Cellular Interior Research Articles

Functional characterization of AVPR2 mutants found in Turkish patients with nephrogenic diabetes insipidus.

Diabetes insipidus is a rare disorder characterized by an impairment in water balance because of the inability to concentrate urine. While central diabetes insipidus is caused by mutations in the AVP, the reason for genetically determined nephrogenic diabetes insipidus can be mutations in AQP2 or AVPR2. After release of AVP from posterior pituitary into blood stream, it binds to AVPR2, which is one of the receptors for AVP and is mainly expressed in principal cells of collecting ducts of kidney. Receptor activation increases cAMP levels in principal cells, resulting in the incorporation of AQP2 into the membrane, finally increasing water reabsorption. This pathway can be altered by mutations in AVPR2 causing nephrogenic diabetes insipidus. In this study, we functionally characterize four mutations (R68W, ΔR67-G69/G107W, V162A and T273M) in AVPR2, which were found in Turkish patients. Upon AVP stimulation, R68W, ΔR67-G69/G107W and T273M showed a significantly reduced maximum in cAMP response compared to wild-type receptor. All mutant receptor proteins were expressed at the protein level; however, R68W, ΔR67-G69/G107W and T273M were partially retained in the cellular interior. Immunofluorescence studies showed that these mutant receptors were trapped in ER and Golgi apparatus. The function of V162A was indistinguishable from the indicating other defects causing disease. The results are important for understanding the influence of mutations on receptor function and cellular trafficking. Therefore, characterization of these mutations provides useful information for further studies addressing treatment of intracellularly trapped receptors with cell-permeable antagonists to restore receptor function in patients with nephrogenic diabetes insipidus.

Microstructure and phase transition of Fe-24Cr-12Co-1.5Si ribbons

Fe-24Cr-12Co-1.5Si ribbons were prepared by simple melt spinning and aging processes, and their microstructure and phase transition were studied. The average grain size of the aged ribbons is ∼4.22 μm, much smaller than that of traditional bulk Fe-Cr-Co alloy. The as-spun ribbons are mainly composed of BCC-type α and FCC-type Cr(M). During aging treatment, the Cr(M) phase firstly partly transforms into (CrSi)0.99(FeCo)1.01 at the initially higher aging temperature, accompanied by the continuous spinodal decomposition of α→α1+α2, and at the lower aging temperature the residual Cr(M) and α turn into Fe(Si,M)2 (M = Cr, Co) and Co2Si rather than (CrSi)0.99(FeCo)1.01. And a new uniform cellular microstructure is formed in the ribbons after multi-stage aging, in which the (Fe,Co)-rich α1 phase is located in the cellular interior, while the Cr-rich α2 phase concentrates in the cellular boundaries.

Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS

The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to ∼80 nm in the cytoplasm of living cells.

Automated patterning and probing with multiple nanoscale tools for single-cell analysis

The nano-manipulation approach that combines Focused Ion Beam (FIB) milling and various imaging and probing techniques enables researchers to investigate the cellular structures in three dimensions. Such fusion approach, however, requires extensive effort on locating and examining randomly-distributed targets due to limited Field of View (FOV) when high magnification is desired. In the present study, we present the development that automates ‘pattern and probe’ particularly for single-cell analysis, achieved by computer aided tools including feature recognition and geometric planning algorithms. Scheduling of serial FOVs for imaging and probing of multiple cells was considered as a rectangle covering problem, and optimal or near-optimal solutions were obtained with the heuristics developed. FIB milling was then employed automatically followed by downstream analysis using Atomic Force Microscopy (AFM) to probe the cellular interior. Our strategy was applied to examine bacterial cells (Klebsiella pneumoniae) and achieved high efficiency with limited human interference. The developed algorithms can be easily adapted and integrated with different imaging platforms towards high-throughput imaging analysis of single cells.

Physicochemical code for quinary protein interactions in Escherichia coli

How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical-chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.

Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells. II. The Influence of Storage.

