Identification of transcriptional and metabolic programs related to mammalian cell size

SummaryBackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing us to study cell size in vivo using transcriptomics and metabolomics.ResultsLarger cells displayed increased expression of cytoskeletal genes but unexpectedly repressed expression of many genes involved in mitochondrial functions. This effect appears to be cell autonomous because cultured Drosophila cells induced to increase cell size displayed a similar gene-expression pattern. Larger hepatocytes also displayed a reduction in the expression of lipogenic transcription factors, especially sterol-regulatory element binding proteins. Inhibition of mitochondrial functions and lipid biosynthesis, which is dependent on mitochondrial metabolism, increased the cell size with reciprocal effects on cell proliferation in several cell lines.ConclusionsWe uncover that large cell-size increase is accompanied by downregulation of mitochondrial gene expression, similar to that observed in diabetic individuals. Mitochondrial metabolism and lipid synthesis are used to couple cell size and cell proliferation. This regulatory mechanism may provide a possible mechanism for sensing metazoan cell size

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes

Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating

Synaptic vesicle biogenesis involves the recycling of synaptic vesicle components by clathrin-mediated endocytosis from the presynaptic membrane. stoned B, a protein encoded by the stoned locus in Drosophila melanogaster has been shown to regulate vesicle recycling by interacting with synaptotagmin. We report here the identification and characterization of a human homolog of stoned B (hStnB). Human stoned B is a brain-specific protein which co-enriches with other endocytic proteins such as AP-2 in a crude synaptic vesicle fraction and at nerve terminals. A domain with homology to the medium chain of adaptor complexes binds directly to both AP-2 and synaptotagmin and competes with AP-2 for the same binding site within synaptotagmin. Finally we show that the µ2 homology domain of hStnB stimulates the uncoating of both clathrin and AP-2 adaptors from clathrin-coated vesicles. We hypothesize that hStnB regulates synaptic vesicle recycling by facilitating vesicle uncoating

Emi2 Is Essential for Mouse Spermatogenesis

The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the cytostatic factor. We generated knockout mice to determine the in vivo functions of Emi2-in particular, its functions in the testis, where Emi2 is expressed at high levels. Male and female Emi2 knockout mice are viable but sterile, indicating that Emi2 is essential for meiosis but dispensable for embryonic development and mitotic cell divisions. We found that, besides regulating cell-cycle arrest in mouse eggs, Emi2 is essential for meiosis I progression in spermatocytes. In the absence of Emi2, spermatocytes arrest in early diplotene of prophase I. This arrest is associated with decreased Cdk1 activity and was partially rescued by a knockin mouse model of elevated Cdk1 activity. Additionally, we detected expression of Emi2 in spermatids and sperm, suggesting potential post-meiotic functions for Emi

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

<div><p>The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (Mastl^NULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in Mastl^NULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, Mastl^NULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of Mastl^NULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using <i>in vitro</i> dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.</p></div

Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2

Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrin-mediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosis-defective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an up-regulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling

Growth analysis of Mastl^NULL MEFs.

<p>(A) Three MEF lines isolated from different embryos were treated with 4-OHT or DMSO to induce Mastl knockout (Mastl^NULL) and their proliferative potential was monitored by Alamar Blue proliferation assays for 8 days. AFU, arbitrary fluorescence units. (B) MEFs were synchronized and recombination in the Mastl and Cdk1 loci were induced as described in the Methods section. Cells were arrested in mitosis for 4 hours using 5μM Eg5 inhibitor S-Trityl-L-cysteine (STLC) starting from 20 hours after release into full growth medium. Still pictures were taken using phase-contrast microscopy. Scale bar 100μm. (C) MEFs were fixed 24 hours after release into full growth medium and stained with anti-phospho-histone H3 Ser10 antibodies (pH3) to quantify mitotic cells using FACS analysis. (D) MEFs expressing the histone H2B-YFP fusion protein were analyzed by time-lapse microscopy. Still images of a dividing Mastl^NULL cell were acquired every 5 minutes. Scale bar 10μm. (E) Quantification of appearance of anaphase bridges in Mastl^FLOX and Mastl^NULL MEFs expressing the H2B-YFP. (F) Mastl^NULL cells were fixed and stained with DAPI 72 hours after synchronization and release into full growth medium. Scale bar 20μm.</p

Kinetochore localization of Mad1 in MEFs in early and late mitosis.

<p>(A) Primary MEFs were treated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#pgen.1006310.g004" target="_blank">Fig 4</a> to induce mitotic arrest and were fixed. Where indicated, cells were treated with 200nM OKA for one hour before fixation. Immunofluorescence analysis was performed using ACA and antibodies against Mad1. Different mitotic phases were determined with early mitotic cells determined by lightly condensed chromosomes that were scattered in the cytoplasm just after NEBD and late mitotic cells displaying a highly condensed chromosome mass typically caused by nocodazole treatment. Insets represent the boxed areas. Scale bar 5 μm. Quantification of kinetochore-localized Mad1 in Mastl^FLOX and Mastl^NULL cells in early prophase (NEBD) (B) or late prometaphase-like state by nocodazole treatment (C) [N>20 cells per each condition, ± standard deviation] as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006310#pgen.1006310.g003" target="_blank">Fig 3B</a> except using kinetochore-localized Mad1. To determine the statistical significance, a Student’s <i>t</i>-test was performed. (n.s. not significance; ***<i>p</i><0.0001, Student’s <i>t</i>-test, unpaired).</p