Imaging cellular ultrastructures using expansion microscopy (U-ExM).

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

Adaptive-illumination STED nanoscopy.

The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to ∼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.

A beamline-compatible STED microscope for combined visible-light and X-ray studies of biological matter.

A dedicated stimulated emission depletion (STED) microscope had been designed and implemented into the Göttingen Instrument for Nano-Imaging with X-rays (GINIX) at the synchrotron beamline P10 of the PETRA III storage ring (DESY, Hamburg). The microscope was installed on the same optical table used for X-ray holography and scanning small-angle X-ray scattering (SAXS). Scanning SAXS was implemented with the Kirkpatrick-Baez (KB) nano-focusing optics of GINIX, while X-ray holography used a combined KB and X-ray waveguide optical system for full-field projection recordings at a defocus position of the object. The STED optical axis was aligned (anti-)parallel to the focused synchrotron beam and was laterally displaced from the KB focus. This close proximity between the STED and the X-ray probe enabled in situ combined recordings on the same biological cell, tissue or any other biomolecular sample, using the same environment and mounting. Here, the instrumentation and experimental details of this correlative microscopy approach are described, as first published in our preceding work [Bernhardt et al. (2018), Nat. Commun. 9, 3641], and the capabilities of correlative STED microscopy, X-ray holography and scanning SAXS are illustrated by presenting additional datasets on cardiac tissue cells with labeled actin cytoskeleton.

Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature.

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (µmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 µm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 µm. The size of the bud linearly depends on µmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on µmax. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the µmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

A 9-pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum.

A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.

3D hybrid modelling of vascular network formation.

We develop an off-lattice, agent-based model to describe vasculogenesis, the de novo formation of blood vessels from endothelial progenitor cells during development. The endothelial cells that comprise our vessel network are viewed as linearly elastic spheres that move in response to the forces they experience. We distinguish two types of endothelial cells: vessel elements are contained within the network and tip cells are located at the ends of vessels. Tip cells move in response to mechanical forces caused by interactions with neighbouring vessel elements and the local tissue environment, chemotactic forces and a persistence force which accounts for their tendency to continue moving in the same direction. Vessel elements are subject to similar mechanical forces but are insensitive to chemotaxis. An angular persistence force representing interactions with the local tissue is introduced to stabilise buckling instabilities caused by cell proliferation. Only vessel elements proliferate, at rates which depend on their degree of stretch: elongated elements have increased rates of proliferation, and compressed elements have reduced rates. Following division, the fate of the new cell depends on the local mechanical environment: the probability of forming a new sprout is increased if the parent vessel is highly compressed and the probability of being incorporated into the parent vessel increased if the parent is stretched. Simulation results reveal that our hybrid model can reproduce the key qualitative features of vasculogenesis. Extensive parameter sensitivity analyses show that significant changes in network size and morphology are induced by varying the chemotactic sensitivity of tip cells, and the sensitivities of the proliferation rate and the sprouting probability to mechanical stretch. Varying the chemotactic sensitivity directly influences the directionality of the networks. The degree of branching, and thereby the density of the networks, is influenced by the sprouting probability. Glyphs that simultaneously depict several network properties are introduced to show how these and other network quantities change over time and also as model parameters vary. We also show how equivalent glyphs constructed from in vivo data could be used to discriminate between normal and tumour vasculature and, in the longer term, for model validation. We conclude that our biomechanical hybrid model can generate vascular networks that are qualitatively similar to those generated from in vitro and in vivo experiments.

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield.

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30-40°C), i.e. above optimal temperature (Topt). The maximal specific growth rate (µmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5-40°C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (Yx/glc(true)), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a 'low' (within 5-31°C) and a 'high' (within 33-40°C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31-32°C in step-wise manner and it was accompanied with onset of suppression of µmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26-31°C. This limit is reflected in the predetermined combination of Yx/glc(true), elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31°C) triggers both an increment in mglc and suppression of µmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40°C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

'Domino' systems biology and the 'A' of ATP.

We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP.

Sharper low-power STED nanoscopy by time gating.

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

Nanoscopic spine localization of Norbin, an mGluR5 accessory protein.

BACKGROUND: Norbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution. RESULTS: Here we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin. CONCLUSIONS: Norbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.

Impact of VP1-specific protein sequence motifs on adeno-associated virus type 2 intracellular trafficking and nuclear entry.

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.

Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy.

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.

Integrating a dynamic central metabolism model of cancer cells with a hybrid 3D multiscale model for vascular hepatocellular carcinoma growth.

We develop here a novel modelling approach with the aim of closing the conceptual gap between tumour-level metabolic processes and the metabolic processes occurring in individual cancer cells. In particular, the metabolism in hepatocellular carcinoma derived cell lines (HEPG2 cells) has been well characterized but implementations of multiscale models integrating this known metabolism have not been previously reported. We therefore extend a previously published multiscale model of vascular tumour growth, and integrate it with an experimentally verified network of central metabolism in HEPG2 cells. This resultant combined model links spatially heterogeneous vascular tumour growth with known metabolic networks within tumour cells and accounts for blood flow, angiogenesis, vascular remodelling and nutrient/growth factor transport within a growing tumour, as well as the movement of, and interactions between normal and cancer cells. Model simulations report for the first time, predictions of spatially resolved time courses of core metabolites in HEPG2 cells. These simulations can be performed at a sufficient scale to incorporate clinically relevant features of different tumour systems using reasonable computational resources. Our results predict larger than expected temporal and spatial heterogeneity in the intracellular concentrations of glucose, oxygen, lactate pyruvate, f16bp and Acetyl-CoA. The integrated multiscale model developed here provides an ideal quantitative framework in which to study the relationship between dosage, timing, and scheduling of anti-neoplastic agents and the physiological effects of tumour metabolism at the cellular level. Such models, therefore, have the potential to inform treatment decisions when drug response is dependent on the metabolic state of individual cancer cells.

