Interlabel Distance Research Articles

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin.

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in several cancers, such as gastric, and breast cancer, and is, therefore, an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness, and is associated with cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) to image specifically labeled SKBR3 breast cancer cells. The breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify cellular regions with spatially correlated actin and ErbB2 expression. For STEM of the intact plasma membrane of whole cells, the cells were fixed and covered with graphene. Spatial distribution patterns of ErbB2 in the actin rich ruffled membrane regions were examined, and compared to adjacent actin-low regions of the same cell, revealing an association of putative signaling active ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, as well as after treatment of cells with Cytochalasin D, which breaks up larger actin filaments. In both latter data sets, a significant inter-label distance of 36 nm was identified, possibly indicating an indirect attachment to helical actin filaments via the formation of heterodimers of ErbB2 with epidermal growth factor receptor (EGFR). The possible attachment to actin filaments was further explored by identifying linear QD-chains in actin-rich regions, which also showed an inter-label distance of 36 nm.

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liquid state with environmental scanning electron microscopy using scanning transmission electron microscopy detection. The locations of individual HER2 positions were determined in a total plasma membrane area of 991 μm2 for several SKBR3 cells and 1062 μm2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 ± 7 and 32 ± 10 nm in SKBR3 and HCC1954 cells, respectively. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the experimental finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.

Alveolar bone loss and mineralization in the pig with experimental periodontal disease

ObjectiveTo address how experimental periodontal disease affects alveolar bone mass and mineral apposition in a young pig model. Materials and methodsSeven three-month-old pigs were periodically inoculated with 4 types of periodontal bacteria, along with a ligature around the last maxillary deciduous molar for 8 weeks to induce periodontal disease (PG). Eight same-aged pigs served as the control (CG). Segmentations of 3D cone-beam CT images were performed to quantify volumes of the total alveolar bone, alveolar ridge, and all roots of the target molar. Calcein and alizarin were administered for labeling mineral apposition before euthanasia. The harvested molar blocks were sectioned and examined under epifluorescence. The inter-label distance between the two vital markers at regional bone surfaces were measured and mineral apposition rate (MAR) was calculated. ResultsA significant reduction of total alveolar bone volume was seen in PG with the major loss at the alveolar ridge. MAR was significantly higher at the root furcation region than those at both buccal and palatal ridges in CG. Compared with CG, PG animals showed more interrupted labeled bands with significantly lower MAR at the furcation region. MARs were positively associated with both the volumes of total alveolar bone and ridge in CG, but only with the total alveolar bone in PG. ConclusionsIn young growing pigs, mineral apposition is region specific. The experimental periodontal disease not only leads to alveolar bone loss, but also perturbs mineral apposition for new bone formation, thus impairing the homeostasis of alveolar bone remodeling.

Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer.

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ∼35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.

Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Directly Regulates Osteogenesis

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na(+)/H(+) exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na(+)/H(+) exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.

Ultrasound mimics the effect of mechanical loading on bone formation in vivo on rat ulnae

While the effect of ultrasound as an extreme example of low-magnitude high-frequency stimulation has been explored in the response of bone to injury, little is known about its effect on normal bone. This experiment was designed to test the hypothesis that ultrasound exerts a similar influence on bone as mechanical stimulation at a physiological level. Three groups of female Wistar rats were anaesthetised (6 per group). In one group, the left ulna was loaded cyclically in vivo 40 times, repeated on a further 5 occasions on alternate days. In a second group, transcutaneous low-intensity pulsed ultrasound stimulation was applied to the left ulnae for the same duration as the period of loading. In a third group, loading and ultrasound stimulation were applied concurrently. The right ulna served as non-loaded control in each animal. At the end of the experiment after 14 days, both ulnae were removed. Induced bone formation was assessed by measuring the proportion of medial periosteal bone surface with double label (dLS/BS, %) and by calculation of mineral apposition rate (MAR) from the inter-label distance. All three treatments induced a significant periosteal response, increasing dLS/BS values from <10% in control limbs to >80% in treated limbs. Increases in MAR of experimental ulnae versus contralateral control ulnae were 2.9 (±0.9), 8.6 (±2.4) and 8.7 μm (±3.2) for the ultrasound only, ultrasound and load, and load only groups, respectively. The effects of loading plus ultrasound were not significantly different from ultrasound alone. These data suggest that ultrasound is able to induce changes in bone that share at least some features with mechanical loading.

