correlative-microscopy-for-3d-structural-analysis-dynamic

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8.

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

Effective immunotherapy against murine gliomas using type 1 polarizing dendritic cells--significant roles of CXCL10.

In einem Versuch, wirksame Impfstoffe gegen Tumoren des zentralen Nervensystems (ZNS) zu entwickeln, wir evaluieren die F&auml;higkeit von Impfstoffen mit Norm dendritische Zellen (DC) im Vergleich zu Typ 1 Polfilter DCs (DC1), Gliom-spezifischen Typ 1 CTLs veranlassen mit CNS Tumor-relevanten zielsuchende Eigenschaften und ihrem Wirkungsmechanismus. C57BL/6 Maus abgeleitet Knochenmarkzellen wurden mit Maus-Granulozyten/Makrophagen-Kolonie-stimulierende-Faktor (GM-CSF) f&uuml;r 6 Tage kultiviert und CD11c(+) Zellen wurden anschlie&szlig;end mit GM-CSF, RmIFN-Gamma, RmIFN-Alpha RmIL-4 und Polyinosinic Polycytidylic S&auml;ure stabilisiert durch Lysin und Carboxymethylcellulose f&uuml;r 24 Stunden zum Generieren von DC1s kultiviert. In Analogie zu ihren menschlichen Pendants Maus DC1s Oberfl&auml;che Markierung Profile Reifen DCS ausgestellt und produziert hohe IL-12 und CXCL10. Wichtig ist f&uuml;r ihre Anwendung als Impfstoffen gegen Krebs, solche DC1s stabil beibehalten ihre Typ-1-Ph&auml;notyp auch wenn Typ 2-F&ouml;rderung oder regulatorische T-Zellen (Treg) ausgesetzt-Umgebungen zu f&ouml;rdern. Konsequent, induzierte Maus DC1s Antigen-spezifischen Typ 1 CTLs effizienter als in-vitro-Nichtpolarisierter DCs. DC1s gegeben s.c. Entw&auml;sserung Lymphknoten migriert, induzierte Antigen-spezifischen CTLs und unterdr&uuml;ckt Treg Akkumulation. Dar&uuml;ber hinaus s.c. Immunisierung mit DC1s geladen mit Gliom-assoziierte Antigen (GAA)-abgeleitete Peptide der CTL-Epitope verl&auml;ngert das &Uuml;berleben der CNS GL261 Gliom-Lager M&auml;use, die verbunden war mit effizienten CNS Gliom Radarst&ouml;rsender von Antigen-spezifischen CTLs. intratumorale Injektionen von GAA Peptid belasteter DC1s verst&auml;rkt die Anti-CNS Gliom-Auswirkungen der DC1-basierte s.c. Immunisierung. Interessanterweise wurden die antitumor-Funktionen zusammen mit CXCL10(-/-) Maus-abgeleitete DC1s. aufgehoben diese Ergebnisse zeigen die Anti-CNS Gliom-Auswirkungen der Therapie auf der Basis von DC1 und eine neuartige Rolle der CXCL10 in der immunologischen und therapeutische Aktivit&auml;t von Impfstoffen gegen Krebs DC-basierte.

Wirksame Immuntherapie gegen murine Gliomen mit Typ 1 polarisierende dendritischen Zellen--wesentliche Rollen von CXCL10.

Sepsis is characterized by a systemic inflammatory response caused by infection, and can result in organ failure and death. Removal of inflammatory mediators such as cytokines from the circulating blood is a promising treatment for severe sepsis. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using biocompatible, polymer sorbent beads. In this study, we used confocal laser scanning microscopy (CLSM) to directly examine adsorption dynamics of a cytokine (IL-6) within hemoadsorption beads. Fluorescently labeled IL-6 was incubated with sorbent particles, and CLSM was used to quantify spatial adsorption profiles of IL-6 within the sorbent matrix. IL-6 adsorption was limited to the outer 15 microm of the sorbent particle over a relevant clinical time period, and intraparticle adsorption dynamics was modeled using classical adsorption/diffusion mechanisms. A single model parameter, alpha = q(max) K/D, was estimated by fitting CLSM intensity profiles to our mathematical model, where q(max) and K are Langmuir adsorption isotherm parameters, and D is the effective diffusion coefficient of IL-6 within the sorbent matrix. Given the large diameter of our sorbent beads (450 microm), less than 20% of available sorbent surface area participates in cytokine adsorption. Development of smaller beads may accelerate cytokine adsorption by maximizing available surface area per bead mass.

