Advanced confocal microscopy

Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool, facilitating fluores- cence methods such as Fluorescence Correlation Spectroscopy (FCS) or single-molecule Förster Resonance Energy Transfer (FRET). It is heavily used to investigate a wide range of biological problems. This holds true especially for protein properties such as ligand binding, complex formation, conformational changes, or the intracellular distribution of the species in question. In this work, I will describe the assembly of two instruments: The first is a multi- parameter fluorescence detection (MFD) setup. It is a purely spectroscopic tool that offers the capability to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion in free solution. When the molecule of interest is labelled with two fluorophores, additional information, like the energy transfer in-between them, becomes accessible and the correct distance between these two fluorophores can be calculated. If the two fluorophores are attached to different molecules, the MFD setup can detect interactions of these molecules in the range from pM up to μM with the help of Fluorescence Cross-Correlation Spectroscopy (FCCS). The second instrument, a stimulated emission depletion setup, combines some of the mentioned techniques, like FCS, with the superior image capability of a confocal micro- scope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission in order to circumvent the diffraction bar- rier and allows images with a three-fold resolution increase, down to 75nm. These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series, like sodium perchlorate or guanidinium chloride. The results show that certain salts, which are commonly summarized as denaturing agents, achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state. 7 The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). Although highly used in biochemistry and biophysics, for example to read out the expression level of genes, it is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used. In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell di- vision. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appear- ance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS. The last application will utilize the STED setup in order to carry out the first steps towards the investigation of the nucleation and branching behavior of actin in cooperation with the actin related protein 2/3 (ARP2/3). This protein complex preferentially attaches to actin filaments that are located at the leading edge of a cell and forms branched filamentous structures. The exact conditions under which this process occurs are not well characterized. This part of the work will deal with the steps that are necessary to follow the polymerization process on the STED setup

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment

Electrostatics control actin filament nucleation and elongation kinetics.

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods