functional-assessment-kinesin-7-cenp-e-spermatocytes-using-vivo

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 ...

generation-centromere-associated-protein-e-cenp-e-knockout-cell-lines

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 ...

Fujian Obstetrics and Gynecology Hospital

Affiliated Hospital of Fujian Medical University

Fujian Medical University

Fujian Maternity and Child Health Hospital

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.

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

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.

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

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

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

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 cell division, gastrulation and organogenesis in development.

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.

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

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.

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

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

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-14 KIFC1 modulates spindle assembly and chromosome segregation in mouse 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-5 Eg5 mediates centrosome separation to control spindle assembly in 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-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes.

Bepridil is a commonly used medication for arrhythmia and heart failure. It primarily exerts hemodynamic effects by inhibiting Na/K movement and regulating the Na/Ca exchange. In comparison to other Ca inhibitors, bepridil has a long half-life and a complex pharmacology. Additionally, it is widely used in antiviral research and the treatment of various diseases. However, the toxicity of this compound and its other possible effects on embryonic development are unknown. In this study, we investigated the toxicity of bepridil on rat myocardial H9c2 cells. After treatment with bepridil, the cells became overloaded with Ca and entered a state of cytoplasmic vacuolization and nuclear abnormality. Bepridil treatment resulted in several morphological abnormalities in zebrafish embryo models, including pericardium enlargement, yolk sac swelling, and growth stunting. The hemodynamic effects on fetal development resulted in abnormal cardiovascular circulation and myocardial weakness. After inhibiting the Ca transmembrane, the liver of zebrafish larvae also displayed an ectopic and deficient spatial location. Additionally, the results of the RNA-seq analysis revealed the detailed gene expression profiles and metabolic responses to bepridil treatment in zebrafish embryonic development. Taken together, our study provides an important evaluation of antiarrhythmic agents for clinical use in prenatal heart patients.

Effects of bepridil on early cardiac development of zebrafish.

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

Congenital heart disease (CHD) is the most common type of birth defect and the main noninfectious cause of death during the neonatal stage. The non-POU domain containing, octamer-binding gene, NONO, performs a variety of roles involved in DNA repair, RNA synthesis, transcriptional and post-transcriptional regulation. Currently, hemizygous loss-of-function mutation of NONO have been described as the genetic origin of CHD. However, essential effects of NONO during cardiac development have not been fully elucidated. In this study, we aim to understand role of Nono in cardiomyocytes during development by utilizing the CRISPR/Cas9 gene editing system to deplete Nono in the rat cardiomyocytes H9c2. Functional comparison of H9c2 control and knockout cells showed that Nono deficiency suppressed cell proliferation and adhesion. Furthermore, Nono depletion significantly affected the mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, resulting in H9c2 overall metabolic deficits. Mechanistically we demonstrated that the Nono knockout impeded the cardiomyocyte function by attenuating phosphatidyl inositol 3 kinase-serine/threonine kinase (Pi3k/Akt) signaling via the assay for transposase-accessible chromatin using sequencing in combination with RNA sequencing. From these results we propose a novel molecular mechanism of Nono to influence cardiomyocytes differentiation and proliferation during the development of embryonic heart. We conclude that NONO may represent an emerging possible biomarkers and targets for the diagnosis and treatment of human cardiac development defects.

ArticleTotal : 2	Year
Publication title Cited by 1	2021
Publication title	2023

Article	Year
Kinesin-6 family motor KIF20A regulates central spindle assembly and acrosome biogenesis in mouse spermatogenesis. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 31884069	2020
Kinesin-5 Eg5 is essential for spindle assembly and chromosome alignment of mouse spermatocytes. Cell division\| PubMed ID: 32165913	2020
Kinesin-7 CENP-E regulates chromosome alignment and genome stability of spermatogenic cells. Cell death discovery\| PubMed ID: 32351712	2020
Kinesin-8 motors: regulation of microtubule dynamics and chromosome movements. Chromosoma\| PubMed ID: 32417983	2020
Kinesin-7 CENP-E regulates cell division, gastrulation and organogenesis in development. European journal of cell biology\| PubMed ID: 32800279	2020
Kinesin-7 CENP-E regulates the formation and structural maintenance of the acrosome. Cell and tissue research\| PubMed ID: 33237480	2021
Male reproductive systems of Macaca mulatta: Gonadal development, spermatogenesis and applications in spermatogonia stem cell transplantation. Research in veterinary science\| PubMed ID: 33965833	2021
Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly. Biological reviews of the Cambridge Philosophical Society\| PubMed ID: 31343816	2019
Kinesin-14 KIFC1 modulates spindle assembly and chromosome segregation in mouse spermatocytes. Experimental cell research\| PubMed ID: 35259401	2022
Kinesin-5 Eg5 mediates centrosome separation to control spindle assembly in spermatocytes. Chromosoma\| PubMed ID: 35437661	2022
Kinesin-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 35680098	2022
Effects of bepridil on early cardiac development of zebrafish. Cell and tissue research\| PubMed ID: 36422735	2023
Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development. Cell death discovery\| PubMed ID: 36513626	2022
Nono deficiency impedes the proliferation and adhesion of H9c2 cardiomyocytes through Pi3k/Akt signaling pathway. Scientific reports\| PubMed ID: 37130848	2023

Ya-Lan Wei

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