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 Medical University

Fujian Province University

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.

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.

Meng-Fei Xu

Department of Cell Biology and Genetics,
The School of Basic Medical Sciences,
Department of Cell Biology and Genetics, The School of Basic Medical Sciences

Fujian Medical University

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

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

Meng-Fei Xu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Genetics,The School of Basic Medical Sciences,Department of Cell Biology and Genetics, The School of Basic Medical Sciences

Fujian Medical University

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

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

Department of Cell Biology and Genetics,
The School of Basic Medical Sciences,
Department of Cell Biology and Genetics, The School of Basic Medical Sciences

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