Electronic Microscopy Facility
Electronic Microscopy Facility
Mazurek S.,University of Lausanne |
Mazurek S.,Wrocław University |
Garroum I.,University of Lausanne |
Daraspe J.,Electronic Microscopy Facility |
And 6 more authors.
Plant Physiology | Year: 2017
The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the n(C=O) and n(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development. © 2017 American Society of Plant Biologists. All rights reserved.
Zaldivar M.M.,RWTH Aachen |
Pauels K.,RWTH Aachen |
Von Hundelshausen P.,RWTH Aachen |
Berres M.-L.,RWTH Aachen |
And 9 more authors.
Hepatology | Year: 2010
Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the plateletderived chemokine (C-X-C motif) ligand 4(CXCL4)as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4-/- and wild-type mice were subjected to two models of chronic liver injury (CCl4 and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus-induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl4 and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf-β[transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4-/- mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8+ T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. Conclusion: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies. Copyright © 2010 by the American Association for the Study of Liver Diseases.
Liao X.,Case Cardiovascular Research Institute |
Zhang R.,Case Cardiovascular Research Institute |
Lu Y.,Case Cardiovascular Research Institute |
Prosdocimo D.A.,Case Cardiovascular Research Institute |
And 16 more authors.
Journal of Clinical Investigation | Year: 2015
Mitochondrial homeostasis is critical for tissue health, and mitochondrial dysfunction contributes to numerous diseases, including heart failure. Here, we have shown that the transcription factor Kruppel-like factor 4 (KLF4) governs mitochondrial biogenesis, metabolic function, dynamics, and autophagic clearance. Adult mice with cardiac-specific Klf4 deficiency developed cardiac dysfunction with aging or in response to pressure overload that was characterized by reduced myocardial ATP levels, elevated ROS, and marked alterations in mitochondrial shape, size, ultrastructure, and alignment. Evaluation of mitochondria isolated from KLF4-deficient hearts revealed a reduced respiration rate that is likely due to defects in electron transport chain complex I. Further, cardiac-specific, embryonic Klf4 deletion resulted in postnatal premature mortality, impaired mitochondrial biogenesis, and altered mitochondrial maturation. We determined that KLF4 binds to, cooperates with, and is requisite for optimal function of the estrogen-related receptor/PPARγ coactivator 1 (ERR/PGC-1) transcriptional regulatory module on metabolic and mitochondrial targets. Finally, we found that KLF4 regulates autophagy flux through transcriptional regulation of a broad array of autophagy genes in cardiomyocytes. Collectively, these findings identify KLF4 as a nodal transcriptional regulator of mitochondrial homeostasis.
PubMed | Boston Childrens Hospital, Beth Israel Deaconess Medical Center and Electronic Microscopy Facility
Type: | Journal: The Journal of thoracic and cardiovascular surgery | Year: 2016
To demonstrate the clinical efficacy of autologous mitochondrial transplantation in preparation for translation to human application using an invivo swine model.A left mini-thoracotomy was performed on Yorkshire pigs. The pectoralis major was dissected, and skeletal muscle tissue was removed and used for the isolation of autologous mitochondria. The heart was subjected to regional ischemia (RI) by temporarily snaring the circumflex artery. After 24minutes of RI, hearts received 80.1mL injections of vehicle (vehicle-only group; n=6) or vehicle containing mitochondria (mitochondria group; n=6) into the area at risk (AAR), and the snare was released. The thoracotomy was closed, and the pigs were allowed to recover for 4weeks.Levels of creatine kinase-MB isoenzyme and cardiac troponin I were significantly increased (P=.006) in the vehicle-only group compared with the mitochondria group. Immune, inflammatory, and cytokine activation markers showed no significant difference between groups. There was no significant between-group difference in the AAR (P=.48), but infarct size was significantly greater in the vehicle group (P=.004). Echocardiography showed no significant differences in global function. Histochemistry and transmission electron microscopy revealed damaged heart tissue in the vehicle group that was not apparent in the mitochondria group. T2-weighted magnetic resonance imaging and histology demonstrated that the injected mitochondria were present for 4weeks.Autologous mitochondrial transplantation provides a novel technique to significantly enhance myocardial cell viability following ischemia and reperfusion in the clinically relevant swine model.
