Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Evidence that meiotic sex chromosome inactivation is essential for male fertility

The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal meiosis. This asynapsis triggers inactivation of X- and Y-linked genes, or meiotic sex chromosome inactivation (MSCI). Whether MSCI is essential for male meiosis is unclear. Pachytene arrest and apoptosis is observed in mouse mutants in which MSCI fails, e.g., Brca1(-/-), H2afx(-/-), Sycp1(-/-), and Msh5(-/-). However, these also harbor defects in synapsis and/or recombination and as such may activate a putative pachytene checkpoint. Here we present evidence that MSCI failure is sufficient to cause pachytene arrest. XYY males exhibit Y-Y synapsis and Y chromosomal escape from MSCI without accompanying synapsis/recombination defects. We find that XYY males, like synapsis/recombination mutants, display pachytene arrest and that this can be circumvented by preventing Y-Y synapsis and associated Y gene expression. Pachytene expression of individual Y genes inserted as transgenes on autosomes shows that expression of the Zfy 1/2 paralogs in XY males is sufficient to phenocopy the pachytene arrest phenotype; insertion of Zfy 1/2 on the X chromosome where they are subject to MSCI prevents this response. Our findings show that MSCI is essential for male meiosis and, as such, provide insight into the differential severity of meiotic mutations' effects on male and female meiosis.     

MNS1 is essential for spermiogenesis and motile ciliary functions in mice

During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1-deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1-deficient sperm flagella, the characteristic arrangement of "9+2" microtubules and outer dense fibers are completely disrupted. In addition, MNS1-deficient mice display situs inversus and hydrocephalus. MNS1-deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.     

MEKK1 is essential for DT40 cell apoptosis in response to microtubule disruption

Vinblastine and other microtubule-damaging agents, such as nocodazole and paclitaxel, cause cell cycle arrest at the G2/M transition and promote apoptosis in eukaryotic cells. The roles of these drugs in disrupting microtubule dynamics and causing cell cycle arrest are well characterized. However, the mechanisms by which these agents promote apoptosis are poorly understood. We disrupted the MEKK1 kinase domain in chicken bursal B-cell line DT40 by homologous recombination and have shown that it is essential for both vinblastine-mediated apoptosis and vinblastine-mediated c-Jun N-terminal protein kinase activation. In addition, our data indicate that vinblastine-mediated apoptosis in DT40 cells requires new protein synthesis but does not require G2/M arrest, suggesting that vinblastine-mediated cell cycle arrest and apoptosis are two independent processes.     

Cadherin-mediated regulation of microtubule dynamics

Epithelial polarization and neuronal outgrowth require the assembly of microtubule arrays that are not associated with centrosomes. As these processes generally involve contact interactions mediated by cadherins, we investigated the potential role of cadherin signalling in the stabilization of non-centrosomal microtubules. Here we show that expression of cadherins in centrosome-free cytoplasts increases levels of microtubule polymer and changes the behaviour of microtubules from treadmilling to dynamic instability. This effect is not a result of cadherin expression per se but depends on the formation of cell-cell contacts. The effect of cell-cell contacts is mimicked by application of beads coated with stimulatory anti-cadherin antibody and is suppressed by overexpression of the cytoplasmic cadherin tail. We therefore propose that cadherins initiate a signalling pathway that alters microtubule organization by stabilizing microtubule ends.     

Accessory protein regulation of microtubule dynamics throughout the cell cycle

A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle.     

Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

Nuages are found in the germ cells of diverse organisms. However, nuages in postnatal male germ cells of mice are poorly studied. Previously, we cloned a germ cell-specific gene named Rnf17, which encodes a protein containing both a RING finger and tudor domains. Here, we report that RNF17 is a component of a novel nuage in male germ cells--the RNF17 granule, which is an electron-dense non-membrane bound spherical organelle with a diameter of 0.5 mum. RNF17 granules are prominent in late pachytene and diplotene spermatocytes, and in elongating spermatids. RNF17 granules are distinguishable from other known nuages, such as chromatoid bodies. RNF17 is able to form dimers or polymers both in vitro and in vivo, indicating that it may play a role in the assembly of RNF17 granules. Rnf17-deficient male mice were sterile and exhibited a complete arrest in round spermatids, demonstrating that Rnf17 encodes a novel key regulator of spermiogenesis. Rnf17-null round spermatids advanced to step 4 but failed to produce sperm. These results have shown that RNF17 is a component of a novel germ cell nuage and is required for differentiation of male germ cells.     

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.     

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.     

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88^Tg737Rpw mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88^−/− mice are completely sterile. They produce ∼350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.     

Gamma-tubulin is essential for microtubule organization and development in Arabidopsis

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. gamma-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, gamma-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.     

ASK1 physically interacts with COI1 and is required for male fertility in Arabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. The COI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with the Arabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action in planta. Functional analysis by antisense strategy showed that ASK1 is involved in male fertility.     

Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view

Assembly of mitotic and meiotic spindles into an elliptical bipolar shape is an example of morphogenetic processes that involve local chromosomal regulation of microtubule dynamics for proper spatial microtubule assembly. Global microtubule dynamics during the cell cycle and local microtubule dynamics during spindle assembly are regulated by a balance between microtubule stabilizing and destabilizing factors. How a chromosome-induced phosphorylation gradient may be generated and modulate spindle microtubule assembly through balanced regulation of the activity of microtubule-associated proteins and Stathmin/Op 18 is analyzed.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

Coupling cell division and cell death to microtubule dynamics

The mitotic spindle is a self-organizing structure that is constructed primarily from microtubules. Among the most important spindle microtubules are those that bind to kinetochores and form the fibers along which chromosomes move. Chemotherapeutics such as taxol and the vinca alkaloids perturb kinetochore—microtubule attachment and disrupt chromosome segregation. This activates a checkpoint pathway that delays cell cycle progression and induces programmed cell death. Recent work has identified at least four mammalian spindle assembly checkpoint proteins.     

Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.     

SirT1 catalytic activity is required for male fertility and metabolic homeostasis in mice

The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.