Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.     

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.     

Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na⁺ than for Li⁺. Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance.     

A New Paradigm for MAPK: Structural Interactions of hERK1 with Mitochondria in HeLa Cells

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.     

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson's disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr-Tyr (dityrosine) dimers through -C-C- bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.     

Nucleosome-like complex of the histone from the hyperthermophile Methanopyrus kandleri (MkaH) with linear DNA

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw or=9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)(2) tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.     

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.