Publikationen / Publications 2011
F. Feil, S. Naumov, J. Michaelis, R. Valiullin, D. Enke, J. Kärger, and C. Bräuchle
Single-Particle and Ensemble Diffusivities - Test of Ergodicity
Angew. Chem. Intern. Edition 51 (2012) 1152-1155
Angew. Chem. 124 (2012) 1178-1181
A. Muschielok, J. Michaelis
Application of the Nano-Positioning System to the Analysis of Fluorescence Resonance Energy Transfer Networks
Single-molecule fluorescence resonance energy transfer (sm-FRET) has been recently applied to distance and position estimation in macromolecular complexes. Here, we generalize the previously published Nano-Positioning System (NPS), a probabilistic method to analyze data obtained in such experiments, which accounts for effects of restricted rotational freedom of fluorescent dyes, as well as for limited knowledge of the exact dye positions due to attachment via flexible linkers. In particular we show that global data analysis of complete FRET networks is beneficial and that the measurement of FRET anisotropies in addition to FRET efficiencies can be used to determine accurately both position and orientation of the dyes. This measurement scheme improves localization accuracy substantially, and we can show that the improvement is a consequence of the more precise information about the transition dipole moment orientation of the dyes obtained by FRET anisotropy measurements. We discuss also rigid body docking of different macromolecules by means of NPS, which can be used to study the structure of macromolecular complexes. Finally, we combine our approach with common FRET analysis methods to determine the number of states of a macromolecule.
Journal physical chemistry B 115 (2011) 11927-11937
B. Treutlein, J. Michaelis
Direct Observation of Single RNA Polymerase Processing through a Single Endogenous Gene in a Living Yeast Cell
Rapid advances in live-cell imaging have now enabled direct observation of the transcription of single nascent mRNA molecules from an endogenous yeast gene. A novel quantitative fluctuation analysis of fluorescently labeled mRNA revealed the kinetics of transcription initiation and the dynamics of elongation and termination (see picture; GFP=green fluorescent protein, PP7 is a bacteriophage coat protein, RNAPII=RNA polymerase II, TF=transcription factor).
Angew. Chem. Int. Ed. 50 (2011) 9788-9790
Direkte Beobachtung einzelner RNA-Polymerasen beim Ablesen eines endogenen Gens in einer lebenden Hefezelle
Neueste technologische Fortschritte in der Abbildung lebender Zellen haben es nun ermöglicht, die Entstehung einzelner mRNA-Moleküle bei der Transkription eines endogenen Hefegens zu beobachten. Eine quantitative Fluktuationsanalyse der fluoreszenzmarkierten mRNA-Moleküle gibt Einblicke in die Kinetik der Transkriptionsinitiation und die Dynamik von mRNA-Verlängerung und Termination (siehe Bild; GFP = Grün fluoreszierendes Protein, PP7 ist ein Bakteriophagen-Hüllprotein, RNAPII = RNA Polymerase II, TF = Transkriptionsfaktor).
Angew. Chem. 123 (2011) 9962-9964
D. Grohmann, J. Nagy, A. Chakraborty, D. Klose, D. Fielden, R.H. Elbright, J. Michaelis, and F. Werner
The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation
TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP ‘‘clamp’’ region as a regulatory hot spot for both transcription initiation and transcription elongation.
Molecular Cell 43 263-274, July 22,2011
Molecular Cell, Abstract
J.R. Moffitt, C. Osseforth, and J. Michaelis
Time-gating improves the spatial resolution of STED microscopy
Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.
Optics Express 19 (2011) 4242-4254
T. Lebold, J. Michaelis, and C. Bräuchle
The complexity of mesoporous silica nanomaterials unravelled by single molecule microscopy
Mesoporous silica nanomaterials are a novel class of materials that offer a highly complex porous network with nanometre-sized channels into which a wide amount of differently sized guests can be incorporated. This makes them an ideal host for various applications for example in catalysis, chromatography and nanomedicine. For these applications, analyzing the host properties and understanding the complicated host–guest interactions is of pivotal importance. In this perspective we review some of our recent work that demonstrates that single molecule microscopy techniques can be utilized to characterize the porous silica host with unprecedented detail. Furthermore, the single molecule studies reveal sample heterogeneities and are a highly efficient tool to gain direct mechanistic insights into the host–guest interactions. Single molecule microscopy thus contributes to a thorough understanding of these nanomaterials enabling the development of novel tailor-made materials and hence optimizing their applicability significantly.
Phys. Chem. Chem. Phys., DOI: 10.1039/c0cp02210a
Jiang, X., Musyanovych, A., Röcker, C., Landfester, K., Mailänder, V., Nienhaus, G. U.
Specific Effects of Surface Carboxyl Groups on Anionic Polystyrene Particles in their Interactions with Mesenchymal Stem Cells
Nanoscale 3 (2011) 2028-2035
Lunov, O., Zablotskii, V., Syrovets, T. Röcker, C., Tron, K., Nienhaus, G. U., & Simmet, T.
Modeling Receptor-mediated Endocytosis of Polymer-functionalized Iron Oxide Nanoparticles by Human Macrophages
Biomaterials 32 (2011) 547-55
Wacker, S. A., Alvarado, C., von Wichert, G., Knippschild, U., Wiedenmann, J., Clauss, K., Nienhaus, G. U., Hameister, H., Baumann, B., Borggrefe, T., Knöchel, W., Oswald, F.
RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J
EMBO J. 30 (2011) 43-56
J. Stigler, F. Ziegler, A. Gieseke, J.C.M. Gebhardt, and M. Rief
The Complex Folding Network of Single Calmodulin Molecules
Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.
Science 28 (2011) 512-516