IL-6 adsorption dynamics in hemoadsorption beads studied using confocal laser scanning microscopy.

Sepsis ist gekennzeichnet durch eine systemische Entz&uuml;ndungsreaktion, die durch Infektion verursacht und Organversagen und Tod f&uuml;hren kann. Entfernung von entz&uuml;ndlichen Mediatoren wie Zytokine aus dem zirkulierenden Blut ist eine vielversprechende Behandlung bei schwerer Sepsis. Wir entwickeln eine extrakorporale Hemoadsorption Ger&auml;t zum Entfernen von Zytokinen aus dem Blut mit biokompatiblen, Polymer Sorbens Perlen. In dieser Studie haben wir konfokalen Laser scanning Mikroskopie (CLSM) um Adsorption Dynamik von einem Cytokine (IL-6) in Hemoadsorption Perlen direkt zu untersuchen. Leuchtstoff mit der Bezeichnung IL-6 wurde mit Sorbens Teilchen inkubiert und CLSM wurde verwendet, um r&auml;umliche Adsorption Profile von IL-6 innerhalb der Sorbens Matrix quantifizieren. IL-6-Adsorption beschr&auml;nkte sich auf die &auml;u&szlig;eren 15 Microm des Sorbens Teilchen einen relevanten klinischen Zeitraum und Intraparticle Adsorption Dynamik war mit klassischen Adsorption/Verbreitung Mechanismen modelliert. Ein einzelnes Modell-Parameter, alpha = q(max) K/D, wurde durch den Einbau CLSM Intensit&auml;t Profile, um unser mathematisches Modell, wo q(max) und K Langmuir-Adsorptions-Isotherme-Parameter, und D ist die effektive Diffusionskoeffizient von IL-6 innerhalb der Sorbens Matrix gesch&auml;tzt. Angesichts den gro&szlig;en Durchmesser der unsere Sorbens Perlen (450 Microm), beteiligt weniger als 20 % der verf&uuml;gbaren Sorbens Fl&auml;che sich Cytokine Adsorption. Entwicklung von kleineren Perlen kann Cytokine Adsorption durch die Maximierung der verf&uuml;gbaren Fl&auml;che pro Perlen beschleunigen mass.

IL-6-Adsorption Dynamik im Hemoadsorption Perlen mit der konfokalen Laser scanning Mikroskopie untersucht.

The LIM-domain containing transcription factor, Lhx1, is involved in the regulation of early gastrulation cell movements, kidney organogenesis and other processes in vertebrate model organisms. To follow the expression of this gene in live embryos, we created transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of lhx1a regulatory regions. Tg(lhx1a:EGFP)(pt303) recapitulates the expression of endogenous lhx1a beginning at early gastrula stages through 72 hours of development with only few exceptions. In addition, over-expression of the Nodal ligand, ndr1, results in the concomitant expansion of the transgene and endogenous lhx1a expression. Treatment of Tg(lhx1a:EGFP)(pt303) embryos with the small molecule SB-431542, an inhibitor of Nodal signaling, results in the loss of both transgene and endogenous lhx1a expression. These experiments suggest that Tg(lhx1a:EGFP)(pt303) is regulated in a manner similar to endogenous lhx1a. Therefore, this reporter can be utilized not only for monitoring lhx1a expression, but also for numerous applications, including chemical genetics screening.