Satir P.,Yeshiva University |
Heuser T.,Electronic Microscopy Facility |
Sale W.S.,Emory University
BioScience | Year: 2014
The motile cilium is a mechanical wonder, a cellular nanomachine that produces a high-speed beat based on a cycle of bends that move along an axoneme made of 9+2 microtubules. The molecular motors, dyneins, power the ciliary beat. The dyneins are compacted into inner and outer dynein arms, whose activity is highly regulated to produce microtubule sliding and axonemal bending. The switch point hypothesis was developed long ago to account for how sliding in the presence of axonemal radial spoke-central pair interactions causes the ciliary beat. Since then, a new genetic, biochemical, and structural complexity has been discovered, in part, with Chlamydomonas mutants, with high-speed, high-resolution analysis of movement and with cryoelectron tomography. We stand poised on the brink of new discoveries relating to the molecular control of motility that extend and refine our understanding of the basic events underlying the switching of arm activity and of bend formation and propagation. © 2014 The Author(s).
Bierwagen J.,Max Planck Institute for Biophysical Chemistry |
Testa I.,Max Planck Institute for Biophysical Chemistry |
Folling J.,Max Planck Institute for Biophysical Chemistry |
Wenzel D.,Electronic Microscopy Facility |
And 3 more authors.
Nano Letters | Year: 2010
We demonstrate far-field optical imaging at the nanoscale with unlabeled samples. Subdiffraction resolution images of autofluorescent samples are obtained by depleting the ground state of natural fluorophores by transferring them to a metastable dark state and simultaneously localizing those fluorophores that are transiently returning. Our approach is based on the insight that nanoscopy methods relying on stochastic single-molecule switching require only a single fluorescence on-off cycle to yield an image, a condition fulfilled by various biomolecules. The method is exemplified by recording label-free nanoscopy images of thylakoid membranes of spinach chloroplasts. © 2010 American Chemical Society.
Fuest M.,University Hospital Freiburg |
Willim K.,University Hospital Freiburg |
Macnelly S.,University Hospital Freiburg |
Fellner N.,Electronic Microscopy Facility |
And 3 more authors.
Hepatology | Year: 2012
Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N-terminal kinase (JNK). The AP-1 (activating protein 1) transcription factor c-Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis. Because the functions of c-Jun during the ER stress response are poorly understood, we addressed this issue in primary hepatocytes and livers of hepatocyte-specific c-Jun knockout mice. ER stress was induced pharmacologically in vitro and in vivo and resulted in a rapid and robust induction of c-Jun protein expression. Interestingly, ER-stressed hepatocytes lacking c-Jun displayed massive cytoplasmic vacuolization due to ER distension. This phenotype correlated with exacerbated and sustained activation of canonical ER stress signaling pathways. Moreover, sustained ER stress in hepatocytes lacking c-Jun resulted in increased cell damage and apoptosis. ER stress is also a strong inducer of macroautophagy, a cell-protective mechanism of self-degradation of cytoplasmic components and organelles. Interestingly, autophagosome numbers in response to ER stress were reduced in hepatocytes lacking c-Jun. To further validate these findings, macroautophagy was inhibited chemically in ER-stressed wildtype hepatocytes, which resulted in cytoplasmic vacuolization and increased cell damage closely resembling the phenotypes observed in c-Jun-deficient cells. Conclusion: Our findings indicate that c-Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death and provide evidence that c-Jun functionally links ER stress responses and macroautophagy. © 2011 American Association for the Study of Liver Diseases.