Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

Loss-of-function of kinesin-5 KIF11 causes microcephaly, chorioretinopathy, and developmental disorders through chromosome instability and cell cycle arrest.

Kinesin motors play a fundamental role in development by controlling intracellular transport, spindle assembly, and microtubule organization. In humans, patients carrying mutations in KIF11 suffer from an autosomal dominant inheritable disease called microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR). While mitotic functions of KIF11 proteins have been well documented in centrosome separation and spindle assembly, cellular mechanisms underlying KIF11 dysfunction and MCLMR remain unclear. In this study, we generate KIF11-inhibition chick and zebrafish models and find that KIF11 inhibition results in microcephaly, chorioretinopathy, and severe developmental defects in vivo. Notably, loss-of-function of KIF11 causes the formation of monopolar spindle and chromosome misalignment, which finally contribute to cell cycle arrest, chromosome instability, and cell death. Our results demonstrate that KIF11 is crucial for spindle assembly, chromosome alignment, and cell cycle progression of progenitor stem cells, indicating a potential link between polyploidy and MCLMR. Our data have revealed that KIF11 inhibition cause microcephaly, chorioretinopathy, and development disorders through the formation of monopolar spindle, polyploid, and cell cycle arrest.

Kinesin-5 Eg5 mediates centrosome separation to control spindle assembly in spermatocytes.

Timely and accurate centrosome separation is critical for bipolar spindle organization and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is essential for centrosome separation and spindle organization in somatic cells; however, the detailed functions and mechanisms of Eg5 in spermatocytes remain unclear. In this study, we show that Eg5 proteins are located at spindle microtubules and centrosomes in spermatocytes both in vivo and in vitro. We reveal that the spermatocytes are arrested at metaphase I in seminiferous tubules after Eg5 inhibition. Eg5 ablation results in cell cycle arrest, the formation of monopolar spindle, and chromosome misalignment in cultured GC-2 spd cells. Importantly, we find that the long-term inhibition of Eg5 results in an increased number of centrosomes and chromosomal instability in spermatocytes. Our findings indicate that Eg5 mediates centrosome separation to control spindle assembly and chromosome alignment in spermatocytes, which finally contribute to chromosome stability and faithful cell division of the spermatocytes.

Kinesin-14 KIFC1 modulates spindle assembly and chromosome segregation in mouse spermatocytes.

Kinesin-14 KIFC1 regulates spindle assembly and centrosome clustering in diverse organisms during cell division. KIFC1 proteins are essential for spindle assembly and chromosome alignment in mitosis. However, the roles and mechanisms of KIFC1 proteins in male spermatocytes remain largely unknown. In this study, we reveal that KIFC1 proteins mainly accumulate at the centrosomes and central spindle in mouse spermatocytes both in vitro and in vivo. We utilize two KIFC1 specific inhibitors, AZ82 and CW069, for the inhibition of KIFC1 in mouse spermatogenic cells and cultured GC-2 spd(ts) cells. We find that KIFC1 inhibition results in the increase of spermatocytes with micronuclei, the disorganization of the meiotic spindles, and the formation of multiple centrosomes. Furthermore, we demonstrate that KIFC1 inhibition leads to spindle defects, chromosome misalignment and the formation of aneuploidy in cultured GC-2 spd(ts) cells. In this study, we reveal that KIFC1 proteins are critical for centrosome maintenance and chromosome stability in mouse spermatocytes.

Kinesin-8 motors: regulation of microtubule dynamics and chromosome movements.

Microtubules are essential for intracellular transport, cell motility, spindle assembly, and chromosome segregation during cell division. Microtubule dynamics regulate the proper spindle organization and thus contribute to chromosome congression and segregation. Accumulating studies suggest that kinesin-8 motors are emerging regulators of microtubule dynamics and organizations. In this review, we provide an overview of the studies focused on kinesin-8 motors in cell division. We discuss the structures and molecular kinetics of kinesin-8 motors. We highlight the essential roles and mechanisms of kinesin-8 in the regulation of microtubule dynamics and spindle organization. We also shed light on the functions of kinesin-8 motors in chromosome movement and the spindle assembly checkpoint during the cell cycle.

Kinesin-7 CENP-E regulates the formation and structural maintenance of the acrosome.

The acrosome is a special organelle that develops from the Golgi apparatus and the endolysosomal compartment in the spermatids. Centromere protein E (CENP-E) is an essential kinesin motor in chromosome congression and alignment. This study is aimed at investigating the roles and mechanisms of kinesin-7 CENP-E in the formation of the acrosome during spermatogenesis. Male ICR mice are injected with GSK923295 for long-term inhibition of CENP-E. Chemical inhibition and siRNA-mediated knockdown of CENP-E are carried out in the GC-2 spd cells. The morphology of the acrosomes is determined by the HE staining, immunofluorescence, and transmission electron microscopy. We have identified CENP-E is a key factor in the formation and structural maintenance of the acrosome during acrosome biogenesis. Long-term inhibition of CENP-E by GSK923295 results in the asymmetric acrosome and the dispersed acrosome. CENP-E depletion leads to the malformation of the Golgi complex and abnormal targeting of the PICK1- and PIST-positive Golgi-associated vesicles. Our findings uncover an essential role of CENP-E in membrane trafficking and structural organization of the acrosome in the spermatids during spermatogenesis. Our results shed light on the molecular mechanisms involved in vesicle trafficking and architecture maintenance of the acrosome.

Mechanisms of kinesin-7 CENP-E in kinetochore-microtubule capture and chromosome alignment during cell division.

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere-associated protein E (CENP-E), a plus-end-directed kinesin motor, is required for congression of pole-proximal chromosomes in metaphase. CENP-E accumulates at the outer plate of kinetochores and mediates the kinetochore-microtubule capture. CENP-E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP-E interacts with Bub1-related kinase, Aurora B and core kinetochore components during kinetochore-microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin-7 CENP-E. We highlight the complicated interactions between CENP-E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP-E in mitosis and meiosis, including the kinetochore-microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP-E in tumourigenesis and CENP-E's specific inhibitors.

Sry and SoxE genes: How they participate in mammalian sex determination and gonadal development?

In mammals, sex determination defines the differentiation of the bipotential genital ridge into either testes or ovaries. Sry, the mammalian Y-chromosomal testis-determining gene, is a master regulator of male sex determination. It acts to switch the undifferentiated genital ridge towards testis development, triggering the adoption of a male fate. Sry initiates a cascade of gene networks through the direct regulation of Sox9 expression and promotes supporting cell differentiation, Leydig cell specification, vasculature formation and testis cord development. In the absence of Sry, alternative genetic cascades, including female sex-determining genes RSPO1, Wnt4/ß-catenin and Foxl2, are involved in the formation of female genitalia and the maintenance of female ovarian development. The mutual antagonisms between male and female sex-determining pathways are crucial in not just the initiation but also the maintenance of the somatic sex of the gonad throughout the organism's lifetime. Any imbalances in above sex-determining genes can cause disorders of sex development in humans and mice. In this review, we provide a detailed summary of the expression profiles, biochemical properties and developmental functions of Sry and SoxE genes in embryonic testis development and adult gonadal development. We also briefly summarize the dedicate balances between male and female sex-determining genes in mammalian sex development, with particular highlights on the molecular actions of Sry and Sox9 transcription factors.

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation.

During eukaryote cell division, molecular motors are crucial regulators of microtubule organization, spindle assembly, chromosome segregation and intracellular transport. The kinesin-14 motors are evolutionarily conserved minus-end-directed kinesin motors that occur in diverse organisms from simple yeasts to higher eukaryotes. Members of the kinesin-14 motor family can bind to, crosslink or slide microtubules and, thus, regulate microtubule organization and spindle assembly. In this Commentary, we present the common subthemes that have emerged from studies of the molecular kinetics and mechanics of kinesin-14 motors, particularly with regard to their non-processive movement, their ability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing center proteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors have recently been implicated in the regulation of microtubule nucleation. We also discuss recent progress in our current understanding of the multiple and fundamental functions that kinesin-14 motors family members have in important aspects of cell division, including the spindle pole, spindle organization and chromosome segregation.

A novel role of KIF3b in the seminoma cell cycle.

KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed that the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division.

Kinesin-7 CENP-E in tumorigenesis: Chromosome instability, spindle assembly checkpoint, and applications.

Kinesin motors are a large family of molecular motors that walk along microtubules to fulfill many roles in intracellular transport, microtubule organization, and chromosome alignment. Kinesin-7 CENP-E (Centromere protein E) is a chromosome scaffold-associated protein that is located in the corona layer of centromeres, which participates in kinetochore-microtubule attachment, chromosome alignment, and spindle assembly checkpoint. Over the past 3 decades, CENP-E has attracted great interest as a promising new mitotic target for cancer therapy and drug development. In this review, we describe expression patterns of CENP-E in multiple tumors and highlight the functions of CENP-E in cancer cell proliferation. We summarize recent advances in structural domains, roles, and functions of CENP-E in cell division. Notably, we describe the dual functions of CENP-E in inhibiting and promoting tumorigenesis. We summarize the mechanisms by which CENP-E affects tumorigenesis through chromosome instability and spindle assembly checkpoints. Finally, we overview and summarize the CENP-E-specific inhibitors, mechanisms of drug resistances and their applications.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

The SOX gene family: function and regulation in testis determination and male fertility maintenance.

The Sox (Sry-type HMG box) genes encode a group of proteins characterized by the existence of an SRY (sex-determining region on Y chromosome) box, a 79 amino acid motif that encodes an HMG (high mobility group) domain which can bind and bend DNA, which is the only part in SRY that is conserved between species. The Sox gene family functions in many aspects in embryogenesis, including testis development, CNS neurogenesis, oligodendrocyte development, chondrogenesis, neural crest cell development and other respects. The Sox gene family was originally identified through homology with Sry. The Sry gene is the mammalian testis-determining gene. It functions to open the testis determination pathway directly and close the ovary pathway indirectly. Sry and Sox9 are the most important two genes expressed during testis determination. Besides, researchers have found that Sox8 and Sox9 have functions in the male fertility maintenance after birth. In this review, information was evaluated from mouse or from human if not mentioned otherwise.

Kinesin-14 KIFC1 promotes acrosome formation and chromatin maturation during mouse spermiogenesis.

KIFC1, a member of kinesin-14 subfamily motors, is essential for meiotic cell division and acrosome formation during spermatogenesis. However, the functions of KIFC1 in the formation and maintenance of the acrosome in male germ cells remain to be elucidated. In this study, we report the structural deformities of acrosomes in the in vivo KIFC1 inhibition mouse models. The proacrosomal vesicles diffuse into the cytoplasm and form atypical acrosomal granules. This phenotype is consistent with globozoospermia patients and probably results from the failure of the Golgi-derived vesicle trafficking and actin filament organization. Moreover, the multinucleated and undifferentiated spermatogenic cells in the epidydimal lumen after KIFC1 inhibition reveal the specific roles of KIFC1 in regulating post-meiotic maturation. Overall, our results uncover KIFC1 as an essential regulator in the trafficking, fusion and maturation of acrosomal vesicles during spermiogenesis.

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Kinesin-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes.

Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.

Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development.

Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor protein, which is essential for spindle pole separation and chromosome alignment in mitosis. Heterozygous Eg5 mutations cause autosomal-dominant microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome in humans. However, the developmental roles and cellular mechanisms of Eg5 in organogenesis remain largely unknown. In this study, we have shown that Eg5 inhibition leads to the formation of the monopolar spindle, chromosome misalignment, polyploidy, and subsequent apoptosis. Strikingly, long-term inhibition of Eg5 stimulates the immune responses and the accumulation of lymphocytes in the mouse spleen through the innate and specific immunity pathways. Eg5 inhibition results in metaphase arrest and cell growth inhibition, and suppresses the formation of somite and retinal development in zebrafish embryos. Our data have revealed the essential roles of kinesin-5 Eg5 involved in cell proliferation, chromosome stability, and organogenesis during development. Our findings shed a light on the cellular basis and pathogenesis in microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome of Eg5-mutation-positive patients.

Male reproductive systems of Macaca mulatta: Gonadal development, spermatogenesis and applications in spermatogonia stem cell transplantation.

Rhesus macaque (Macaca mulatta) is widely applied in animal model construction of infertility, spermatogonia stem cell transplantation and male reproductive diseases. In this review, we describe the seasonal changes of the reproductive system in rhesus macaques, the regular pattern of spermatogenesis and spermatozoa maturation, and the differentiation of spermatogonia and spermatocytes. The duration of the M. mulatta spermatogenesis is approximately 10 days and seminiferous epithelium cycles mainly consist of 12 stages, which provide a suitable model for reproductive studies in non-human primates. Here, we summarize the features of gonadal development and sperm maturation in the rhesus monkeys, which provide important information in the studies of reproductive biology. Rhesus macaque is an excellent animal model in spermatogonia stem cell transplantation. We discuss the applications and progresses of assisted reproductive technologies in sperm liquefaction, semen cryopreservation and spermatogonia stem cell transplantation of rhesus macaques. Besides, we sort out recent proteomic analyses of male reproductive systems and semen samples in rhesus macaques. This review mainly focuses on male reproductive biology and application studies using M. mulatta, which would promote the development of new therapeutic interventions on assisted reproduction and reproductive disease studies in the future.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes using In Vivo Inhibition, Immunofluorescence and Flow Cytometry.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Kinesin-7 CENP-E regulates cell division, gastrulation and organogenesis in development.

Kinesin-7 CENP-E motor protein is essential for chromosome alignment and kinetochore-microtubule attachment in cell division. Human CENP-E has recently identified to be linked with the microcephalic primordial dwarfism syndromes associated with a smaller head, brain malformations and a prominent nose. However, the roles of CENP-E in embryonic development remain largely unknown. In this study, we find that zebrafish CENP-E inhibition results in defects in early zygote cleavage, including asymmetric cell division, cell cycle arrest and the developmental abnormalities. We also demonstrate that CENP-E ablation in cultured cells leads to chromosome misalignment, spindle abnormalities and interruptions of the cell cycle. These observations suggest that CENP-E plays a key role in early cell division and cell cycle progression. Furthermore, we also find that CENP-E inhibition results in the defects in the epiboly, the developmental arrest, the smaller head and the abnormal embryo during zebrafish embryogenesis. Our data demonstrate new functions of CENP-E in development and provide insights into its essential roles in organogenesis.