Chromosome segregation impacts on cell growth and division site selection in Corynebacterium glutamicum.

Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

Abstract Background Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. Results In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. Conclusions The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions. See Commentary: http://www.biomedcentral.com/1741-7007/8/9

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.S.E.M. and D.J.O. are funded by a Wellcome Trust Fellowship (to D.J.O. no. 090909/Z). N.A.B. is funded by MRC grant MR/M010007/1, and S.H. is funded by a grant from the German Science Foundation (SFB 635, TP A3). D.S. and S.M. acknowledge financial support from the Lundbeck Foundation and the Danish Councils for Independent and Strategic Research. C.J.M. and F.P. were funded by the Fondation pour la Recherche Medicale.This is the final published version. It first appeared at http://www.cell.com/developmental-cell/fulltext/S1534-5807%2815%2900144-6

Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection—A Prospective Pilot Study

Background: Blood or tissue culture or histology prove invasive Candida infection, but long time to result, limited feasibility and sensitivity call for new approaches. In this pilot project, we describe the diagnostic potential of quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes in blood of patients with invasive Candida infection.Methods: We used flow cytometry quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes from peripheral blood of patients with invasive Candida infection, from patients at risk and healthy volunteers as controls.Results: Elevated levels of Candida-reactive lymphocytes were measured in 13 patients with proven invasive Candida infection and in one patient with probable hepatosplenic candidiasis. Results of three candidemia patients were uninterpretable due to autofluorescence of samples. Twelve of 13 patients had Candida identified to species level by conventional methods, and T cell reactivity correctly identified Candida species in 10 of 12 patients. Nine hematological high-risk patients and 14 healthy donors had no elevated Candida-reactive T cell counts.Conclusions: This Candida-reactive lymphocyte assay correctly identified the majority of patients with invasive Candida infection and the respective species. Our assay has the potential to support diagnosis of invasive Candida infection to species level and to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative

CLUH regulates mitochondrial metabolism by controlling translation and decay of target mRNAs

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal–neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.Peer reviewe

Chromosome segregation defects lead to altered growth in <i>C. glutamicum</i>.

<p>(<b>A</b>) Distribution of elongation rates of wild type, DivIVA-mCherry, Δ<i>parA</i> DivIVA-mCherry and Δ<i>parB</i> DivIVA-mCherry mutant cells. (<i>n</i> ≥300). (<b>B</b>) Association of birth lengths and elongation lengths in DivIVA-mCherry, Δ<i>parA</i> DivIVA-mCherry and Δ<i>parB</i> DivIVA-mCherry. In wild type and DivIVA-mCherry cells the birth length and elongation length are associated. In the absence of <i>parA</i> or <i>parB</i>, more variation in the length of the cell at birth and the cell length before division is observed. Negative correlation between birth and elongation lengths indicates a size-based cell cycle regulatory mechanism (grey line). Regression line slope is shown on top right. (<i>n</i> ≥300).</p

Anucleate cells and polar divisions in wild type <i>C. glutamicum</i> and <i>par</i> mutants.

<p>Anucleate cells and polar divisions in wild type <i>C. glutamicum</i> and <i>par</i> mutants.</p

In the absence of <i>parB</i>, division septa constrict over the nucleoid, guillotining part of the chromosome.

<p>Shown is a time lapse series of DivIVA-mCherry expressing cell in the absence of <i>parB</i>. Images were acquired every 5 minutes and time points are indicated for each time frame. DNA is stained with Hoechst (blue) and DivIVA-mCherry is shown in red. DivIVA-mCherry is recruited to the division septum, which has assembled over the chromosome (white arrowhead). The chromosome is pumped in one direction, into the cell half containing the bulk of the chromosome. The division septum constricts directly over part of the chromosome, leading to guillotining part of the chromosome (black arrowhead). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055078#pone.0055078.s006" target="_blank">Movie S5</a>.</p

Defining the action spectrum of potential PGC-1 activators on a mitochondrial and cellular level in vivo

Previous studies have demonstrated a therapeutic benefit of pharmaceutical PGC-1 activation in cellular and murine model of disorders linked to mitochondrial dysfunction. While in some cases, this effect seems to be clearly associated with boosting of mitochondrial function, additional alterations as well as tissue- and cell-type-specific effects might play an important role. We initiated a comprehensive analysis of the effects of potential PGC-1-activating drugs and pharmaceutically targeted the PPAR (bezafibrate, rosiglitazone), AMPK (AICAR, metformin) and Sirt1 (resveratrol) pathways in HeLa cells, neuronal cells and PGC-1-deficient MEFs to get insight into cell type specificity and PGC-1 dependence of their working action. We used bezafibrate as a model drug to assess the effect on a tissue-specific level in a murine model. Not all analyzed drugs activate the PGC pathway or alter mitochondrial protein levels. However, they all affect supramolecular assembly of OXPHOS complexes and OXPHOS protein stability. In addition, a clear drug- and cell-type-specific influence on several cellular stress pathways as well as on post-translational modifications could be demonstrated, which might be relevant to fully understand the action of the analyzed drugs in the disease state. Importantly, the effect on the activation of mitochondrial biogenesis and stress response program upon drug treatment is PGC-1 dependent in MEFs demonstrating not only the pleiotropic effects of this molecule but points also to the working mechanism of the analyzed drugs. The definition of the action spectrum of the different drugs forms the basis for a defect-specific compensation strategy and a future personalized therapeutic approach