Characterization of an lhx1a transgenic reporter in zebrafish.

LIM-Dom&auml;ne enthaltenden Transkriptionsfaktors, Lhx1, ist die Regulierung der fr&uuml;hen Gastrulation Zelle Bewegungen, Niere Organogenese und andere Prozesse in vertebrate Modellorganismen beteiligt. Um den Ausdruck des Gens in live Embryonen zu folgen, haben wir unter der Kontrolle der lhx1a regelnde Regionen transgene Zebrafisch Ausdr&uuml;cken verbessertes gr&uuml;nes fluoreszierendes Protein (EGFP) erstellt. TG(lhx1a:EGFP)(pt303) rekapituliert den Ausdruck des endogenen lhx1a Anfang im Fr&uuml;hstadium der Gastrula bis 72 Stunden nach der Entwicklung mit nur wenigen Ausnahmen. Dar&uuml;ber hinaus f&uuml;hrt &Uuml;berexpression des Nodal Liganden, ndr1, die damit einhergehende Erweiterung des Transgens und endogene lhx1a Ausdruck. Behandlung von Tg(lhx1a:EGFP)(pt303) Embryonen mit dem kleinen Molek&uuml;l SB-431542, ein Inhibitor der Nodal Signalling, f&uuml;hrt zum Verlust des Transgens und endogene lhx1a Ausdruck. Diese Experimente deuten darauf hin, dass Tg(lhx1a:EGFP)(pt303) &auml;hnlich wie bei endogenen lhx1a geregelt ist. Daher kann diese Reporter nicht nur f&uuml;r die &Uuml;berwachung der lhx1a Ausdruck, sondern auch f&uuml;r zahlreiche Anwendungen, einschlie&szlig;lich Chemische Genetik Screening genutzt werden.

Charakterisierung eines transgenen lhx1a-Reporter im Zebrafisch.

Recent findings have implicated tight junction (TJ) protein Occludin (OCLN) as an essential factor for hepatitis C virus (HCV) to enter human hepatocytes. To gain insights into OCLN-mediated HCV entry, we created a panel of OCLN deletion mutants and found that without impairing OCLN's cell surface localization, removal of the extracellular loop 2 (EL2) from OCLN abolished both its ability to mediate HIV-HCV pseudotypes' (HCVpp) entry as well as its ability to coprecipitate HCV glycoprotein E2. Recombinant OCLN EL2, however, failed to robustly bind soluble E2 (sE2) in pull-down assays. Subsequent studies revealed that OCLN formed complex with Dynamin II, an important GTPase for endocytosis, in an EL2-dependent fashion. HCVpp, as well as cell culture grown HCV (HCVcc), was sensitive to Dynamin knockdown or inhibition. We conclude that OCLN EL2 dictates the Dynamin-dependent HCV entry. Furthermore, OCLN could function to bridge virions to Dynamin-dependent endocytic machineries.

The second extracellular loop dictates Occludin-mediated HCV entry.

Neuere Befunde haben tight Junction (TJ) Protein verwickelt Occludin (OCLN) als einen wesentlichen Faktor f&uuml;r Hepatitis-C-Virus (HCV) menschliche Hepatozyten eingeben. Um tiefere Einblicke in OCLN-vermittelte HCV-Eintrag, wir erstellt ein Panel von OCLN L&ouml;schung Mutanten und festgestellt, dass ohne Beeintr&auml;chtigung OCLNs Zelle Oberfl&auml;che Lokalisierung, Entfernung der extrazellul&auml;re Schleife 2 (EL2) aus OCLN beide seine F&auml;higkeit zur HIV-HCV-Pseudotypes (HCVpp) vermitteln abgeschafft Einreise sowie seine F&auml;higkeit, HCV-Glykoprotein E2 coprecipitate. Rekombinante OCLN EL2, konnte jedoch robust binden l&ouml;sliche E2 (sE2) bei Pulldown-Assays. Sp&auml;tere Studien ergab, dass OCLN Komplex mit Dynamin II, eine wichtige GTPase f&uuml;r Endozytose, EL2-abh&auml;ngigen Weise gebildet. HCVpp sowie Zelle Kultur angebaut HCV (HCVcc), war empfindlich auf Dynamin Niederschlag oder Hemmung. Wir schlie&szlig;en, dass OCLN EL2 Dynamin-abh&auml;ngige HCV-Eintrag diktiert. Dar&uuml;ber hinaus konnte OCLN funktionieren, um Virionen zu Dynamin-abh&auml;ngige endocytic Maschinerien zu &uuml;berbr&uuml;cken.

Die zweite extrazellul&auml;re Schleife diktiert Occludin-vermittelte HCV-Eintrag.

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4RÎ±/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c(+) alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.

Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.

Dendritische Zellen (DCs) sind die potentesten APCs. W&auml;hrend unreife DCs-ab-T-Zell-Antworten regulieren so induzieren/Pflege der immunologischen Toleranz, F&ouml;rderung Reife DCs Immunit&auml;t. Um ihre Funktionen zu verst&auml;rken, kommunizieren DCs mit benachbarten DCs durch l&ouml;sliche Mediatoren, Zell-Zell-Kontakt und Vesikel Exchange. &Uuml;bertragung von Nanovesicles (&lt; 100 nm), abgeleitet von der endocytic Weg (genannt Exosomes) stellt einen neuen Mechanismus der DC-DC-Kommunikation. Die Tatsache, dass Exosomes Exosome-Shuttle MiRNAs enthalten und DC-Funktionen durch exogene MiRNAs geregelt k&ouml;nnen, schlagen vor, da&szlig; DC / DC-Interaktionen durch Exosome-Shuttle MiRNAs, eine Hypothese vermittelt werden k&ouml;nnten, die noch getestet werden. Wichtig ist, ist der Mechanismus der Transfer von Exosome-Shuttle MiRNAs aus der Exosome-Lumen in das Zytosol der Zielzellen unbekannt. Hier zeigen wir, dass DCs Exosomes mit verschiedenen MiRNAs je nach der Reifung der DCS freigeben. Durch die Visualisierung spontane &Uuml;bertragung von Exosomes zwischen DCs, zeigen wir, dass Exosomes mit den Ziel-DCs, Letzteres verschmolzen, gefolgt von Freigabe des Exosome Inhalts in das Zytosol DC. Exosome-Shuttle MiRNAs sind funktionell, weil sie deiner mRNAs der Akzeptor DCs unterdr&uuml;cken. Unsere Erkenntnisse enth&uuml;llen einen Mechanismus der &Uuml;bertragung von Exosome-Shuttle MiRNAs zwischen DCs und seine Rolle als Mittel der Kommunikation und Posttranscriptionale Regulation zwischen DCs.

Mechanismus der &Uuml;bertragung von funktionalen MicroRNAs zwischen Maus dendritische Zellen &uuml;ber Exosomes.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.

Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography.

Cryo-Elektron Tomographie (CryoET) erm&ouml;glicht die 3D Visualisierung von Zellstrukturen Molekulare Aufl&ouml;sung in einem Ende-zu-nativ-Zustand und hat somit das Potenzial, zur fr&uuml;hen Ereignisse von HIV-1 Infektion in Wirtszellen zu erhellen. Jedoch wurden strukturelle Details f&uuml;r die HIV-1 Infektion nicht, aufgrund der technologischen Herausforderungen im Umgang mit seltenen und dynamische HIV-1-Partikel in den menschlichen Zellen beobachtet. Hier berichten wir Strukturanalyse von HIV-1 und Host-Zell-Interaktionen durch eine Korrelative Hochgeschwindigkeits-3D live-Cell-Imaging und CryoET Methode. Mit dieser Methode, unter fast Native Bedingungen zeigten wir, dass intakte Hyperstable werden mutant HIV-1-Kerne in das Zytoplasma der Wirtszellen abgegeben. Wir erhielten weitere direkte Hinweise darauf, dass ein Hyperstable mutant Kapsid, E45A, zeigte im Vergleich zu Wildtyp-Kapsid verz&ouml;gerte Kapsid-Demontage. Diese Ergebnisse werden gemeinsam die Vorteile unserer Korrelative Leben-Zelle und CryoET Ansatz f&uuml;r imaging-dynamische Prozesse, wie virale Infektion.

