Within my work, it became clear that the pair-wise interactions between Hsp90, nucleotides, co-chaperones and clients do not occur independently, but interfere with each other’s effects by more than just competitive binding. Hence, the complexity of the machine is much higher than usually assumed in interpretations that aimed to describe the complete machine in a model – and the prediction quality of those models is therefore very limited.
It is therefore necessary to cope with the complete system of the Hsp90 chaperone machine in order to predict effects of e.g. inhibitors or mutations in vivo. The reconstitution of the machine in vitrofrom the bottom-up is still challenging, because the numer and exact stoichiometries of the single parts (i.e. co-chaperones, clients)in vivo always depend on the dynamic interplay between a cell and its environment.
A complementary, top-down approach is able to access the complete machine as it is, where it is – in the cell. This is the logical next step towards a more thorough understanding of Hsp90. The challenges and preliminary results of my efforts solving them are briefly presented in the following.
5.1 Challenges for smFRET in living cells
I identified the following challenges to investigations on the dynamics of a protein by smFRET in living cells:
• The imaging of the cell to determine the localization of the studied single molecule.
• The background fluorescence, which impedes the S/N ratio due to the auto-fluorescence of metabolites in a cell that are present at nano- to micromolar concentration and not necessarily isotropically distributed (e.g. NAD(P)H or flavines [179,180]).
• The signal intensity depending on the employed fluorophores. Intrinsic labeling by fluorescent protein labels come with the disadvantage of decreased photo-stability and lower quantum yield, when compared to organic dyes, impeding the S/N ratio.
• Only a very low number of labeled proteins (𝑐 ≈ pM) must be present to ensure separation of single molecules.
• Site-specific labeling with one donor and one acceptor dye must be ensured.
• The diffusion of the protein must be hindered in order to access the dynamics of single proteins on the second to minute timescale.
My work on the issues presented above and and the results are briefly summarized. This work is a collaborative work together with Fernando Aprile-Garcia.21
21 Laboratory of Ritwick Sawarkar at the Max Planck Institute for epigenetics, Freiburg, Germany.
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5.2 Towards smFRET in living cells
To decrease the background fluorescence, I construct a microscope with highly inclined laminar optical sheet (HILO) illumination in the red and near-infrared (NIR) spectrum.22 Auto-fluorescence is mainly observed at wavelengths between 400 and 600 nm [180], thus we use lasers of the wavelengths 635 nm and 728 nm for the excitation of fluorescent dyes.
In HILO illumination, only a section of a few 𝜇m within the cell is illuminated, which additionally reduces background fluorescence.
The fluorescent background in the two channels (R/NIR) differs, with a significant lower background in the NIR channel. The cell nucleus seems in general much better suited for future smFRET studies in vivo, because the background fluorescence is much weaker and homogenous (c.f. Appendix D.3, p.140).
The injection of recombinantly expressed, purified and labeled protein circumvents further problems as detailed in the following. The S/N ratio is increased by employing organic fluorophores. The number of fluorescent molecules is controlled by the respective injection method. Site-specific labeling at two distinct positions can be achieved by the creation of hetero-dimers via monomer-exchange of zipped Hsp90 prior to the insertion into the cell.
Figure 5.1: dsDNA labeled with one Cy7 fluorophore is successfully transfected into a HeLa cell and resolved as single molecule. The cellular and nuclear membrane are indicated in black.
The inset shows the time dependent fluorescence at 10 Hz at the indicated spot in the cytosole.
One single bleaching step indicates that this is indeed a single molecule. A control experiment in absence of transfection agent did not results in such fluorescent spots in the NIR channel.
Further single molecule traces are shown in Fig.D.8on page142.
The organic dyes Atto647N and Cy7 as FRET donor and acceptor fluorophores have been employed in earlier smFRET studies to their relative high photo-stability, high quantum yield and high extinction coefficients [181, 182]. While Atto647N has been characterized in the previous smFRET studies, I find the NIR dyes Cy7 and Alexa750 being functional as FRET acceptors on DNA and yeast Hsp90in vitro (c.f. AppendixD.1 and AppendixD.2 on page 137 ff.). However, the dyes remain photo-stable only in presence of an oxygen scavenger system. The redox potential in a cell implies a much lower oxygen concentration than in buffer solutions and should at least partly substitute for an artificial scavenger system, but scavenger systems for fluorescence experiments in cell is also available [183].
The previously characterized kinetic 4-state model of Hsp90 is successfully reproduced with the FRET pair Atto647N and Alexa750 on different positions of yeast Hsp90 AppendixD.2, proving functionality of the labeled Hsp90.
Furthermore, labeled DNA is successfully transfected into HeLa cells and traces from
22 Detailed in Section2.1.2on page22.
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single molecules can be observed (see Fig. 5.1).
A third excitation color at 458 nm is included in the setup, which is used to excite the green fluorescent protein (GFP) and derivatives. GFP-fusion protein (e.g. GFP-actin) can be constantly expressed within the cell of interest and thereby enable imaging of the whole cell by fluorescence. GFP does not fluoresce at R/NIR wavelengths and thus does not produce crosstalk into the detection channels for smFRET. A stable HeLA cell line expressing GFP-actin fusion protein could be imaged in the GFP detection channel (c.f.
AppendixD.4, p.141). Thus, imaging GFP fluorescence works as sensitive substitute for bright-field imaging.
So far, the remaining challenges are the incorporation of labeled protein into cells, because transfection with the DNA transfection kit produces too much background of protein stuck to the cell membrane and the localization of the protein. While the first can be solved by testing of alternative transfection methods (see also AppendixD.5, p.141), we tackle the latter by biochemical tags for Hsp90. We created an Hsp90 tagged C-terminally with a nucleus localization sequences (NLS) that should direct Hsp90 into the nucleus.
Further localization would be possible by introducing e.g. a DNA binding tag. However, we found already single molecule traces from transfected short double stranded DNA that did not feature a localization tag. This suggests that at least a fraction of inserted molecules might exhibit hindered diffusion and a localization tag is rather optional.
These first results demonstrate that our approach essentially works and thereby opens the route towards the first smFRET study on Hsp90 in vivo.