Three-dimensional organization of microfilaments and microtubules in the cytoskeleton : Immunoperoxidase labelling and stereo-electron microscopy of detergent-extracted cells

Stereo-electron microscopy has been combined with indirect immunoperoxidase labelling to describe the three-dimensional organization of microfilaments and microtubules in spreading cells of a cultured cell line with fibroblastic morphology. Labelling was carried out after extraction of the cells with a non-ionic detergent in a buffer system allowing retention of many of the cytoskeletal elements. Preservation of the three-dimensional organization was ensured by critical point drying. The peroxidase reaction product is readily detectable in electron micrographs both at high and low magnification. Thus, visualization of the three-dimensional organization of the labelled cytoskeletal elements is possible at a magnification where entire cells or large parts of them can be examined in whole mounts. The microfilament system is shown to constitute a continuous, three-dimensional sheath enclosing the bulk of the cytoplasm and most of the microtubular system. In cytoskeletons labelled with actin antibodies, the unlabelled intermediate filaments (10 nm filaments) can be identified by their size and morphology. They constitute a network throughout the cytoplasm which is in part interwoven with the large actin cables located near the lower surface of the cell.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Effects of three-dimensional and color patterns on nest location and progeny mortality in alfalfa leafcutting bee (Hymenoptera: Megachilidae)

ABSTRACT In alfalfa, Medicago sativa L., seed production where high bee densities are released, alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), females may enter several nesting holes before locating their nests. Such levels of "wrong hole" visits lead to an increase in the time spent by females locating their own nests, thereby decreasing alfalfa pollination efficiency and possibly healthy brood production. The objectives of this study were to determine the effect of different nesting board configurations in commercial alfalfa leafcutting bee shelters (separating nesting boards, applying a three-dimensional pattern to the boards, applying a color contrast pattern, or applying a combination of three-dimensional and color contrast patterns) on nest location performance, on the incidence of chalkbrood disease, and on the incidence of broodless provisions. Separating the nesting boards inside shelters improved the ability of females to locate their nests. An increase in nest location performance also occurred in boards with the three-dimensional pattern and the combined three-dimensional and color contrast pattern, compared with the uniform board (a standard configuration currently used commercially). The percentage of provisioned cells that were broodless was not statistically different between treatments, but the percentage of larvae infected with chalkbrood decreased by half in the three-dimensional board design, compared with the uniform board.     

Low-level aspects of segmentation and recognition.

This paper discusses two problems related to three-dimensional object recognition. The first is segmentation and the selection of a candidate object in the image, the second is the recognition of a three-dimensional object from different viewing positions. Regarding segmentation, it is shown how globally salient structures can be extracted from a contour image based on geometrical attributes, including smoothness and contour length. This computation is performed by a parallel network of locally connected neuron-like elements. With respect to the effect of viewing, it is shown how the problem can be overcome by using the linear combinations of a small number of two-dimensional object views. In both problems the emphasis is on methods that are relatively low level in nature. Segmentation is performed using a bottom-up process, driven by the geometry of image contours. Recognition is performed without using explicit three-dimensional models, but by the direct manipulation of two-dimensional images.     

Mapping three-dimensional stress and strain fields within a soft hydrogel using a fluorescence microscope

Three-dimensional cell culture is becoming mainstream as it is recognized that many animal cell types require the biophysical and biochemical cues within the extracellular matrices to perform truly physiologically realistic functions. However, tools for characterizing cellular mechanical environment are largely limited to cell culture plated on a two-dimensional substrate. We present a three-dimensional traction microscopy that is capable of mapping three-dimensional stress and strain within a soft and transparent extracellular matrix using a fluorescence microscope and a simple forward data analysis algorithm. We validated this technique by mapping the strain and stress field within the bulk of a thin polyacrylamide gel layer indented by a millimeter-size glass ball, together with a finite-element analysis. The experimentally measured stress and strain fields are in excellent agreements with results of the finite-element simulation. The unique contributions of the presented three-dimensional traction microscopy technique are: 1), the use of a fluorescence microscope in contrast with the confocal microscope that is required for the current three-dimensional traction microscopes in the literature; 2), the determination of the pressure field of an incompressible gel from strains; and 3), the simple forward-data-analysis algorithm. Future application of this technique for mapping animal cell traction in three-dimensional nonlinear biological gels is discussed.     

