Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example-a general stress-response-controlled switch to errorprone DNA break (double-strand break, DSB) repair-was suggested to be peculiar to an Escherichia coli F′ conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F − E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation-and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS generalstress-response activator is expressed in unstressed cells, illustrating a stress-response-controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repairdependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment.antibiotic resistance | cancer | genome evolution | rapid evolution | stressinduced mutagenesis H ow, when, and where mutations form underpins understanding pathogen-host interactions, antibiotic resistance, aging, cancer progression and therapy resistance, and evolution generally. Initial models of mutagenesis that drives evolution imagined random stochastic processes, roughly constant with time, and blind to selective environments (1). In contrast, bacterial, yeast, and human cells appear to possess mechanisms that induce mutation pathways specifically during stress, under the control of stress responses (2) (stress-induced mutagenesis or SIM). These pathways suggest mechanisms by which genetic diversity could be generated preferentially when cells are maladapted to their environment (i.e., are stressed), potentially accelerating evolution responsively to environments, a major departure from classic views (1). However, the significance of such mechanisms has been debated, as have the mechanisms themselves.First, mutagenesis associated with the DNA-damage response has been argued to be an unavoidable consequence of induced DNA repair (3, 4), not an evolutionary engine (5, 6). Second, the strongest support for the idea of increas...

Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.

Summary A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.

Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension.

Background: Huntington disease patients show an accumulation of oligomers and fibrillar species of mutant huntingtin (mHTT). Results: Cryoelectron tomography and subvolume averaging visualizes heterogeneous mHTT oligomeric species inside the chaperonin-like CCT5 cavity. Conclusion:The structural basis of mHTT aggregation inhibition by CCT5 is through capping of fibrils and encapsulation of oligomers. Significance: These structural mechanisms inspire the development of new strategies for inhibiting mHTT aggregation.

Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.

Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens ( super SIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, s uper SIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.