Direkte Visualisierung der HIV-1 mit Korrelative Mikroskopie und Cryo-Elektron Tomographie von Leben-Zelle.

Gradual collagen recruitment has been hypothesized as the underlying mechanism for the mechanical stiffening with increasing stress in arteries. In this work, we investigated this hypothesis in eight rabbit carotid arteries by directly measuring the distribution of collagen recruitment stretch under increasing circumferential loading using a custom uniaxial (UA) extension device combined with a multi-photon microscope (MPM). This approach allowed simultaneous mechanical testing and imaging of collagen fibers without traditional destructive fixation methods. Fiber recruitment was quantified from 3D rendered MPM images, and fiber orientation was measured in projected stacks of images. Collagen recruitment was observed to initiate at a finite strain, corresponding to a sharp increase in the measured mechanical stiffness, confirming the previous hypothesis and motivating the development of a new constitutive model to capture this response. Previous constitutive equations for the arterial wall have modeled the collagen contribution with either abrupt recruitment at zero strain, abrupt recruitment at finite strain or as gradual recruitment beginning at infinitesimal strain. Based on our experimental data, a new combined constitutive model was presented in which fiber recruitment begins at a finite strain with activation stretch represented by a probability distribution function. By directly including this recruitment data, the collagen contribution was modeled using a simple Neo-Hookean equation. As a result, only two phenomenological material constants were required from the fit to the stress stretch data. Three other models for the arterial wall were then compared with these results. The approach taken here was successful in combining stress-strain analysis with simultaneous microstructural imaging of collagen recruitment and orientation, providing a new approach by which underlying fiber architecture may be quantified and included in constitutive equations.

A theoretical and non-destructive experimental approach for direct inclusion of measured collagen orientation and recruitment into mechanical models of the artery wall.

ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection.

Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes.

ATM ist eine Proteinkinase, die eine gut charakterisierten signalisierende Kaskade in Zellen, die ionisierende Strahlung (IR) initiiert. Die Rolle f&uuml;r ATM bei der Koordinierung der kritischen Proteininteraktionen und nachtr&auml;glicher Umtausch innerhalb von DNA-Sch&auml;den-Reaktion (DDR)-komplexe ist jedoch unbekannt. Wir kombinierten SILAC-basierten tandem Massenspektrometrie und ein Subzellulare Fraktionierung-Protokoll, das Proteom der bestrahlten Zellen zu verh&ouml;ren behandelt, mit oder ohne der ATM-Kinase-Hemmer KU55933. Wir entwickelten eine integratives Netzwerk-Analyse zu identifizieren und priorisieren Proteine, waren empf&auml;nglich f&uuml;r KU55933, speziell im Chromatin und, die auch f&uuml;r k&ouml;rperliche Interaktionen mit bekannten DNA-Reparatur-Proteinen bereichert wurden. Diese Analyse identifiziert, 53BP1 und annexin A1 (ANXA1) als starke Kandidaten. Einsatz von Fluoreszenz Recovery after Photobleaching, fanden wir, dass der Austausch von GFP-53BP1 in DDR-komplexen mit KU55933 abgenommen. Dar&uuml;ber hinaus fanden wir, dass ANXA1 Niederschlag &uuml;ber einen Mechanismus zu IR Zellen sensibilisiert, die nicht durch KU55933 potenzierte war. Unsere Studie zeigt eine Rolle f&uuml;r ATM-Kinase-Aktivit&auml;t in den dynamischen Austausch von Proteinen im DDR-komplexe und identifiziert eine Rolle f&uuml;r ANXA1 in zellul&auml;ren Strahlenschutz.

Quantitative Proteomik offenbaren ATM Kinase-abh&auml;ngigen Exchange in DNA-Sch&auml;den-Response-komplexen.

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we have identified that inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by hyperfused mitochondrial structure and unscheduled expression of cyclin E in G2 phase. This persistent replication stress then induces an ATM-dependent activation of G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are the causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and ROS generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.

Mitochondrial hyperfusion induced by loss of fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress.

Mitochondriale Spalt- und Zyklen mit Zellzyklusprogression integriert sind. Hier haben wir festgestellt, dass die Hemmung der mitochondrialen Kernspaltung Protein Drp1 G2/M Zellzyklusprogression und Aneuploidie eine unerwartete Verz&ouml;gerung verursacht. Bei der Untersuchung der zugrunde liegenden molekularen Mechanismen, ergeben wir, dass die Hemmung Drp1 Replikation Stress, l&ouml;st die durch mitochondriale Hyperfused-Struktur und ungeplante Ausdruck von cyclin E in G2 Phase vermittelt wird. Dieser anhaltende Replikation Stress induziert dann eine ATM-abh&auml;ngige Aktivierung von G2 M &Uuml;bergang Zellzyklus Checkpoint. Niederschlag von ATR, eine wesentliche Kinase verhindern Replikation Stress, verbessert erheblich DNA-Sch&auml;den und Zelle Tod Drp1-Deficienct-Zellen. Persistente mitochondriale Hyperfusion induziert auch centrosomal Overamplification und chromosomalen Instabilit&auml;t, die die Ursachen der Aneuploidie sind. Mit Zellen ersch&ouml;pft der mitochondrialen DNA Analyse ergab, dass diese Ereignisse nicht von den M&auml;ngeln in mitochondriale ATP-Produktion und ROS-Generation vermittelt werden. So induziert gest&ouml;rte mitochondriale Kernspaltung direkt Genom Instabilit&auml;t durch Replikation Stress, der dann DNA Sch&auml;den Reaktion initiiert. Unsere Ergebnisse stellen einen neuen Mechanismus, der zur zellul&auml;ren Dysfunktion und Krankheiten im Zusammenhang mit ver&auml;nderten mitochondriale Dynamik beitr&auml;gt.

Mitochondriale Hyperfusion induziert durch Verlust von Kernspaltung Protein Drp1 verursacht ATM-abh&auml;ngige G2/M-Verhaftung und Aneuploidie durch DNA-Replikation-Stress.

Article	Year
Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 16713360	2006
Effective immunotherapy against murine gliomas using type 1 polarizing dendritic cells--significant roles of CXCL10. Cancer research\| PubMed ID: 19190335	2009
IL-6 adsorption dynamics in hemoadsorption beads studied using confocal laser scanning microscopy. Journal of biomedical materials research. Part B, Applied biomaterials\| PubMed ID: 19904819	2010
Characterization of an lhx1a transgenic reporter in zebrafish. The International journal of developmental biology\| PubMed ID: 20209443	2010
The second extracellular loop dictates Occludin-mediated HCV entry. Virology\| PubMed ID: 20822789	2010
Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype. PloS one\| PubMed ID: 21246055	2011
Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood\| PubMed ID: 22031862	2012
Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography. Structure (London, England : 1993)\| PubMed ID: 22078557	2011
A theoretical and non-destructive experimental approach for direct inclusion of measured collagen orientation and recruitment into mechanical models of the artery wall. Journal of biomechanics\| PubMed ID: 22305290	2012
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes. Journal of proteome research\| PubMed ID: 22909323	2012
Mitochondrial hyperfusion induced by loss of fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress. Journal of cell science\| PubMed ID: 23015593	2012

Gregory A. Gibson

Biography

JoVE Journal Publications

Other Publications