Microporosity Affects Bioactivity of Macroporous Hydroxyapatite Bone Graft Substitutes

This paper describes an investigation into the influence of microporosity on early osseointegration within porous hydroxyapatite scaffolds. Two batches of phase pure porous hydroxyapatite were produced with total porosities of approximately 80%, but with varied levels of microporosity such that the strut porosity of the two batches were 10 and 20%. Cylindrical specimens 4.5mm in diameter were implanted in the distal femur of 6 month New Zealand White rabbits and retrieved for histological and histomorphometric analysis at 1 and 3 weeks. Optical microscopy demonstrated variation in the degree of capillary penetration and bone morphology within scaffolds at 1 and 3 weeks, respectively. Moreover, histomorphometry demonstrated that there was significantly more bone ingrowth within HA80-2 scaffolds and that the rate of bone formation within these scaffolds was significantly faster. These results indicate that the bioactivity of porous hydroxyapatite scaffolds may be improved by increasing the level of microporosity within the ceramic struts. Introduction The combined affects of an ageing population and greater expectations in quality of life have resulted in an increasing global demand for orthopaedic implants for the replacement or augmentation of damaged bones and joints. In bone grafting current ‘gold standards’ include the use of autograft (living bone from the patient) or allograft (dead, sterilised bone from bone banks) but these methods are increasingly recognised as non-ideal due to limitations in supply and consistency [1]. Porous ceramics have been considered for use as bone graft substitutes in the treatment of bone defects for over 30 years [2]. In particular, calcium phosphates such as hydroxyapatite (HA) have been promoted as a result of their osteoconductive properties. However, while it is well recognised that both the rate of integration and the final volume of regenerated bone may be primarily dependent on various features of the macro-porosity, such as volume fraction, pore size and pore connectivity [3], recent in vitro studies have demonstrated bone cell sensitivity to the level of microporosity within the ceramic struts [4]. The aim of this study was to investigate the influence of microporosity on early osseointegration within porous HA (PHA) scaffolds. Materials and Methods Two batches of phase pure PHA were produced using a novel slip foaming technique [5], both had total porosities of approximately 80%, but varied in the volume fraction of porosity distributed 2 Title of Publication (to be inserted by the publisher) Figure 1 – (a) Porosity distribution within batches HA80-1 and HA80-2. Micropore morphology within the struts of batches (b) HA80-1 and (c) HA80-2. Bar = 50 μm between their macropore (>50μm) and micropore (<20μm) populations (Fig. 1). This variation was such that the strut porosity of batches HA80-1 and HA80-2 were 10 and 20%, respectively. Morphological characterisation of both the macroand microporosity was performed through a combination of immersion densitometry and image analysis of serial sections using a Zeiss Axioskop optical microscope linked to a KS300 image analyser. Macropore size, interconnection size and connectivity index, a measure of inter-pore connectivity, were all measured. Both the open and closed % of microporosity within the struts was quantified. Cylindrical specimens 4.5mm in diameter were implanted in the distal femur of 6 month New Zealand White rabbits and retrieved for histological and histomorphometric analysis at 1 and 3 weeks. The volume of new bone ingrowth was calculated using a Weibel grid and the mineral apposition rate (MAR) was determined through the administration of fluorochrome labels at 1 and 2 weeks and measurement of the inter-label distance using a Zeiss Axioskop optical microscope with a UV light source, linked to KS300 image analyser. Figure 2 – (a) Macropore interconnection data and (b) porosity distribution of open and closed microporosity within batches HA80-1 and HA80-2. 0 1 2 3 4 5 6 7 8 HA80-1 HA80-2 M ic ro po ro si ty (% ) Open Closed (b) (a) 0 10 20 30 40 50 60 70 80 90 HA80-1 HA80-2 P or os ity (% ) Microporosity Macroporosity (a) (b) (c) 0 100 200 300 400 500 600 Interconnection Size Connectivity Index HA80-1 HA80-2 Figure 3 – Variation in ingrowth morphology within batches (a) HA80-1 (Goldner’s trichrome) and (b) HA80-2 (Toluidine blue). Bar = 100 μm

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR.

We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2'FUTGGC)*d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2'FU7-pS19 inter-label distance, was 9.4 +/- 0.7 A, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 A decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 A, consistent with the results of a previous solution NMR study of the 2:1 complex. These 31P-19F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7-14 A in DNA-drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.

Extracellular matrix heparin induces alteration of the cell adhesion during brain development

The studies of neuronal cell-glycosaminoglycan interactions indicate an increasing interest in the question of how heparin can mediate adhesion properties of the cell. We have found that high levels of both N-CAM concentration and heparin-binding activity were noticed in the early stages of brain formation. According to electron microscopy data, an elevation of free heparin in the substratum leads to a decrease of the N-CAM content and changing of its distribution on the membrane of cultured hippocampal neurons. Spatial arrangement of immunogold labelled N-CAM molecules in plasma membrane profiles of cultured neurones was quantified with image analysis software using an interlabel distance estimate. To convert these estimates into two dimensional (2D) quantities, namely the 2D pattern and density of labelling, a computer simulation technique was used. Heparin added to the substratum in a concentration of 40 μg/ml decreased the 2D N-CAM labelling density by 50% — 39.8 labels/μm 2 compared with the control values of 88.9 labels/μm 2.

