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Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.

The Federal Company for Radioactive Waste Disposal (BGE) is responsible for the search for a site with the best possible safety for the disposal of high-level radioactive waste in Germany. The site selection procedure is regulated in a law that was adopted by the German Federal Parliament (Repository Site Selection Act – StandAG, 2017, last updated 2020) and aims to be a participatory, transparent, learning, and self-questioning process based on scientific expertise. The first step of the first phase of the site selection procedure was completed in September 2020 and resulted in the identification of sub-areas that give reason to expect favorable geological conditions for the long-term storage of nuclear waste in the subsurface. These sub-areas cover approximately 54 % of Germany and are located in three different host rocks: rock salt – halite, claystone, and crystalline rock. The challenge for the next step is to find suitable siting regions within the previously determined sub-areas that are then considered further in the next phase of the site selection procedure. In the following, the methodology of the so-called representative preliminary safety analyses is described, which constitute one of the tools to identify siting regions, and some first insight on how they are planned to be implemented in practice is given.

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Forster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

Actin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

Actin nucleation is a fundamental biological process involved in several cellular events. Actin filament nucleation requires several hundred nano-molars to micro-molar amounts of polymerizable actin monomers. This requirement makes the impossible use of standard single molecule techniques. Here, we use zero-mode waveguides to reduce the observation volume and monitor, with single molecule resolution, the initial steps in actin filament formation. We immobilized gelsolin, a capping and nucleating protein at the bottom of the waveguides and added up to several micro-molar amounts of fully labeled actin monomers in polymerization conditions. We observe the first steps during filament formation as step-wise increases in detected fluorescence intensity. Mean reaction rates are then estimated for each oligomer size. Arrival time distribution of individual steps reveals the presence of kinetic intermediates. Furthermore, analysis of oligomer size distribution as a function of actin concentration reveals transitions at around 600 and 900 nM where the system dynamics change concomitant with the appearance of filament elongation. The change in dynamics at the transition is associated with an increase in dissociation rates. Such transition is blocked by Latrunculin A, suggesting a functional role for ATP hydrolysis and/or monomer flattening. Our results point towards a gelsolin-mediated actin filament nucleation mechanism where protein-protein interactions trigger ‘nuclei’ instability necessary for filament elongation.