Christian Frei

DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1

To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra‐S phase checkpoint responds to stalled replication forks by downregulating late‐firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases α and ϵ are stabilized for 40–60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM‐related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single‐stranded DNA that allows RP‐A and Mec1 to promote DNA polymerase association.

Structural Maintenance of Chromosomes Protein C-terminal Domains Bind Preferentially to DNA with Secondary Structure

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains ofSaccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with theirin vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Drosophila cyclin D/Cdk4 requires Hif-1 prolyl hydroxylase to drive cell growth

The Drosophila cyclin-dependent protein kinase complex Cyclin D/Cdk4 induces cell growth (accumulation of mass) as well as proliferation (cell cycle progression). To understand how CycD/Cdk4 promotes growth, we performed a screen for modifiers of CycD/Cdk4-driven overgrowth in the eye. Loss-of-function mutations in Hif-1 prolyl hydroxylase (Hph), an enzyme involved in the cellular response to hypoxic stress, dominantly suppress the growth but not the proliferation function of CycD/Cdk4. hph mutant cells are defective for growth, and, remarkably, ectopic expression of Hph is sufficient to increase cellular growth. Epistasis analysis places Hph downstream of CycD/Cdk4. Overexpressed CycD/Cdk4 causes an increase in Hph protein in tissues where Hph induces growth, suggesting a mechanism whereby Hph levels are regulated posttranscriptionally in response to CycD/Cdk4. Our data suggest that Hph, in addition to its function in hypoxic response, is a regulator of cellular growth and that it is a key mediator for CycD/Cdk4.

The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling

Mitochondrial mass and activity must be adapted to tissue function, cellular growth and nutrient availability. In mammals, the related transcriptional coactivators PGC‐1α, PGC‐1β and PRC regulate multiple metabolic functions, including mitochondrial biogenesis. However, we know relatively little about their respective roles in vivo. Here we show that the Drosophila PGC‐1 family homologue, Spargel, is required for the expression of multiple genes encoding mitochondrial proteins. Accordingly, spargel mutants showed mitochondrial respiration defects when complex II of the electron transport chain was stimulated. Spargel, however, was not limiting for mitochondrial mass, but functioned in this respect redundantly with Delg, the fly NRF‐2α/GABPα homologue. More importantly, in the larval fat body, Spargel mediated mitochondrial activity, cell growth and transcription of target genes in response to insulin signalling. In this process, Spargel functioned in parallel to the insulin‐responsive transcription factor, dFoxo, and provided a negative feedback loop to fine‐tune insulin signalling. Taken together, our data place Spargel at a nodal point for the integration of mitochondrial activity to tissue and organismal metabolism and growth.

The Drosophila mitochondrial ribosomal protein mRpL12 is required for Cyclin D/Cdk4-driven growth

The Drosophila melanogaster cyclin‐dependent protein kinase complex CycD/Cdk4 stimulates both cell cycle progression and cell growth (accumulation of mass). CycD/Cdk4 promotes cell cycle progression via the well‐characterized RBF/E2F pathway, but our understanding of how growth is stimulated is still limited. To identify growth regulatory targets of CycD/Cdk4, we performed a loss‐of‐function screen for modifiers of CycD/Cdk4‐induced overgrowth of the Drosophila eye. One mutation that suppressed CycD/Cdk4 was in a gene encoding the mitochondrial ribosomal protein, mRpL12. We show here that mRpL12 is required for CycD/Cdk4‐induced cell growth. Cells homozygous mutant for mRpL12 have reduced mitochondrial activity, and exhibit growth defects that are very similar to those of cdk4 null cells. CycD/Cdk4 stimulates mitochondrial activity, and this is mRpL12 dependent. Hif‐1 prolyl hydroxylase (Hph), another effector of CycD/Cdk4, regulates growth and is required for inhibition of the hypoxia‐inducible transcription factor 1 (Hif‐1). Both functions depend on mRpL12 dosage, suggesting that CycD/Cdk4, mRpL12 and Hph function together in a common pathway that controls cell growth via affecting mitochondrial activity.

