Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window.

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophile' and 'polyextremophile'; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

In situ electron microscopy studies of calcium carbonate precipitation from aqueous solution with and without organic additives.

For the understanding of mineral formation processes from solution it is important to obtain a deeper insight into the dynamics of crystal growth. In this study we applied for this purpose a novel atmospheric scanning electron microscope that allows the investigation of CaCO3 particle formation in solution under atmospheric conditions with a resolution of approximately 10nm. Furthermore it permits the in situ observation of the dynamics of crystal evolution. With this tool the precipitation of CaCO3 was studied in the absence and presence of additives, namely poly(acrylic acid) and poly(styrene sulfonate-co-maleic acid) which are known to influence the crystal growth rate and morphology. We determined particle growth rates and investigated the formation and dissolution dynamics of an observed transient phase, believed to be amorphous calcium carbonate. This technique also enabled us to study the depletion zones, areas of lower intensity due to reduced ion concentrations. Ion flux rates were obtained from the depletion zone width, which amounted to several µm assuming the formation and dissolution dynamics of amorphous calcium carbonate being the rate determining process. This assumption was confirmed since the obtained fluxes were found to be in good agreement with fluxes derived from the experimentally observed crystal growth rates.

Interaction of HLA-DR and CD74 at the cell surface of antigen-presenting cells by single particle image analysis.

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Stochastic modeling of protein motions within cell membranes.

A simple model in which immobilizing events are imposed onto otherwise free Brownian diffusion [R. Metzler and J. Klafter, Phys. Rep. 339, 1 (2000) and a recent adaptation due to S. Khan and A. M. Reynolds, Physica A 350, 183 (2005)] is shown to encapsulate the peculiar transport characteristics of individual cell receptors within plasma membranes observed in single-particle tracking (SPT) experiments. These characteristics include the occurrence of normal diffusion; non-Gaussian subdiffusion; confined diffusion; a superdiffusive mode of transport that is not due to flow of the membrane or molecular motor attachment; and the occurrence of transitions between these transport modes. Model predictions are shown to be in close agreement with a reanalysis of existing SPT data.

Interaction of HLA-DR with actin microfilaments.

Capping of HLA-DR on the surface of a human lymphoblastoid cell line (RAJI) and a transfectant human fibroblast cell line (M1DR1) was studied by confocal microscopy. Capping was induced at 22 degrees C after treating cells with an HLA-DR specific monoclonal antibody, L243, followed by a secondary antibody conjugated with FITC. Cytoskeletal actin filaments (F-actin) accumulated under the caps were detected by rhodamine-phalloidin fluorescence. Two processes appear to take place: in the round lymphoblastoid cells, actin, initially distributed uniformly at the cell periphery, redistributes and becomes concentrated underneath HLA-DR patches or caps. In the non-round, substrate-attached fibroblasts, actin was organized in tightly packed filaments along the plasma membrane. It was observed that crosslinked HLA-DR receptors were associated with these filaments and were dragged toward the perinuclear area of the cells, where they coalesce to form a cap. The cytoskeleton-disrupting drugs that inhibit actin polymerisation were used to investigate the mechanism of capping of HLA-DR molecules. Sodium nitroprusside, a nitric oxide releasing agent, cytochalasin D both inhibited the percentage of capping in a dose-dependent manner. These data suggest that on antigen presenting cells, such as B cells and fibroblasts, actin microfilaments acts as a regulator of the movement and capping of HLA-DR receptors.

Atmospheric scanning electron microscope for correlative microscopy.

The JEOL ClairScope is the first truly correlative scanning electron and optical microscope. An inverted scanning electron microscope (SEM) column allows electron images of wet samples to be obtained in ambient conditions in a biological culture dish, via a silicon nitride film window in the base. A standard inverted optical microscope positioned above the dish holder can be used to take reflected light and epifluorescence images of the same sample, under atmospheric conditions that permit biochemical modifications. For SEM, the open dish allows successive staining operations to be performed without moving the holder. The standard optical color camera used for fluorescence imaging can be exchanged for a high-sensitivity monochrome camera to detect low-intensity fluorescence signals, and also cathodoluminescence emission from nanophosphor particles. If these particles are applied to the sample at a suitable density, they can greatly assist the task of perfecting the correlation between the optical and electron images.

Digital fluorescence analysis of trafficking of single endosomes containing low-density lipoprotein in adrenocortical cells: facilitation of centripetal motion by adrenocorticotropic hormone.

Imaging of trafficking of endosomes containing low-density lipoprotein (LDL) is useful to analyze cholesterol transport in adrenocortical cells. At 60 min after the application of fluorescently labeled LDL to adrenocortical cells, individual endosomes containing LDL were demonstrated to undergo frequent switching between forward and reverse movement and immobility. The population of moving endosomes (>or=0.065 microm/s) was approximately 75% in control cells. The remaining endosomes were either slowly moving or temporarily immobile. At 3h after the LDL addition, endosomes were concentrated around the circumference of the cell nuclei. The endosome movement was inhibited by nocodazole, implying that endosomes undergo movement along microtubule networks. Anti-dynein antibodies inhibited the motion of endosomes towards the nucleus, and anti-kinesin antibodies inhibited peripherally directed motion. These results imply that both dynein-like and kinesin-like motor proteins bind to the same endosome, resulting in saltatory movements with centripetal or peripherally directed direction, depending on which motor binds to microtubules. Though the dynein and kinesin motors drive the endosomes very rapidly (microm/s), frequent saltatory motions of single endosomes may induce the very slow net centripetal motion (microm/h).The application of adrenocorticotropic hormone (ACTH) resulted in a facilitation of the centripetal motion of endosomes, resulting in the establishment of the concentration of endosomes around cell nuclei within 1 h.

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression.

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging.

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.

Id proteins negatively regulate basic helix-loop-helix transcription factor function by disrupting subnuclear compartmentalization.

Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect.