The spatio-temporal organization of mitochondrial F1FO ATP synthase in cristae depends on its activity mode.

F1FO ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy. Under normal conditions, complex V was mainly restricted to cristae indicated by orthogonal trajectories along the cristae membranes. In addition confined trajectories that are quasi immobile exist. By inhibiting glycolysis with 2-DG, the activity and mobility of complex V was altered. The distinct cristae-related orthogonal trajectories of complex V were obliterated. Moreover, a mobile subpopulation of complex V was found in the inner boundary membrane. The observed changes in the ratio of dimeric/monomeric complex V, respectively less mobile/more mobile complex V and its activity changes were reversible. In IF1-KO cells, in which ATP hydrolysis is not inhibited by IF1, complex V was more mobile, while inhibition of ATP hydrolysis by BMS-199264 reduced the mobility of complex V. Taken together, these data support the existence of different subpopulations of complex V, ATP synthase and ATP hydrolase, the latter with higher mobility and probably not prevailing at the cristae edges. Obviously, complex V reacts quickly and reversibly to metabolic conditions, not only by functional, but also by spatial and structural reorganization.This work was supported by the DFG (INST 190/167-2). K. Busch is associated with the CiM (Cells in Motion cluster, Munster).S

Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain

6 páginas, 2 figurasReprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.This study was supported by research funding from the IMF at University Hospital Münster to GH (I-HA-111219) and from the DFG to TK (SFB-TRR128-B7).Peer reviewe

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.close1

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

peer reviewedIn vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Multi-functional DNA nanostructures that puncture and remodel lipid membranes into hybrid materials

DNA nanopores can span lipid bilayers but how they interact with lipids is not known. Here the authors establish at single-molecule level the insertion mechanism and show that DNA nanopores can locally cluster and remodel membranes, and stabilize autonomously formed lipid nanotubes

Three-Dimensional Interfacing of Cells with Hierarchical Silicon Nano/Microstructures for Midinfrared Interrogation of In Situ Captured Proteins

Label-free optical detection of biomolecules is currently limited by a lack of specificity rather than sensitivity. To exploit the much more characteristic refractive index dispersion in the mid-infrared (IR) regime, we have engineered three-dimensional IR-resonant silicon micropillar arrays (Si-MPAs) for protein sensing. By exploiting the unique hierarchical nano- and microstructured design of these Si-MPAs attained by CMOS-compatible silicon-based microfabrication processes, we achieved an optimized interrogation of surface protein binding. Based on spatially resolved surface functionalization, we demonstrate controlled three-dimensional interfacing of mammalian cells with Si-MPAs. Spatially controlled surface functionalization for site-specific protein immobilization enabled efficient targeting of soluble and membrane proteins into sensing hotspots directly from cells cultured on Si-MPAs. Protein binding to Si-MPA hotspots at submonolayer level was unambiguously detected by conventional Fourier transform IR spectroscopy. The compatibility with cost-effective CMOS-based microfabrication techniques readily allows integration of this novel IR transducer into fully fledged bioanalytical microdevices for selective and sensitive protein sensing

Mitochondria ultrastructure in pre-implantation embryos.

<p>Representative micrographs show mitochondria ultrastructure in mouse wild-type SCNT and ICSI embryos, throughout pre-implantation development. nucleus: n; mitochondria: d, developed; md, medium developed; ud, undeveloped. Mitochondria morphology and analysis are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036850#s4" target="_blank">Materials and Methods</a>. Magnification: 12200X.</p

Model of post-transcriptional regulation after SCNT.

<p>Interlinked events at the metabolic level, disturbed after SCNT, are proposed to impact on reprogramming and embryo development outcome. SCNT manipulation alters mitochondrial membrane potential (Δψ), triggers cytosolic Ca²⁺ concentration ([Ca²⁺]_c) increase, ATP decrease and reactive oxygen species (ROS) increase. Increased cytosolic Ca²⁺-induced retrograde response, and ROS-induced redox imbalance, could induce transcriptional regulation. Oxidative stress and decreased ATP could induce cellular responses driving post-transcriptional regulation. Proposed effects could be direct (bold arrows), or follow after a cascade of events (indirect; dashed arrows).</p

Transcription and translation in mouse blastocysts.

<p>mRNA levels were determined for pools of six ICSI and SCNT blastocysts (in duplicate), by quantitative RT-PCR, over two independent experiments. Technical replicates were determined three times for each sample, and results were normalized to Flag-Oct4 mRNA. (A) dCT values for transcripts in mtGFP-Tg SCNT, compared to ICSI. (B) dCT values for Gapdh and Actb mRNA, as well as protein levels (by immunocytochemistry), in wild-type SCNT relative to ICSI. Gapdh and Actb levels were significantly different, both for mRNA (t-test, <i>p</i> = 0,001 and <i>p</i> = 1,29E-08, respectively) and protein (t-test, <i>p</i> = 0.006 and <i>p</i> = 0.04, respectively). Box plot: fluorescence intensities distribution in the blastocyst; top and bottom lines: inter-quartile range; middle line: median; whiskers: range of variation limited to 1.5 times inter-quartile range.</p