High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy

Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.     

RoGFP1 is a quantitative biosensor in maize cells for cellular redox changes caused by environmental and endogenous stimuli

Reduction–oxidation-sensitive green fluorescent proteins (roGFPs) have been demonstrated to be valuable tools in sensing cellular redox changes in mammalian cells and model plants, yet have not been applied in crops such as maize. Here we report the characteristics of roGFP1 in transiently transformed maize mesophyll protoplasts in response to environmental stimuli and knocked-down expression of ROS-scavenging genes. We demonstrated that roGFP1 in maize cells ratiometrically responds to cellular redox changes caused by H₂O₂ and DTT, as it does in mammalian cells and model plants. Moreover, we found that roGFP1 is sensitive enough to cellular redox changes caused by genetic perturbation of single ROS genes, as exemplified by knocked-down expression of individual ZmAPXs, in maize protoplasts under controlled culture conditions and under stress conditions imposed by H₂O₂ addition. These data provide evidence that roGFP1 functions in maize cells as a biosensor for cellular redox changes triggered by genetic lesion of single ROS genes even under stress conditions, and suggest a potential application of roGFP1 in large-scale screening for maize mutants of ROS signaling involved in development and stress resistance.     

Measuring E(GSH) and H2O2 with roGFP2-based redox probes

Redox biochemistry plays an important role in a wide range of cellular events. However, investigation of cellular redox processes is complicated by the large number of cellular redox couples, which are often not in equilibrium with one another and can vary significantly between subcellular compartments and cell types. Further, it is becoming increasingly clear that different redox systems convey different biological information; thus it makes little sense to talk of an overall "cellular redox state". To gain a more differentiated understanding of cellular redox biology, quantitative, redox couple-specific, in vivo measurements are necessary. Unfortunately our ability to investigate specific redox couples or redox-reactive molecules with the necessary degree of spatiotemporal resolution is very limited. The development of genetically encoded redox biosensors offers a promising new way to investigate redox biology. Recently developed redox-sensitive green fluorescent proteins (roGFPs), genetically fused to redox-active proteins, allow rapid equilibration of the roGFP moiety with a specific redox couple. Two probes based on this principle are now available: Grx1-roGFP2 for the measurement of glutathione redox potential (E(GSH)) and roGFP2-Orp1 for measuring changes in H(2)O(2) concentration. Here we provide a detailed protocol for the use of these probes in both yeast and mammalian systems using either plate-reader- or microscopy-based measurements.     

Live monitoring of plant redox and energy physiology with genetically encoded biosensors

Genetically encoded biosensors pave the way for understanding plant redox dynamics and energy metabolism on cellular and subcellular levels.

ADVANCES

• Methodological advances in fluorescent protein-based in vivo biosensing have been instrumental for several paradigm shifts in our understanding of cell physiology, metabolism and signaling.
• An increasing number of genetically encoded biosensors has been used to dissect the dynamics of several distinct redox couples and energy physiology in plants.
• In vivo monitoring using biosensors has pioneered the simultaneous read-out of different physiological parameters in different subcellular locations by parallelized plate reader-based, multiwell fluorimetry, or expression strategies for multiple sensors in parallel.
• Sensing dynamic changes in hydrogen peroxide levels is possible with sensors of the HyPer family, or roGFP fusion variants with a thiol peroxidase.
• Peredox and SoNar family sensors enable direct visualization of NADH/NAD⁺, while iNAP family sensors respond to NADPH concentration in plants.
• Sensor variants with different sensitivity ranges enable use of the most appropriate variant for the specific in vivo environment or experimental scope.

     

Monitoring Oxidative Stress and DNA Damage Induced by Heavy Metals in Yeast Expressing a Redox-Sensitive Green Fluorescent Protein

Reactive oxygen species (ROS) has been proposed to play an important role in heavy metal-associated toxicity and pathology. Conventional methods for determining ambient redox state in cells are usually labor-intensive, precluding real-time or single-cell monitoring changes in intracellular redox poise resulting from either metabolic processes or environmental influences. Redox-sensitive green fluorescent protein (roGFP) was expressed in Saccharomyces cerevisiae and recombinant cells were evaluated in monitoring the changes in the redox state of living cells when challenged with toxicologically relevant metal ions. roGFP expressed in yeast responded not only to typical membrane-permeant oxidants and reductants, but also to toxicological metal ion-induced intracellular redox changes. Moreover, exposure of yeast cells to NaAsO₂ or Pb(NO₃)₂ at concentrations that induced redox changes reported by roGFP caused up to two- to three-fold increases in DNA mutation frequency. This mutagenic effect was largely caused by oxidative stress since blocking the production of hydryl radicals significantly reduced the mutation rate as well as delayed the cell death.     