Parallelized STED fluorescence nanoscopy.

We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m.

Prediction of kinetic parameters from DNA-binding site sequences for modeling global transcription dynamics in Escherichia coli.

The majority of dynamic gene regulatory network (GRN) models are comprised of only a few genes and do not take multiple transcription regulation into account. The models are conceived in this way in order to minimize the number of kinetic parameters. In this paper, we propose a new approach for predicting kinetic parameters from DNA-binding site sequences by correlating the protein-DNA-binding affinities with nucleotide sequence conservation. We present the dynamic modeling of the cra modulon transcription in Escherichia coli during glucose-limited fed-batch cultivation. The concentration of the Cra regulator protein inhibitor, fructose 1,6-bis(phosphate), decreases sharply, eventually leading to the repression of transcription. Total RNA concentration data indicate a strong regulation of transcription through the availability of RNA polymerase. A critical assessment of the results of the model simulations supports this finding. This new approach for the prediction of transcription dynamics may improve the metabolic engineering of gene regulation processes.

Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation.

Stimulated emission depletion (STED) microscopy usually employs a scanning excitation beam that is superimposed by a donut-shaped STED beam for keeping the fluorophores at the periphery of the excitation spot dark. Here, we introduce a simple birefringent device that produces a donut-shaped focal spot with suitable polarization for STED, while leaving the excitation spot virtually intact. The device instantly converts a scanning (confocal) microscope with a co-aligned STED beam into a full-blown STED microscope. The donut can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing subdiffraction resolution images of molecular orientation.

Quantifying cytosolic messenger RNA concentrations in Escherichia coli using real-time polymerase chain reaction for a systems biology approach.

Current messenger RNA (mRNA) quantification methods are sophisticated tools for the analysis of gene regulation. However, these methods are not suitable for more complex quantitative approaches such as the mathematical modeling of the in vivo regulation of transcription where dynamic cytosolic mRNA concentrations need to be taken into consideration. In the current study, the "standard curve method" for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) was extended by including an internal RNA standard. This standard enables transcript losses that occur during the process, as well as variations resulting from nonquantitative processes, to be accounted for. The use of an internal standard yielded transcript concentration estimates that were on average seven times higher than those in cases where an internal standard is omitted. Choosing the cra modulon in Escherichia coli as an example, the method applied shows that the regulation of the Cra protein, as well as the growth rate-dependent regulation, need to be taken into consideration. The new method, which enables the determination of cytosolic mRNA concentrations, allows the quantitative representation of transcriptional dynamics. This is an important aspect of the analysis of the complex interactions of metabolism and regulation and in the application of mathematical modeling for systems biology.

Stochastic simulation of signal transduction: impact of the cellular architecture on diffusion.

The transduction of signals depends on the translocation of signaling molecules to specific targets. Undirected diffusion processes play a key role in the bridging of spaces between different cellular compartments. The diffusion of the molecules is, in turn, governed by the intracellular architecture. Molecular crowding and the cytoskeleton decrease macroscopic diffusion. This article shows the use of a stochastic simulation method to study the effects of the cytoskeleton structure on the mobility of macromolecules. Brownian dynamics and single particle tracking were used to simulate the diffusion process of individual molecules through a model cytoskeleton. The resulting average effective diffusion is in line with data obtained in the in vitro and in vivo experiments. It shows that the cytoskeleton structure strongly influences the diffusion of macromolecules. The simulation method used also allows the inclusion of reactions in order to model complete signaling pathways in their spatio-temporal dynamics, taking into account the effects of the cellular architecture.

Quantification of statin effects on hepatic cholesterol synthesis by transient (13)C-flux analysis.

The present work is the first to deal with the determination of cholesterol synthesis rates in primary rat hepatocytes using transient (13)C-flux analysis. The effects of statins on cholesterol biosynthesis and central carbon fluxes were quantified at a therapeutic concentration of 50 nM atorvastatin using carbon-labeled glutamine. The flux through the cholesterol pathway decreased from 0.27 to 0.08 mmol/l(cv)h in response to the administration of the hypolipidemic drug. Isotopic steady state was reached within 4h in the central carbon metabolism but not in the cholesterol pathway, regardless of whether atorvastatin was administered or not. Marked channeling was observed for the symmetrical tricarboxylic acid cycle intermediates, succinate and fumarate. Non-stationary (13)C-based flux identification delivers both intracellular fluxes and intermediate levels, which was for the first time utilized for investigating systems-level effects of the administered drug by quantifying the flux control of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells.

The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mg L(-1) homogentisate and 84 microg L(-1) geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 microg g(-1)). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound-delta-tocotrienol (15 microg g(-1))-in E. coli.