Two- and three-dimensional 1H NMR studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure.

Two- and three-dimensional 1H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy proved to be a very efficient method for resonance assignments of protein 1H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by 3J alpha NH coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.     

A physiologic three-dimensional cell culture system to investigate the role of decorin in matrix organisation and cell survival

In vivo cells exist in a three-dimensional environment generated and maintained by multiple cell-cell and cell-matrix interactions. Proteoglycans, like decorin, affect these complex interactions. Thus, we sought to investigate the role of decorin in a three-dimensional environment where the matrix was generated over time by decorin-deficient fibroblasts in the presence of L-ascorbic acid 2-phosphate. The cells were viable and proliferated in response to FGF2. Decorin was incorporated in the matrix and caused a approximately 2 nm shift in the average diameter of the collagen fibrils, and the range and distribution of the fibrils became narrower and more uniform. Although there were no appreciable changes in collagen composition, we found that exogenous decorin induced the de novo synthesis of collagen I and V and cross-linked beta(I). In the early phases of the three-dimensional culture, decorin reduced apoptosis. However, following the establishment of a three-dimensional matrix, the cells did not require decorin for their survival.     

One- and three-dimensional pathways for proteins to reach specific DNA sites

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one-dimensional diffusion along the DNA, or through three-dimensional space via multiple dissociation/re-associations. To distinguish these routes, reactions of the ECO:RV endonuclease were studied on substrates with two ECO:RV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one-dimensional diffusion. The variation in processivity with inter-site spacing shows instead that protein moves from one site to another through three-dimensional space, by successive dissociation/re-associations, though each re-association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA-binding proteins are usually thought to find their target sites by one-dimensional pathways, three-dimensional routes may be more common than previously anticipated.     

Automated Correlation and Averaging of Three-Dimensional Reconstructions Obtained by Electron Tomography

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from anin situspecimen containingEscherichia coli70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.     

Stereo immunofluorescence microscopy: I. Three-dimensional arrangement of microfilaments, microtubules and tonofilaments.

An easy manipulation of the commercial fluorescence microscope allows stereo pairs of pictures to be taken, which when examined with a stereo viewer, give a strong three-dimensional impression. The procedure is described in detail. Its use allows the documentation by immunofluorescence microscopy of the three-dimensional display and organization of microfilament bundles, microtubules and tonofilaments in some well characterized tissue culture cell lines.     

Generation and visualization of root-like structures in a three-dimensional space

In his Notebooks Leonardo da Vinci describes his diameter squared rule for the branching of above-ground tree parts. We now apply this branching-rule to the study of root-branching. Repeated application of this rule in a recursive computer program (ArtRoot) produces the diameters of the branches. An algorithm in this recursive program calculates the orientation of the branches in a three-dimensional space. From these data three-dimensional root-like structures are visualized by a computer program (PLUTON) designed to draw three-dimensional molecular structures. The visualization shows that starting from a relatively simple, symmetrical structure of dichotomous branching, more complex, asymmetrical root-like structures arise even if only a few parameter values are changed. The introduction of randomness into parameter values produces structures that differ considerably in their architecture. An extra force has been added in order to influence the orientation of new branches in space and to increase the flexibility of the structure formation.     

Protein sequence and structure relationship ARMA spectral analysis: application to membrane proteins.

If it is assumed that the primary sequence determines the three-dimensional folded structure of a protein, then the regular folding patterns, such as alpha-helix, beta-sheet, and other ordered patterns in the three-dimensional structure must correspond to the periodic distribution of the physical properties of the amino acids along the primary sequence. An AutoRegressive Moving Average (ARMA) model method of spectral analysis is applied to analyze protein sequences represented by the hydrophobicity of their amino acids. The results for several membrane proteins of known structures indicate that the periodic distribution of hydrophobicity of the primary sequence is closely related to the regular folding patterns in a protein's three-dimensional structure. We also applied the method to the transmembrane regions of acetylcholine receptor alpha subunit and Shaker potassium channel for which no atomic resolution structure is available. This work is an extension of our analysis of globular proteins by a similar method.     