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.     

Real-time imaging of the intracellular glutathione redox potential

Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E(GSH)) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

In vivo Architecture of the Polar Organizing Protein Z (PopZ) Meshwork in the Alphaproteobacteria Magnetospirillum gryphiswaldense and Caulobacter crescentus

The polar organizing protein Z (PopZ) forms a polar microdomain that is inaccessible to larger macromolecules such as ribosomes, and selectively sequesters proteins crucial for cell cycle control and polar morphogenesis in various Alphaproteobacteria. However, the in vivo architecture of this microdomain has remained elusive. Here, we analyzed the three-dimensional ultrastructural organization of the PopZ network in Magnetospirillum gryphiswaldense and Caulobacter crescentus by Volta phase plate cryo-electron tomography, which provides high spatial resolution and improved image contrast. Our results suggest that PopZ forms a porous network of disordered short, flexible, and branching filaments.     

OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (E_GSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) and roGFP2-Orp1 (for H₂O₂) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Methyl viologen-induced photo-oxidative stress increases hydrogen peroxide and oxidation of glutathione in chloroplasts, cytosol, and mitochondria, as well as autonomous oxidation in mitochondria.     

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80?K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae

Genetically encoded fluorescent H₂O₂ probes continue to advance the field of redox biology. Here, we compare the previously established peroxiredoxin-based H₂O₂ probe roGFP2-Tsa2ΔC_R with the newly described OxyR-based H₂O₂ probe HyPer7, using yeast as the model system. Although not as sensitive as roGFP2-Tsa2ΔC_R, HyPer7 is much improved relative to earlier HyPer versions, most notably by ratiometric pH stability. The most striking difference between the two probes is the dynamics of intracellular probe reduction. HyPer7 is rapidly reduced, predominantly by the thioredoxin system, whereas roGFP2-Tsa2ΔC_R is reduced more slowly, predominantly by the glutathione system. We discuss the pros and cons of each probe and suggest that future side-by-side measurements with both probes may provide information on the relative activity of the two major cellular reducing systems.     

The thioredoxin-mediated recycling of Arabidopsis thaliana GRXS16 relies on a conserved C-terminal cysteine

Background

Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate iron?sulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a ¹⁵⁸CGFS signature.

Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer

The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.     

Dynamics of the Bacterial Intermediate Filament Crescentin In Vitro and In Vivo

Background

Crescentin, the recently discovered bacterial intermediate filament protein, organizes into an extended filamentous structure that spans the length of the bacterium Caulobacter crescentus and plays a critical role in defining its curvature. The mechanism by which crescentin mediates cell curvature and whether crescentin filamentous structures are dynamic and/or polar are not fully understood.

Methodology/Principal Findings

Using light microscopy, electron microscopy and quantitative rheology, we investigated the mechanics and dynamics of crescentin structures. Live-cell microscopy reveals that crescentin forms structures in vivo that undergo slow remodeling. The exchange of subunits between these structures and a pool of unassembled subunits is slow during the life cycle of the cell however; in vitro assembly and gelation of C. crescentus crescentin structures are rapid. Moreover, crescentin forms filamentous structures that are elastic, solid-like, and, like other intermediate filaments, can recover a significant portion of their network elasticity after shear. The assembly efficiency of crescentin is largely unaffected by monovalent cations (K⁺, Na⁺), but is enhanced by divalent cations (Mg²⁺, Ca²⁺), suggesting that the assembly kinetics and micromechanics of crescentin depend on the valence of the ions present in solution.

Conclusions/Significance

These results indicate that crescentin forms filamentous structures that are elastic, labile, and stiff, and that their low dissociation rate from established structures controls the slow remodeling of crescentin in C. crescentus.