Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane‐nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ‐tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo‐like kinase Cdc5 and the meiosis‐specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase‐promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking‐activation mechanism ensures that only single‐copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II‐specific processes.     

Mitochondrial Calcium uniporters are essential for meiotic progression in mouse oocytes by controlling Ca2+ entry

ObjectivesThe alteration of bioenergetics by oocytes in response to the demands of various biological processes plays a critical role in maintaining normal cellular physiology. However, little is known about the association between energy sensing and energy production with energy‐dependent cellular processes like meiosis.Materials and methodsWe demonstrated that cell cycle‐dependent mitochondrial Ca²⁺ connects energy sensing to mitochondrial activity in meiosis progression within mouse oocytes. Further, we established a model in mouse oocytes using siRNA knockdowns that target mitochondrial calcium uniporters (MCUs) in order to inhibit mitochondrial Ca²⁺ concentrations.ResultsDecreased numbers of oocytes successfully progressed to the germinal vesicle stage and extruded the first polar body during in vitro culture after inhibition, while spindle checkpoint‐dependent meiosis was also delayed. Mitochondrial Ca²⁺ levels changed, and this was followed by altered mitochondrial masses and ATP levels within oocytes during the entirety of meiosis progression. Abnormal mitochondrial Ca²⁺ concentrations in oocytes then hindered meiotic progress and activated AMP‐activated protein kinase (AMPK) signalling that is associated with gene expression.ConclusionsThese data provide new insight into the protective role that MCU‐dependent mitochondrial Ca²⁺ signalling plays in meiotic progress, in addition to demonstrating a new mechanism of mitochondrial energy regulation by AMPK signalling that influences meiotic maturation.     

RAB14 GTPase is essential for actin‐based asymmetric division during mouse oocyte maturation

ObjectivesRAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation.Materials and methodsMicroinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study.ResultsOur results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection.ConclusionsTaken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.     

Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.     

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

Downregulation of PLK4 expression induces apoptosis and G0/G1‐phase cell cycle arrest in keloid fibroblasts

ObjectivesKeloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids.Materials and MethodsWe evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen‐B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs.ResultsWe discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen‐B suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro.ConclusionsThese findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.

Keloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids. Here, we discovered that PLK4 is a potential target for the treatment of keloids. PLK4 was overexpressed in keloid dermal samples and keloid fibroblasts (KFs) compared with adjacent normal skin samples and normal skin fibroblasts derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by a highly selective inhibitor, centrinone B (Cen‐B), suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase via the p53/p21/Cyclin D1 pathway in vitro. These findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.     

Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.     

LKB1/PAR4 protein is asymmetrically localized in mouse oocytes and associates with meiotic spindle

The mouse secondary oocyte is polarized at the ultrastructural and molecular level, but very little is known about mechanisms involved in the establishment of this polarity. We showed that the LKB1 kinase, a mouse homologue of Caenorhabditis elegans PAR4 protein is asymmetrically localized to the animal pole of the mouse oocyte and during oocyte maturation associates with the microtubules of metaphase I and metaphase II meiotic spindles. Therefore, we suggest that LKB1/PAR4 protein, may participate in the polarization of the oocyte and in the regulation of the asymmetry of meiotic divisions during mouse oogenesis.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

The POLO kinase is required at multiple stages during spermatogenesis in Drosophila melanogaster

The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with α-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis. Received: 5 August 1998; in revised form: 3 September 1998 / Accepted: 3 September, 1998     

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.     

Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes

Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.     

Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans

In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. Although many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. Here we analyze microtubule behavior in Caenorhabditis elegans oocytes at early stages of the meiotic maturation process using confocal microscopy and live-cell imaging. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. We show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. We present evidence that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic G alpha(o/i) and G alpha(s) pathways and gap-junctional communication with somatic cells of the gonad. We propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD.     

KLP-18, a Klp2 kinesin,is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.