Nutrition Controls Mitochondrial Biogenesis in the Drosophila Adipose Tissue through Delg and Cyclin D/Cdk4

Mitochondria are cellular organelles that perform critical metabolic functions: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. Here, we demonstrate that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, we show that Delg (CG6338), the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. Our data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity.

Mitochondria in response to nutrients and nutrient-sensitive pathways.

Mitochondria are abundant cellular organelles, and are required for the generation of energy through oxidative catabolism. Equally important, mitochondria also provide substrates for de novo synthesis of fatty acids and multiple amino acids. Mitochondrial functions must therefore be tightly linked to cellular nutrient availability. This review focuses on the current knowledge of how nutrients affect mitochondria. In particular, we describe how the transcriptional profile of the nucleus is altered to mediate this control, and the transcription factors that are involved. In addition, we summarize recent progress in our understanding of how transcription-independent mechanisms, most notably through the cellular energy sensor mTOR, are used to adapt mitochondrial functions in respect to cellular metabolic needs.

Mammalian cyclin D1/Cdk4 complexes induce cell growth in Drosophila

The Drosophila melanogaster cyclin dependent protein kinase complex CycD/Cdk4 has been shown to regulate cellular growth (accumulation of mass) as well as proliferation (cell cycle progression). In contrast, the orthologous mammalian complex has been shown to regulate cell cycle progression, but possible functions in growth control have not been addressed directly. To test whether mammalian Cyclin D1/Cdk4 complexes are capable of driving cell growth, we expressed such a complex in Drosophila. Using assays that distinguish between mass increase and cell cycle progression, we found that this complex stimulated cell growth, like its Drosophila counterpart. Furthermore, Hif-1 prolyl hydroxylase (Hph) is required for both complexes to drive growth. Our data suggest that the growth-specific function of CycD/Cdk4 is conserved from arthropods to mammals.

Cyclin D/Cdk4: new insights from Drosophila.

The Drosophila cyclin-dependent protein kinase complex CycD/Cdk4 has been known to drive cellular growth (accumulation of mass) as well as proliferation (cell cycle progression). Recent data demonstrate that Hif prolyl hydroxylase (Hph) is required for the induction of growth, but not for the induction of proliferation. Normal levels of Hph are required for cellular growth, demonstrating that Hph, in addition to its known function in the cellular response low oxygen levels, regulates growth. Since Hph’s hydroxylation activity depends on oxygen and possibly the mitochondrial activity, these data provide a link between CycD/Cdk4 and oxygen/energy homeostasis.

Stoichiometry of various Ag(In)SbTe phase change materials (PCMs) determined using LA-ICP-MS

In this study major element concentrations of AgInSbTe phase change materials of different stoichiometry were quantified using UV-ns-LA-ICP-MS. Performing raster ablation sampling, precision and accuracy of the determined concentrations were improved compared to single hole drilling ablation. Using raster mode, possible fractionation effects during the ablation and temporal mass load effects in the ICP were found less variable for the respective materials. Additionally, using raster mode a more representative sampling (mass) was achieved, which results in a more bulk-equivalent description of the sample. An indication for that is given by the improved RSD of the determined concentrations to values as low as 1%. A single calibration material composed of all four elements of interest was applied to determine the major element content in AgInSbTe samples with unknown stoichiometry. A quantification approach based on a 100% normalisation (see ref. 1) was used, due to the similar behaviour of the elements during raster ablation sampling. The high purity of these materials allows using the 100% normalisation, based on the four major elements only. Furthermore, the applicability of a four-component calibration material for the quantification of three-component AgSbTe materials was investigated in terms of precision and accuracy. The stoichiometry-dependent particle size distribution of the laser induced aerosol was studied and is suspected to be the limiting factor for accuracy. It is shown that a matrix-matched calibration material, or at least ablation conditions that provide a particle size-matched aerosol between calibration- and sample-material, provides access to accurate stoichiometry of such materials.