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 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.

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-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.

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.

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 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.

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.

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.

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.

Kinesin-7 CENP-E regulates chromosome alignment and genome stability of spermatogenic cells.

Kinesin-7 CENP-E is an essential kinetochore motor required for chromosome alignment and congression. However, the specific functions of CENP-E in the spermatogenic cells during spermatogenesis remain unknown. In this study, we find that CENP-E proteins are expressed in the spermatogonia, spermatocytes, and the elongating spermatids. CENP-E inhibition by specific inhibitor GSK923295 results in the disruption of spermatogenesis and cell cycle arrest of spermatogenic cells. Both spermatogonia and spermatocytes are arrested in metaphase and several chromosomes are not aligned at the equatorial plate. We find that CENP-E inhibition leads to chromosome misalignment, the spindle disorganization, and the formation of the aneuploidy cells. Furthermore, the inhibition of CENP-E results in the defects in the formation of spermatids, including the sperm head condensation and the sperm tail formation. We have revealed that kinesin-7 CENP-E is essential for chromosome alignment and genome stability of the spermatogenic cells.

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-5 Eg5 is essential for spindle assembly and chromosome alignment of mouse spermatocytes.

BACKGROUND: Microtubule organization is essential for bipolar spindle assembly and chromosome segregation, which contribute to genome stability. Kinesin-5 Eg5 is known to be a crucial regulator in centrosome separation and spindle assembly in mammalian somatic cells, however, the functions and mechanisms of Eg5 in male meiotic cell division remain largely unknown. RESULTS: In this study, we have found that Eg5 proteins are expressed in mouse spermatogonia, spermatocytes and spermatids. After Eg5 inhibition by specific inhibitors Monastrol, STLC and Dimethylenastron, the meiotic spindles of dividing spermatocytes show spindle collapse and the defects in bipolar spindle formation. We demonstrate that Eg5 regulates spindle bipolarity and the maintenance of meiotic spindles in meiosis. Eg5 inhibition leads to monopolar spindles, spindle abnormalities and chromosome misalignment in cultured GC-2 spd cells. Furthermore, Eg5 inhibition results in the decrease of the spermatids and the abnormalities in mature sperms. CONCLUSIONS: Our results have revealed an important role of kinesin-5 Eg5 in male meiosis and the maintenance of male fertility. We demonstrate that Eg5 is crucial for bipolar spindle assembly and chromosome alignment in dividing spermatocytes. Our data provide insights into the functions of Eg5 in meiotic spindle assembly of dividing spermatocytes.

Kinesin-6 family motor KIF20A regulates central spindle assembly and acrosome biogenesis in mouse spermatogenesis.

Kinesin-6 KIF20A is essential for microtubule organization and central spindle assembly during cytokinesis. However, the functions of KIF20A in meiotic division and spermatogenesis remain elusive. Here, we report that kinesin-6 KIF20A locates at the microtubules in mouse spermatogenic cells and co-localizes with the spindle midzone and midbody. We demonstrate that central spindle organization and chromosomal stability are regulated by KIF20A in male meiotic division. KIF20A inhibition leads to the defects in central spindle assembly and cytokinetic abscission, and finally results in the increase of aneuploid cells and the alteration of cell populations in the spermatogenic cells. Furthermore, we have revealed that kinesin-6 KIF20A is associated with the formation and maturation of the acrosomes during spermatogenesis. Our findings have identified the specific roles of KIF20A in central spindle organization in meiotic division.

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly.

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non-motor microtubule-associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule-bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post-translational modifications of Ase1/PRC1 by cyclin-dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo-like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division.

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.

Roles and mechanisms of Kinesin-6 KIF20A in spindle organization during cell division.

Mitotic kinesin is crucial for spindle assembly and chromosome segregation in cell division. KIF20A/MKlp2, a member of kinesin-6 subfamily, plays important roles in the central spindle organization at anaphase and cytokinesis. In this review, we briefly introduce the discovery and classification of kinesin-6 motors in model organisms, and summarize the biochemical features and mechanics of KIF20A proteins. We emphasize the complicated interactions of KIF20A with partner proteins, including MKlp1, Plk1 and Rab6. Particularly, we highlight the regulation of Cdk1 and chromosomal passenger complex on kinesin-6 KIF20A at late stage of mitosis. We summarized the multiple functions of KIF20A in central spindle assembly and the formation of cleavage furrow in both mitosis and meiosis. In addition, we conclude the expression patterns of KIF20A in tumorigenesis and its applications in tumor therapy.