Maintaining an appropriate inventory of packaged blood products is a critical part of modern medicine. Consequently, the assessment of red blood cell (RBC) functionality is instrumental for the monitoring of the quality of stored RBC (sRBC) in the blood bank. We present a comprehensive study of sRBC lesion kinetics in SAGM (saline, adenine, glucose, mannitol) solution, using microwave dielectric spectroscopy (0.5-50 GHz) and cell deformability. As part of the research, we have isolated the microwave dielectric response of cytoplasmic water in sRBC. The extracted dielectric parameters are sensitive to the age of the cells and, in particular, to the critical moment of transition from discocyte to echinocyte. From the analysis of the dielectric relaxation as a function of storage-duration, we postulate that the behavior is rooted in the delicate interplay between bound and bulk water in the cellular interior. In particular, the microwave dielectric response reflects the moment when the continuous diffusion of oxygen to the cell and the oxygenation of hemoglobin affects the role played by water in the maintenance of cell integrity. These results open a possible new avenue for the noninvasive inspection of stored red blood cells, permitting a true inventory system for the modern blood bank.

G protein Signaling, Journeys Beyond the Plasma Membrane

G proteins classically associated with the 7TM or serpentine receptors generally exist as a heterotrimeric complex consisting of alpha, beta and gamma subunits. These proteins serve as transducers of extracellular signal from plasma membrane to cellular interior. Binding of G protein-coupled receptor (GPCR) with a ligand leads to their activation, followed by that of G alpha, which then dissociates from G beta gamma subunits and initiate downstream effects through plasma membrane-localized effectors. This plasma membrane-restricted view of G proteins was challenged when they were found to be present in various intracellular locations such as endomembranes, cytoskeleton, mitochondria, and nucleus thereby opening up newer spatial domains for the action of these signaling molecules. Many recent studies have addressed the spatiotemporal dynamics underlying this atypical distribution and the G proteins have been shown to undergo activation-dependent as well as activation-independent relocalization. This spatially `directed' targeting of G proteins provides them rapid access to intracellular communication network without relying on diffusible second messengers. Here, we present a consolidated review of the existing knowledge about the presence and physiological roles for G proteins in these atypical locations, along with the mechanistic knowhow presently known about underlying processes.

Diffusive Behavior of Mismatch Repair Protein MSH2 in Cells at Different Stages of Cancer

Cellular interiors present media that are very complex as they encompass a mixture of proteins, organelles, viscoelastic filaments, and chromatin structures in the nuclear interior. The behavior of proteins in such environments reflects the cellular complexity. Here we present the results of studies on the mobility, or diffusion, of free eGFP proteins, and of the important nuclear mismatch repair protein, MSH2. MSH2 was labeled with eGFP and the diffusion of both the free eGFP and MSH2-eGFP was measured using Fluorescence Recovery After Photobleaching (FRAP) on four distinct cell types that act as a model for the neoplastic cancer transformation of human mammary epithelial cells from non-cancerous to metastatic. We obtained several interesting results, such as a significant change in behavior for the first stage of cancer (immortal) compared to other stages. We also observed a significantly lower value for MSH2 diffusion (in both cytoplasm and nucleus) than what would be expected by scaling from the eGFP values (using appropriate size ratios of the two proteins). Diffusion coefficients ranged from 14 to 24 μm2/s for EGFP and from 3 to 7 μm2/s for EGFP-MSH2. This material is based upon work supported by the National Science Foundation under Grant Number 1106105 (KB, KS, and MG). We also thank Glen Marrs, director of the WFU Microscopic Imaging Core Facility, and Anita McCauley, for their help with the confocal microscopy setup.

Crowder-Induced Rigidity in a Multidomain Protein: Insights from Solvation.

Global changes in the conformations of proteins in the presence of macromolecular crowding agents have been well documented. Here, we have used solvation dynamics to monitor the changes in a specific domain of the multidomain protein human serum albumin (HSA) in the presence of various crowders. The solvation probe 6-bromoacetyl-2-dimethylaminonaphthalene was site-specifically attached to the cysteine-34 of domain I of HSA. Analyses of the time-resolved Stokes shift of this probe in the presence of crowding agents revealed a significant retardation of the solvent coordinate, particularly in a crowder-dependent manner. We attribute the observed slowing primarily to the increased internal protein friction in the presence of these polymers, implying considerable stiffness of the protein matrix. We have discussed our findings with regard to recent reports on cellular interiors and have also made an attempt to assess the importance of the physiological concentration of macromolecules in protein dynamics and function.

Urea cycle is enhanced by petit-high pressure carbon dioxide stress in yeast Saccharomyces cerevisiae

ABSTRACTIt has been demonstrated that pasteurized effect on microorganisms of petit-high pressure carbon dioxide (p-HPCD) with long time treatment is similar to the effect of HPCD with short time treatment. The ‘petit-high pressure’ refers to a pressure of 1.5–13 atm (standard atmosphere). After 0.5 MPa of CO2 at 25°C for 2 h treatment, specific growth rate of yeast cells in the logarithm phase was decreased by 50% approximately. Under this condition, our study analyzed transcriptional responses of Saccharomyces cerevisiae through the functional genomic approach. Transcription of 837 open reading frames (ORFs) was altered relative to cells without treatment and 476 ORFs were induced after p-HPCD treatment. These selected genes were then categorized by function of gene product using the Munich Information Centre for Protein Sequences database. Genes involved in ‘metabolism of the urea cycle’ were found to be significantly induced. This enhanced metabolic process could help to remove redundant in cellular interior, thereby decrease the production of . Liberation of H+ ion could be decreased along with the inhibition of dissociation.

Electrostatic Contributions to Protein Quinary Structure.

There are four well-known levels of protein structure: primary (amino acid sequence), secondary (helices, sheets and turns), tertiary (three-dimensional structure) and quaternary (specific protein-protein interactions). The fifth level remains largely undefined because characterization of quinary structure, the transient but essential macromolecular interactions that organize the crowded cellular interior, requires the measurement of equilibrium thermodynamic parameters in living cells. We have overcome this challenge by quantifying the pH-dependence of quinary interactions in living Escherichia coli cells using the B1 domain of protein G (GB1, 6.2 kDa). To accomplish this goal, we buffered the cellular interior and used NMR-detected amide proton exchange to quantify the free energy of unfolding in cells. At neutral pH, the unfolding free energy in cells is comparable to that in buffered solution. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes the protein in cells relative to buffer alone. The data show that electrostatic interactions contribute to quinary structure.

Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level

Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions.Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface.Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets).The data suggest that promiscuous interactions are a major factor influencing protein function in the cell.

Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.

Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.

Correlated and Anticorrelated Domain Movement of Human Serum Albumin: A Peek into the Complexity of the Crowded Milieu.

Protein dynamics in cells have been shown to be markedly different from that in dilute solutions because of the highly crowded cellular interior. The volume exclusion arising from the high concentration of macromolecules present can affect both equilibrium and kinetic processes involving protein conformational changes. While global changes in structure leading to modulations in the stability of the protein have been well-documented, local changes that can have a large bearing on the functional aspects of these biomolecules are rare to come across. Using the multidomain serum protein human serum albumin and a fluorescence resonance energy transfer (FRET)-based approach, with fluorescent reporters in each of its three domains, we, in this article, have provided a detailed mapping of variations in the interdomain distances (as a function of pH) in the presence of five macromolecular crowding agents, differing based on their constituent monomers and average molecular weight(s). From the observation of correlated domain movements for dextran based crowding agents to anticorrelated motion induced by Ficoll 70, and both correlated and anticorrelated action for PEG8000 (PEG8), our results reveal the inherent complexity of a crowded milieu with the serum protein serving as an able sensor for decoding such variations. Differences in the manner in which the macromolecular crowders of similar average molecular weights influence the protein conformational ensemble also provide insights into the possible variations at the molecular level that these polymeric molecules possess. Evidence is presented in support of the fact that for the large molecular weight crowding agents and PEG8, soft interactions predominate over hard sphere potentials. Finally, the nature of domain movements encountered for the serum protein are of immense significance with respect to the function of human serum albumin (HSA) as a prolific binder and transporter of small molecules.

