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

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and Recycling.

SummaryClathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact μ-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Benzothiazole derivatives as human DNA topoisomerase IIα inhibitors

Abstract Benzothiazole derivatives resembling the structure of DNA purine bases were tested to determine their topoisomerase inhibition activities. Based on DNA topoisomerase I and II relaxation assay results, all 12 derivatives acted as human topoisomerase IIa inhibitors, whereas only two compounds inhibited Calf thymus topoisomerase I. 3-amino-2-(2-bromobenzyl)-1,3-benzothiazol-3-ium 4-methylbenzensulfonate (BM3) was observed to be the most effective human topoisomerase IIa inhibitor with the lowest IC 50 value of 39 nM. The mechanistic studies suggested that BM3 was neither a DNA intercalator nor a topoisomerase poison, it was only a DNA minor groovebinding agent. BM3 initially bound to the DNA topoisomerase IIa enzyme, then to DNA. As a result, the tested benzothiazole derivatives were obtained as strong topoisomerase IIa inhibitors. The benzothiazole tosylated salt form BM3 was found as the most effective topoisomerase IIa inhibitor. BM3's mechanisms of action might be its direct interaction with the enzyme. BM3's minor groove-binding property might also contribute to this action. Hence, BM3 could be a good candidate as a new anticancer agent

Benzothiazole derivatives as human DNA topoisomerase IIα inhibitors

Benzothiazole derivatives resembling the structure of DNA purine bases were tested to determine their topoisomerase inhibition activities. Based on DNA topoisomerase I and II relaxation assay results, all 12 derivatives acted as human topoisomerase IIα inhibitors, whereas only two compounds inhibited Calf thymus topoisomerase I. 3-amino-2-(2-bromobenzyl)-1,3-benzothiazol-3-ium 4-methylbenzensulfonate (BM3) was observed to be the most effective human topoisomerase IIα inhibitor with the lowest IC50 value of 39 nM. The mechanistic studies suggested that BM3 was neither a DNA intercalator nor a topoisomerase poison, it was only a DNA minor groove-binding agent. BM3 initially bound to the DNA topoisomerase IIα enzyme, then to DNA. As a result, the tested benzothiazole derivatives were obtained as strong topoisomerase IIα inhibitors. The benzothiazole tosylated salt form BM3 was found as the most effective topoisomerase IIα inhibitor. BM3's mechanisms of action might be its direct interaction with the enzyme. BM3's minor groove-binding property might also contribute to this action. Hence, BM3 could be a good candidate as a new anticancer agent. © 2013 Springer Science+Business Media New York

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