Prohibitins are a highly conserved family of proteins that have been implicated in a variety of functions including mitochondrial stress signalling and housekeeping, cell cycle progression, apoptosis, lifespan regulation and many others. The human prohibitins prohibitin 1 and prohibitin 2 have been proposed to act as scaffolds within the mitochondrial inner membrane, but their molecular organization has remained elusive. Here we determined the molecular organization of the human prohibitin complex within the mitochondrial inner membrane using an integrative structural biology approach combining quantitative western blotting, cryo-electron tomography, subtomogram averaging and molecular modelling. The proposed bell-shaped structure consists of 11 alternating prohibitin 1 and prohibitin 2 molecules. This study reveals an average of about 43 prohibitin complexes per crista, covering 1-3% of the crista membrane area. These findings provide a structural basis for understanding the functional contributions of prohibitins to the integrity and spatial organization of the mitochondrial inner membrane.

Mitochondria contain their own DNA (mtDNA) and a dedicated gene expression machinery. As the mitochondrial dimensions are close to the diffraction limit of classical light microscopy, the spatial distribution of mitochondrial proteins and in particular of mitochondrial mRNAs remains underexplored. Here, we establish single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) to visualize individual mitochondrial mRNA molecules and associated proteins. STED nanoscopy reveals the spatial relationships between distinct mRNA species and proteins such as the RNA granule marker GRSF1, demonstrating adaptive changes in mRNA distribution and quantity in challenged mammalian cells and patient-derived cell lines. Notably, STED-smFISH shows the release of mRNAs during apoptosis, while MINFLUX reveals the folding of the mRNAs into variable shapes, as well as their spatial proximity to mitochondrial ribosomes. These protocols are transferable to various cell types and open new avenues for understanding mitochondrial gene regulation in health and disease.

The study of mitochondria is a formidable challenge for super-resolution microscopy due to their dynamic nature and complex membrane architecture. In this issue, Ren et al. introduce HBmito Crimson, a fluorogenic and photostable mitochondrial probe for STED microscopy and investigate how mitochondrial dynamics influence the spatial organization of mitochondrial DNA.

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1 year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 – 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

AbstractMitochondria are essential cellular organelles, which supply eukaryotic cells with the universal energy carrier adenosine triphosphate. These organelles feature a unique double-membrane architecture, which is formed by a smooth outer membrane and a highly folded inner membrane. Harnessing super-resolution light and electron microscopy, we investigate the role of MICOS, a large mitochondrial protein complex, in determining the complex folding of the inner membrane.

Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microscopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments.

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F1Fo-ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae andde novoformation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F1Fo-ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.

Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microscopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments.

Coordinate-targeted super-resolution techniques distinguish fluorophores within a diffraction-limited zone by differing molecular states (typically on vs. off) that are imprinted with light. In STED microscopy, state contrast is generated via stimulated emission. For strong spatial confinement, this requires high STED light intensities. Here we show that enhanced state contrast can be achieved by combining multiple off-state transitions, specifically by combining stimulated emission with reversible photoswitching in "protected STED". This directly translated into improved resolution and contrast while fluorophores were protected from photobleaching by high STED light intensities by virtue of an additional off-state (J. G. Danzl, S. C. Sidenstein et al., Nature Photonics 10, 122 (2016)). It enabled repeated imaging and 3D-diffraction unlimited imaging of living cells and tissues with a genetically encoded marker.

The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted super-resolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy.

RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

Fusarium graminearum is one of the most destructive pathogens of cereals and a threat to food and feed production worldwide. Infection especially of wheat leads to yield losses and mycotoxin contamination. Deoxynivalenol is a hazardous mycotoxin inhibiting the biosynthesis of proteins. Its production by the fungus is a prerequisite for the colonization of the wheat spike. Combining molecular genetics with bioimaging techniques allows a fascinating insight into the pathobiology of this cereal pathogen.

The fungal pathogen Fusarium graminearum is the most common agent of Fusarium head blight (FHB) in small grain cereals and cob rot of maize. The threat posed by this fungus is due to a decrease in yield and, additionally, mycotoxin contamination of the harvested cereals. Among the mycotoxins, trichothecenes influence virulence of F. graminearum in a highly complex manner that is strongly host- as well as chemotype-specific. The factors inducing mycotoxin production during plant infection are still unknown. To evaluate the induction of the trichothecene pathway, the green fluorescence protein (GFP) gene was fused to the promoter of the TRI5 gene coding for the trichodiene synthase and integrated into the genome by homologous integration. The resulting mutant contains a fully functional TRI5 gene ensuring virulence on wheat and exhibits GFP driven by the endogenous TRI5 promoter. We are now able to monitor the induction of trichothecenes under real-time conditions. To localize the fungus in the plant tissue, the dsRed gene was integrated under constitutive control of the glycerol-3-phosphate dehydrogenase (gpdA) promoter. We are now able to show that, first, induction of GFP as well as trichothecene production in the reporter strain reflects TRI5 induction and trichothecene production in the wild type; second, expression of TRI5 is inducible during growth in culture; and, third, trichothecene production is not uniformly induced during the onset of infection but is tissue specific during fungal infection of wheat.

We investigate secreted virulence factors of F. graminearum, namely trichothecenes and lipases. The first gene of the trichothecene pathway, the trichodiene synthase (Tri5), was disrupted in different F. graminearum wild-type strains, producing high or medium amounts of deoxynivalenol (DON) or nivalenol. All disruption mutants ceased to produce trichothecenes and showed a similar reduced virulence on wheat but unchanged virulence to barley. Nivalenol proved to be a weak virulence factor to maize, whereas DON production displayed no influence on maize infection. Therefore, trichothecenes are host specific virulence factors. Mutants lacking DON colonized only the inoculated wheat spikelets. These spikelets showed heavy cell-wall thickening that seemed to prevent the mutant from growing through the rachis node into the rachis. No such symptoms were found in spikelets inoculated with the wild type. Disruption mutants of FGL1, a secreted lipase, showed similar symptoms on wheat spikelets as the Δtri5 mutants. In contrast to the trichothecene mutants, the lipase mutants were unable to colonize barley and maize, defining the fungal lipase a general virulence factor. In wheat, Δfgl1 mutants were able to colonize the inoculated spikelet but could not protrude into the rachis and the rest of the spike. The lipase-mutants produced a higher amount of DON compared to the wild type during initial infection of wheat spikelets, despite its inability to further colonize the spike. It seems that both virulence factors, lipases and trichothecenes, have to be present for an effective colonization of wheat. To gain further knowledge of the circumstances that lead to mycotoxin production we created a mutant that expresses eGFP under control of the endogenous tri5 promoter as well as a fully functional tri5 gene. To help tracing the fungus on the plant dsRed under constitutive control of the gpd promoter was additionally transformed into the mutant. Now, we are able to monitor trichothecene biosynthesis induction on the plant with cellular resolution.