A lightweight, biological structure with tailored stiffness: The feather vane

The flying feathers of birds are keratinous appendages designed for maximum performance with a minimum weight penalty. Thus, their design contains ingenious combinations of components that optimize lift, stiffness, aerodynamics, and damage resistance. This design involves two main parts: a central shaft that prescribes stiffness and lateral vanes which allows for the capture of air. Within the feather vane, barbs branch from the shaft and barbules branch from barbs, forming a flat surface which ensures lift. Microhooks at the end of barbules hold barbs tightly together, providing the close-knit, unified structure of the feather vane and enabling a repair of the structure through the reattachment of un-hooked junctions. Both the shaft and barbs are lightweight biological structures constructed of keratin using the common motif of a solid shell and cellular interior. The cellular core increases the resistance to buckling with little added weight. Here we analyze the detailed structure of the feather barb and, for the first time, explain its flexural stiffness in terms of the mechanics of asymmetric foam-filled beams subjected to bending. The results are correlated and validated with finite element modeling. We compare the flexure of single barbs as well as arrays of barbs and find that the interlocking adherence of barbs to one another enables a more robust structure due to minimized barb rotation during deflection. Thus, the flexure behavior of the feather vane can be tailored by the adhesive hooking between barbs, creating a system that mitigates damage. A simplified three-dimensional physical model for this interlocking mechanism is constructed by additive manufacturing. The exceptional architecture of the feather vane will motivate the design of bioinspired structures with tailored and unique properties ranging from adhesives to aerospace materials. Statement of SignificanceDespite its importance to bird flight, literature characterizing the feather vane is extremely limited. The feather vane is composed of barbs that branch from the main shaft (rachis) and barbules that branch from barbs. In this study, the flexural behavior of the feather barb and the role of barbule connections in reinforcing the feather vane are quantitatively investigated for the first time, both experimentally and theoretically. Through the performed experiments, structure-function relationships within the feather vane are uncovered. Additionally, in the proposed model the sophisticated structure of the barbs and the interlocking mechanism of the feather vane are simplified to understand these processes in order to engineer new lightweight structures and adhesives.

Charged Molecules Modulate the Volume Exclusion Effects Exerted by Crowders on FtsZ Polymerization.

We have studied the influence of protein crowders, either combined or individually, on the GTP-induced FtsZ cooperative assembly, crucial for the formation of the dynamic septal ring and, hence, for bacterial division. It was earlier demonstrated that high concentrations of inert polymers like Ficoll 70, used to mimic the crowded cellular interior, favor the assembly of FtsZ into bundles with slow depolymerization. We have found, by fluorescence anisotropy together with light scattering measurements, that the presence of protein crowders increases the tendency of FtsZ to polymerize at micromolar magnesium concentration, being the effect larger with ovomucoid, a negatively charged protein. Neutral polymers and a positively charged protein also diminished the critical concentration of assembly, the extent of the effect being compatible with that expected according to pure volume exclusion models. FtsZ polymerization was also observed to be strongly promoted by a negatively charged polymer, DNA, and by some unrelated polymers like PEGs at concentrations below the crowding regime. The influence of mixed crowders mimicking the heterogeneity of the intracellular environment on the tendency of FtsZ to assemble was also studied and nonadditive effects were found to prevail. Far from exactly reproducing the bacterial cytoplasm environment, this approach serves as a simplified model illustrating how its intrinsically crowded and heterogeneous nature may modulate FtsZ assembly into a functional Z-ring.

In-cell thermodynamics and a new role for protein surfaces

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

How are molecular crowding and the spatial organization of a biopolymer interrelated.

In a crowded cellular interior, dissolved biomolecules or crowders exert excluded volume effects on other biomolecules, which in turn control various processes including protein aggregation and chromosome organization. As a result of these effects, a long chain molecule can be phase-separated into a condensed state, redistributing the surrounding crowders. Using computer simulations and a theoretical approach, we study the interrelationship between molecular crowding and chain organization. In a parameter space of biological relevance, the distributions of monomers and crowders follow a simple relationship: the sum of their volume fractions rescaled by their size remains constant. Beyond a physical picture of molecular crowding it offers, this finding explains a few key features of what has been known about chromosome organization in an E. coli cell.

Thermodynamics of protein destabilization in live cells

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Hydrogen exchange of disordered proteins in Escherichia coli.

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. We have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY. The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange.