

A protein complex consisting of Mago Nashi and Tsunagi/Y14 is required to establish the major body axes and for the localization of primordial germ cell determinants during Drosophila melanogaster oogenesis. The Mago Nashi:Tsunagi/Y14 heterodimer also serves as the core of the exon junction complex (EJC), a multiprotein complex assembled on spliced mRNAs. In previous studies, reduced function alleles of mago nashi and tsunagi/Y14 were used to characterize the roles of the genes in oogenesis. Here, we investigated mago nashi and tsunagi/Y14 using null alleles and clonal analysis. Germline clones lacking mago nashi function divide but fail to differentiate. The mago nashi null germline stem cells produce clones over a period of at least 11 days, suggesting that mago nashi is not necessary for stem cell self-renewal. However, germline stem cells lacking tsunagi/Y14 function are indistinguishable from wild type. Additionally, in tsunagi/Y14 null germline cysts, centrosomes and oocyte-specific components fail to concentrate within a single cell and oocyte fate is not restricted to a single cell. Together, our results suggest not only that mago nashi is required for germline stem cell differentiation but that surprisingly mago nashi functions independently of tsunagi/Y14 in this process. On the other hand, Tsunagi/Y14 is essential for restricting oocyte fate to a single cell and may function with mago nashi in this process.     

Microtubule-Depolymerizing Kinesin KLP10A Restricts the Length of the Acentrosomal Meiotic Spindle in Drosophila Females

During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.     

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down’s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

Characterization of aurora-a in porcine oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization and cleavage

Aurora-A is a serine/threonine protein kinase that plays important regulatory roles during mitotic cell cycle progression. In this study, Aurora-A expression, subcellular localization, and possible functions during porcine oocyte meiotic maturation, fertilization and early embryonic cleavage were studied by using Western blot, confocal microscopy and drug treatments. The quantity of Aurora-A protein remained stable during porcine oocyte meiotic maturation. Confocal microscopy revealed that Aurora-A distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, Aurora-A concentrated around the condensed chromosomes and the metaphase I spindle, and finally, Aurora-A was associated with spindle poles during the formation of the metaphase II spindle. Aurora-A concentrated in the pronuclei in fertilized eggs. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. In conclusion, Aurora-A may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.