Cohesin Removal along the Chromosome Arms during the First Meiotic Division Depends on a NEK1-PP1γ-WAPL Axis in the Mouse

SummaryMammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, sororin, and PDS5B and direct cleavage by separase. Here, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by protein phosphatase 1 gamma (PP1γ), which both interacts with and is a phosphotarget of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ, leading to the dephosphorylation of WAPL, which, in turn, results in its retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals

NIMA-related kinase 1 (NEK1) regulates meiosis I spindle assembly by altering the balance between α-Adducin and Myosin X

<div><p>NIMA-related kinase 1 (NEK1) is a serine/threonine and tyrosine kinase that is highly expressed in mammalian germ cells. Mutations in <i>Nek1</i> induce anemia, polycystic kidney and infertility. In this study we evaluated the role of NEK1 in meiotic spindle formation in both male and female gametes. Our results show that the lack of NEK1 provokes an abnormal organization of the meiosis I spindle characterized by elongated and/or multipolar spindles, and abnormal chromosome congression. The aberrant spindle structure is concomitant with the disruption in localization and protein levels of myosin X (MYO10) and α-adducin (ADD1), both of which are implicated in the regulation of spindle formation during mitosis. Interaction of ADD1 with MYO10 is dependent on phosphorylation, whereby phosphorylation of ADD1 enables its binding to MYO10 on mitotic spindles. Reduction in ADD1 protein in NEK1 mutant mice is associated with hyperphosphorylation of ADD1, thereby preventing the interaction with MYO10 during meiotic spindle formation. Our results reveal a novel regulatory role for NEK1 in the regulation of spindle architecture and function during meiosis.</p></div

Loss of NEK1 results in disrupted spindle morphology and chromosome congression during meiosis I in spermatocytes.

<p>(a-c) Immunofluorescence (IF) against β-tubulin (green) and DNA staining with DAPI (magenta) showing meiotic spindle morphology (panels a1-c1), with graphic representations below (panels a2-c2). (A) <i>Nek1</i>^<i>+/+</i> spermatocyte shows normal spindle morphology and chromosome congression at the midplate of the spindle; (b, c) Examples of aberrant spindle morphology in spermatocytes from <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice showing monopolar spindles with mislocalized chromosomes resulting from failed chromosome congression (arrowheads) in b and multipolar spindles in c (arrowheads). (d) Quantitation of abnormal spindles in spermatocytes from <i>Nek1</i>^<i>+/+</i> and <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice (n = 100 cells counted for each). (e) Quantitation cells with abnormal spindles by type of defect. Values are percentages ± Standard deviation. * Indicates statistically significant differences (Unpaired t-test p < 0.05).</p

<i>Nek1</i>^{<i>kat2j/kat2j</i>} spermatocytes and oocytes show aberrant localization of Adducin 1 (ADD1) on meiosis I spindles.

<p>(a) IF against ADD1 (red), β-tubulin (green) and DAPI (magenta) on spermatocytes from wildtype (left) and <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice (right) in coronal (top) and transverse (bottom) planes. Arrowheads indicate examples of ADD1 foci. Quantitation of ADD1 focus counts per nucleus are given in panel (b). (c) IF against ADD1 (red), β-tubulin (green) and DAPI (magenta) in oocytes of both wildtype and mutant mice. Arrowheads indicate examples of ADD1 foci. Quantitation of ADD1 focus counts per nucleus are given in panel (d). All values are focus counts per spindle and black bars denote means ± Standard deviation. * Indicates statistically significant differences (Unpaired t test p < 0.05).</p

Loss of NEK1 results in disrupted spindle morphology and chromosome congression during meiosis I in oocytes.

<p>(a-d) Immunofluorescence (IF) against β-tubulin (green) and DNA staining with DAPI (magenta) showing meiotic spindle morphology (panels a1-d1), with graphic representations below (panels a2-d2). (a) <i>Nek1</i>^<i>+/+</i> oocyte shows normal spindle morphology and chromosome congression at the midplate of the spindle; (b-d) Examples of aberrant spindle morphology in oocytes from <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice showing multipolar spindles with or without errors in chromosome congression (arrowheads indicate three extra spindles formed in the oocyte in b and two additional spindles in c). (e) Quantitation of abnormal spindles in oocytes from <i>Nek1</i>^<i>+/+</i> and <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice (n = 100 cells counted for each). (f) Quantitation of cells with abnormal spindles by type of defect. Values are percentages ± Standard deviation. * Indicates statistically significant differences (Unpaired t-test p < 0.05).</p

<i>Nek1</i>^{<i>kat2j/kat2j</i>} spermatocytes and oocytes show aberrant localization of myosin X (MYO10) on meiosis I spindles.

<p>(a) IF against MYO10 (red), β-tubulin (green) and DAPI (magenta) on spermatocytes from wildtype (left) and <i>Nek1</i>^{<i>kat2j/kat2j</i>} mice (right) in coronal (top) and transverse (bottom) planes. Arrowheads indicate examples of MYO10 foci. Quantitation of MYO10 focus counts per nucleus are given in panel (b). (c) IF against MYO10 (red), β-tubulin (green) and DAPI (magenta) in oocytes of both wildtype and mutant mice. Arrowheads indicate examples of MYO10 foci. Quantitation of MYO10 focus counts per nucleus are given in panel (d). All values are focus counts per spindle and black bars denote means ± Standard deviation. * Indicates statistically significant differences (Unpaired t test p < 0.05).</p

Loss of NEK1 during meiosis is associated with hyper-phosphorylation and reduced ADD1 protein in a PP1γ-dependent manner.

<p>(a) Quantitation of ADD1 protein levels in <i>Nek1</i>^+/+ testis extracts relative to GAPDH control. (b) ADD1 and MYO10 protein levels, expressed as a ratio of <i>Nek1</i>^{<i>kat2j/kat2j</i>} / <i>Nek1</i>^<i>+/+</i>, as determined by mass spectrometry. (c) ADD1 phosphopeptide and deaminated peptide sequence determined by mass spectrometry to be higher in <i>Nek1</i>^{<i>kat2j/kat2j</i>} testis extracts. (d) Quantitation by western blotting of ADD1 protein levels in oocytes at 0h and after 6h of culture. (e) Quantification of ADD1 protein levels in wildtype oocytes cultured for 6h in vehicle (ethanol) and the PP1 inhibitor Calyculin A (CLA) (at doses of 2 and 20 nM). All values are means ± Standard deviation. * Indicate statistically significant differences (Unpaired t test p < 0.05 and one-way ANOVA followed by Dunnett’s multiple comparisons test, p < 0.05, for cultures with inhibitor)</p

Lack of NEK1 results in abnormal meiosis I spindle morphology in spermatocytes and oocytes.

<p>(a) Immunofluorescence (IF) against β-tubulin (green) and DNA staining with DAPI (magenta) showing a wildtype (top) and <i>Nek1</i>^{<i>kat2j/kat2j</i>} spermatocytes (bottom), along with measurements of spindle length (b) and width (c). Black bars are means (μm) ± Standard deviation. (d) Immunofluorescence (IF) against β-tubulin (green) and DNA staining with DAPI (magenta) showing a wildtype (top) and <i>Nek1</i>^{<i>kat2j/kat2j</i>} (bottom) oocytes, along with measurements of spindle length (b) and width (c). Black bars are means (μm) ± Standard deviation. * Indicates statistically significant differences (Unpaired t-test p < 0.05).</p