Computer simulation approach to the quantification of immunogold labelling on plasma membrane of cultured neurons

Cell culture is a convenient model system to study the expression of plasma membrane-bound proteins in nerve cells. Analysing it with an ultrastructural detail researchers often apply transmission electron microscopy together with immunogold labelling. Plasma membrane profiles are one-dimensional (1D) and provide little information about the topography of membrane-bound proteins. In order to convert 1D estimates of spatial arrangement for preembedding immunogold labelled proteins into two-dimensional (2D) quantities, namely the 2D pattern and density of labelling, this paper presents a simple computer simulation technique. This technique is based on a mathematical model permitting a simulated immunogold labelled membrane to be sampled in a way similar to microtome sectioning. An interlabel distance (ILD) estimate is used to define the position of immunogold particles in membrane profiles. In order to interpret experimental ILD measurements the simulated distribution best fit to the experimental data is selected and the corresponding 2D density and pattern of particle scattering are considered to explain the real situation. Various parameters including a cell section thickness, immunogold particle size etc can be adjusted to suit the demands of a particular experiment. The technique was applied to quantify the NCAM preembedding immunogold labelling in the plasma membrane of cultured rat hippocampal neurons.

Histomorphometrical analysis of the bone-implant interface: comparison of microradiography and brightfield microscopy

Section thickness has been shown to affect the histomorphometrical measurement of bone-implant contact when analysed under brightfield microscopy. This study investigated whether microradiography of the bone-implant interface eliminated the errors associated with thick section analysis. Seven implant containing sections were utilized. Microradiographs of the thick (~100 μm) sections were taken and the sections were subsequently ground to thicknesses of 50 μm and 25 μm. Photomicrographs were taken of the microradiographs and of the sections at each thickness (100, 50 and 25 μm) under brightfield microscopy. The photomicrographs were analysed for direct bone-implant contact in the cortical passage region and along the total length of the implant. The effect of section thickness on multiple fluorochrome labelling in 10 rabbit femur specimens was also examined. Centre-to-centre interlabel distance was measured for each label pair at a thickness of 100 μm and then again after the sections were ground to 50 μm and 25 μm. The thick (100 μm) sections showed a significantly greater amount of bone-implant contact than either the thin sections or the microradiographs. There was no difference in direct bone-implant contact measured by microradiography or thin sections. However, the microradiographic analysis showed a much lower variability of the bone-implant contact than the sections evaluated under brightfield microscopy. In addition, they have the added benefit of providing information on bone mineral density. Centre-to-centre interlabel distance was not significantly different for any label pair owing to section thickness. Data from this study provides evidence that the use of microradiographs for histomorphometrical analysis of the bone-implant interface is superior to brightfield analysis of thin sections owing to the lower variability of microradiographical data and the ability to obtain bone mineral density measures. Additionally, given that interlabel distance was not significantly affected by section thickness, the use of 100 μm thick sections for analysis of fluorochrome labels in cortical bone is supported.

Light-induced isomerization causes an increase in the chromophore tilt in the M intermediate of bacteriorhodopsin: a neutron diffraction study.

Bacteriorhodopsin (BR) was regenerated with two selectively deuterated retinals, one with 11 deuterons in the beta-ionone ring (D11) and the other with 5 deuterons (D5) at the end of the polyene chain closest to the Schiff base at carbon atoms C-14, C-15, and C-20. Both label positions (centers of deuteration) were obtained from difference Fourier maps of projections onto the plane of the membrane by neutron diffraction at 90 K, both in the light-adapted ground-state BR568 and in the photocycle intermediate M412. To retard the decay of M412, purple membrane films were soaked in 0.1 M or 1 M guanidine hydrochloride at pH 9.6. M412 was produced by illuminating oriented membrane films at physiological temperature (278 K), followed by rapid cooling to 90 K in the absence of light. The results show that in the projected structure the ring position is unaltered during the transition from BR568 to M412, whereas the position of the D5 label shifts by 1.4 +/- 0.9 A toward the ring. The shortened interlabel distance in the projected structure for the M412 state implies that as a result of the all-trans/13-cis isomerization, the C-5 to C-13 part of the polyene chain tilts out of the plane of the membrane toward the cytoplasm by about 11 degrees +/- 6 degrees. Pairwise comparison of data sets with the same retinal for the two photocycle states M412 and BR568 leads to four difference-density maps for the protein, which are in agreement with previous work. They show changes in the protein density near helices G and F.

The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels

Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues, such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching, the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).

Label escape theory revisited: The effects of resting periods and section thickness

A mathematical theory is developed for the effects of section thickness and resting periods on “label escape”—that is, the tendency for some bone formation sites to display only one label when a double fluorochrome marker is given. It is demonstrated that when section thickness is greater than the interlabel distance, many double labels will appear to be fused, so that they will be counted as single labels. Thus, thinner sections allow more accurate measurement of labeled surfaces, but it turns out that thicker sections yield a better estimate of apposition rate. It is also shown that if the refilling of BMU resorption cavities is interrupted by resting periods of arbitrary frequency and duration, the number of single labels will be increased in proportion to the total duration of the interruptions. Equations are derived which allow one to predict the magnitude of these effects, and to calculate the average total duration of resting periods during refilling from the ratio of single to double labeled surfaces. When the theory is applied to existing experimental data, it is found that substantial resting periods must be present to explain the amount of excess single label which is usually observed.