Seminar for PhD students - Max-Planck-Institut für biophysikalische ...

Weitere Magazine

Empfehlungen

Info

Fig. 3: The m-AAA protease processes the cytochrome c peroxidase preferentially at the inner boundary membrane of yeast mitochondria. (a) The submitochondrial distribution of the m-AAA protease was analyzed by fluorescence microscopy in genetically enlarged mitochondria (left) and by quantitative immuno-electron microscopy (right). Thereby, an enrichment of the m-AAA protease at the inner boundary membrane was observed. (b) Processing of the cytochrome c peroxidase (Ccp1) occurs preferentially at the inner boundary membrane. In cells lacking a functional m-AAA protease, precursor Ccp1 (pCcp1) is not further processed. We found that pCcp1 is enriched at the inner boundary membrane (upper panel). Only upon proteolytic processing by the m-AAA protease, the mature Ccp1 is released into the intermembrane space and evenly distributed (lower panel). (c) Model of the import and proteolytic processing of Ccp1. (1) pCcp1 is imported into mitochondria by the TOM complex. (2) The TIM23 complex inserts pCcp1 into the inner membrane. (3) After proteolytic processing by the m-AAA protease, mature Ccp1 is released into the intermembrane space. (4) In the absence of a functional m-AAA protease, pCcp1 is accumulated in the inner boundary membrane. Scale bars: 3 µm (light microscopy), 200 nm (electron microscopy). Abb. 3: Die proteolytische Prozessierung der Cytochrom c-Peroxidase (Ccp1) durch die m-AAA-Protease findet vorrangig an der inneren Grenzflächenmembran statt. (a) Die submitochondriale Verteilung der m-AAA-Protease wurde lichtmikroskopisch in genetisch vergrösserten Mitochondrien (links) und mittels quantitativer Immuno-Elektronenmikroskopie (rechts) analysiert. Dabei wurde eine Anreicherung der m-AAA-Protease in der inneren Grenzflächenmembran festgestellt. (b) Die Prozessierung der Cytochrom c-Peroxidase findet vorrangig an der inneren Grenzflächenmembran statt. In Zellen, die über keine funktionelle m-AAA-Protease verfügen, ist die unprozessierte Vorstufe von Ccp1 (pCcp1) in der inneren Grenzflächenmembran lokalisiert (obere Reihe). Erst nach der Prozessierung wird das maturierte Ccp1 in den Intermembranraum freigesetzt, in dem es sich verteilt (untere Reihe). (c) Modell des Imports und der proteolytischen Prozessierung von Ccp1. (1) pCcp1 wird durch den TOM-Komplex in die Mitochondrien importiert. (2) Der TIM23-Komplex inseriert pCcp1 in die innere Membran. (3) Nach der proteolytischen Prozessierung wird das fertige Ccp1 in den Intermembranraum entlassen. (4) Ohne funktionelle m-AAA-Protease wird pCcp1 nicht prozessiert, und es kommt zu einer Akkumulation von pCcp1 in der inneren Grenzflächenmembran. Größenstandard: 3 µm (Lichtmikroskopie), 200 nm (Elektronenmikroskopie). Fig. 2: Sketch of a mitochondrion. Mitochondria are built up of two membranes: A rather smooth outer membrane (grey) envelopes the organelle; underneath, the highly folded inner membrane (green) is located. It surrounds the largest reaction room of the mitochondrion, the mitochondrial matrix (blue). The inner membrane can be subdivided into two domains: The inner boundary membrane that parallels the outer membrane and the cristae membrane that builds up the characteristic infoldings called cristae. Abb. 2: Aufbau eines Mitochondriums. Mitochondrien besitzen zwei Membranen: Die glatte Außenmembran (grau) begrenzt das Organell. Darunter befindet sich die stark gefaltete Innenmembran (grün). Diese umgibt den größten Reaktionsraum der Mitochondrien, die mitochondriale Matrix (blau). Die Innenmembran kann morphologisch in zwei Bereiche untergliedert werden: die innere Grenzflächenmembran, welche der Außenmembran anliegt, und die Cristaemembran, welche die für Mitochondrien charakteristischen Einstülpungen, die Cristae, bildet. Seite 2
diffraction-limited resolution of fluorescence microscopy of around 250 nm. Hence conventional light microscopy cannot be used to study sub-mitochondrial protein distributions in wild-type cells. Nonetheless, recently two studies using the budding yeast Saccharomyces cerevisiae as a model organism demonstrated conclusively that the IBM and the CM have different protein compositions. Using live cell fluorescence microscopy in conjunction with genetically enlarged mitochondria (a detour to circumvent the problems associated with the diffraction barrier), we (Wurm and Jakobs, 2006) and Andreas Reichert and colleagues with quantitative immunogold electron microscopy (Vogel et al., 2006) demonstrated different protein compositions of the IBM and the CM. However, these initial studies primarily concentrated on proteins involved in protein import and oxidative phosphorylation. Hence we next focused on other cellular processes to analyze differences in the functions of both domains of the inner membrane. The m-AAA protease processes the cytochrome c peroxidase preferentially at the IBM Many proteins of the inner membrane assemble into large protein complexes. Such assembly processes require extensive quality control mechanisms to avoid the accumulation of deleterious malfolded or misassembled proteins. We determined Fig. 4: isoSTED nanoscopy enables the visualization of protein distributions within mitochondria of intact mammalian cells. (a) Distribution of the TOM complex. Shown is a confocal image (at the sides) and the corresponding isoSTED image (middle) taken at the center of the mitochondria of a PtK2 cell. The cell was decorated with antibodies against Tom20. The isoSTED image reveals that the TOM complexes line the envelope of the organelles (b) Two-color isoSTED imaging. The cells were decorated with antisera against Tom20 and mtHsp70. The proteins have distinct distributions. Displayed is an optical section (thickness ~50 nm) at the equatorial planes of the mitochondria. Scale bars, 500 nm (a) and 1 µm (b). Abb. 4: isoSTED-Nanoskopie ermöglicht die Abbildung von Proteinverteilungen innerhalb der Mitochondrien von Säugerzellen. (a) Verteilung des TOM-Komplexes. Gezeigt ist eine konfokale Abbildung (an den Seiten) und die entsprechende isoSTED-Abbildung (in der Mitte) der Mitochondrien einer PtK2-Zelle. Die Zelle wurde mit Antikörpern gegen das Protein Tom20 markiert. Das isoSTED-Bild zeigt, dass die TOM-Komplexe in der äußeren Membran aufgereiht sind. (b) Zweifarben-isoSTED-Nanoskopie. Die Zellen wurden mit Antikörpern gegen Tom20 und mtHsp70 markiert. Die isoSTED- Bilder belegen, dass beide Proteine unterschiedliche Verteilungen haben. Abgebildet ist ein optischer Schnitt (Dicke ~ 50 nm) in der Mitte der Mitochondrien. Größenstandards, 500 nm (a) und 1 µm (b). the inner membrane distribution of a central component of the proteolytic control system, namely the m-AAA protease, and its substrate cytochrome c peroxidase (Ccp1) within budding yeast mitochondria (Suppanz et al., 2009). For this study, we used the approaches mentioned above, namely fluorescence microscopy in conjunction with genetically enlarged mitochondria and quantitative immunogold electron microscopy. The m-AAA protease is a hetero-oligomeric complex composed of the homologous subunits AFG3L2 and paraplegin in humans and Yta10 (Afg3) and Yta12 (Rca1) in yeast. We found that both subunits of the m-AAA protease are preferentially, but not exclusively, localized in the IBM (Fig. 3a). Likewise, the membrane-anchored precursor form of Ccp1 accumulates in the IBM of mitochondria lacking a functional m-AAA protease (Fig. 3b, top). Only upon proteolytic cleavage the mature form mCcp1 moves into the cristae space (Fig. 3b, bottom). The preferential localization of the m-AAA protease in the IBM points to the intriguing idea that the insertion, assembly, and quality control of the proteins of the mitochondrial inner membrane may predominantly take place in the IBM, Seite 3 suggesting that the functional differences of the IBM and the CM are more significant than previously anticipated. Concomitantly this study clearly demonstrated the limitations of the utilized methods for analyzing submitochondrial structures, most notably the time-consuming and painstaking sample preparation for electron microscopy, the difficulties when using two labels and the lack of a possibility to image submitochondrial dynamics. Many of these problems would be alleviated by light microscopy; however, wild-type mitochondria are too small, even for the best conventional fluorescence microscopes. Resolving inner-mitochondrial protein distributions using isoSTED nanoscopy To facilitate a high (diffraction-unlimited) optical resolution in all three room dimensions at the same time, recently an isoSTED fluorescence nanoscope enabling a nearly spherical focal spot of 40-45 nm (l/16) in diameter was introduced (Schmidt et al., 2008). In the isoSTED nanoscope, the effective focus is produced by overlapping the excitation spot of two opposing oil immersion lenses with a hollow sphere of light for switching off the fluorescence by Stimulated Emission Depletion
Seite 1: Berichte aus den Abteilungen Focusi
Seite 5 und 6: Christian Wurm studied biology at t
Seite 7 und 8: Aktuelle Pressemeldungen Flinke Ver
Seite 9 und 10: Erfolgreiche Zusammenarbeit - Metho
Seite 11 und 12: A winning team - magnetic resonance
Seite 13 und 14: Mehdi Pirouz FG Entwicklungsbiologi
Seite 15 und 16: Publikationen Chinesisches Emeritus
Seite 17 und 18: So haben die Richter geurteilt Aufl
Seite 19 und 20: Prof. H. Jäckle Seite 19 Dr. H. Os
Seite 21 und 22: Doktorandenseminar - Seminar for Ph
Seite 23 und 24: Jörg Mittelstät Physical Biochemi

Seminar for PhD students - Max-Planck-Institut für biophysikalische ...

Sie wollen auch ein ePaper? Erhöhen Sie die Reichweite Ihrer Titel.

Template löschen?

Als Template speichern?