Anja Seybert

Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re‐localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re‐targeted to alternative sites such as the inner membrane in the presence of site‐specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage‐free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

Correlative light- and electron microscopy with chemical tags.

Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP‐dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading.

Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures

Yeast prions constitute a “protein-only” mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI+], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI+] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo–electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.

Communication between Subunits within an Archaeal Clamp-Loader Complex.

We have investigated the communication between subunits in replication factor C (RFC) from Archaeoglobus fulgidus. Mutation of the proposed arginine finger in the small subunits results in a complex that can still bind ATP but has impaired clamp‐loading activity, a process that normally only requires binding of nucleotide. The small subunit alone forms a hexameric ring that is six‐fold symmetric in the absence of ATP. However, this symmetry is broken when the nucleotide is bound to the complex. A conformational change associated with nucleotide binding may relate to the opening of PCNA rings by RFC during the loading reaction. The structures also reveal the importance of the N‐terminal helix of each subunit at the ATP‐binding site. Analysis of mutant protein complexes containing subunits lacking this N‐terminal helix reveals key distinct regulatory roles during clamp loading that are different for the large and small subunits in the RFC complex.

Structure of RNA polymerase I transcribing ribosomal DNA genes.

RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a ‘connector’ element and an expanded cleft containing the active centre in an inactive conformation. The central bridge helix was unfolded and a Pol-I-specific ‘expander’ element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 Å resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 Å resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium

Mycoplasma genitalium, the causative agent of non‐gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo‐electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub‐tomogram averages of free and cell‐attached NAPs respectively, revealing a tetrameric complex with two‐fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative‐stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.

The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for selective binding of PA over phosphatidylserine (PS). The insertion of the AH into the membrane core renders Opi1 sensitive to the lipid acyl chain composition and provides a means to adjust membrane biogenesis. By rational design of the AH, we tune the membrane-binding properties of Opi1 and control its responsiveness in vivo. Using extensive molecular dynamics simulations, we identify two PA-selective three-finger grips that tightly bind the PA phosphate headgroup while interacting less intimately with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

Cryo-electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium : Terminal organelle of M. genitalium

The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron‐dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double‐spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.

Reprint of "Fiducial-less alignment of cryo-sections" [J. Struct. Biol. 159 (2007) 413-423].

Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.