血管内皮细胞和心脏组织块的立体培养

本文旨在对比研究二维平面与三维立体培养模式下,内皮细胞和心脏组织形态学的差异。采用胶内、胶上、三明治模式、玻片培养小室模型等多种I型胶原立体培养模型,通过免疫荧光技术及显微形态学观察组织和细胞的生长情况。在二维平面培养中,原代心脏血管内皮细胞呈铺路石样排列;而在三维胶原培养模式中,内皮细胞呈长梭状形态,并迁入胶原培养介质中,和体内血管新生及血管生成过程中的内皮细胞活化表型相似。加入血管内皮生长因子(vascular endo- thelial growth factor VEGF)能增强内皮细胞管状结构的形成。在三维胶原中,心脏组织块生长良好,迁出的细胞将相邻组织块连接起来,组织块有自发的搏动。本工作表明,改进的薄层胶原培养、玻片培养小室模型和动脉条模型是较好的研究血管生成和血管新生的工具。在三维培养的情况下,内皮细胞通过空间增殖、迁移和锚定,可形成管状结构,比二维平面培养更适合用于血管新生的研究。不同的立体培养模型可用于不同目的的研究。     

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.     

Distribution of Electromechanical Delay in the Heart: Insights from a Three-Dimensional Electromechanical Model

In the intact heart, the distribution of electromechanical delay (EMD), the time interval between local depolarization and myocyte shortening onset, depends on the loading conditions. The distribution of EMD throughout the heart remains, however, unknown because current experimental techniques are unable to evaluate three-dimensional cardiac electromechanical behavior. The goal of this study was to determine the three-dimensional EMD distributions in the intact ventricles for sinus rhythm (SR) and epicardial pacing (EP) by using a new, to our knowledge, electromechanical model of the rabbit ventricles that incorporates a biophysical representation of myofilament dynamics. Furthermore, we aimed to ascertain the mechanisms that underlie the specific three-dimensional EMD distributions. The results revealed that under both conditions, the three-dimensional EMD distribution is nonuniform. During SR, EMD is longer at the epicardium than at the endocardium, and is greater near the base than at the apex. After EP, the three-dimensional EMD distribution is markedly different; it also changes with the pacing rate. For both SR and EP, late-depolarized regions were characterized with significant myofiber prestretch caused by the contraction of the early-depolarized regions. This prestretch delays myofiber-shortening onset, and results in a longer EMD, giving rise to heterogeneous three-dimensional EMD distributions.     

Validation of in vivo assessment of facial soft-tissue volume changes and clinical application in midfacial distraction: a technical report

The purpose of this study was to validate the assessment of visible volume changes of the facial soft tissue with an optical three-dimensional sensor and to introduce new parameters for the evaluation of the soft-tissue shape achieved from three-dimensional data of selected cases of midfacial distraction. Images of a truncated cone of known volume were assessed repeatedly with an optical three-dimensional sensor based on phase-measuring triangulation to calculate the volume. Two cubic centimeters of anesthetic solution was injected into the right malar region of 10 volunteers who gave their informed consent. Three-dimensional images were assessed before and immediately after the injections for the assessment of the visible volume change. In five patients who underwent midfacial distraction after a high quadrangular Le Fort I osteotomy, three-dimensional scans were acquired before and 6 and 24 months after the operation. The visible soft-tissue volume change in the malar-midfacial area and the mean distance of the accommodation vector that transformed the preoperative into the postoperative surface were calculated. The volume of the truncated cone was 235.26 +/- 1.01 cc, revealing a measurement uncertainty of 0.4 percent. The injections of anesthetic solution into the malar area resulted in an average visible volume change of 2.06 +/- 0.06 cc. The measurement uncertainty was 3 percent. In the five patients, the average distance of maxillary advancement was 6.7 +/- 2.3 mm after 6 months and 5.4 +/- 3.0 mm after 2 years. It was accompanied by a mean visible volume increase of 8.92 +/- 5.95 cc on the right side and 9.54 +/- 4.39 cc on the left side after 6 months and 3.54 +/- 3.70 cc and 4.80 +/- 3.47 cc, respectively, after 2 years. The mean distance of the accommodation vector was 4.41 +/- 1.94 mm on the right side and 4.74 +/- 1.32 mm on the left side after 6 months and 1.62 +/- 1.96 mm and 2.16 +/- 1.52 mm, respectively, after 2 years. The assessment of visible volume changes by optical three-dimensional images can be carried out with considerable accuracy. The determination of volume changes and accompanying accommodation vectors completes the cephalometric analysis during the follow-up of patients undergoing midfacial distraction. The new parameters will help to assess normative soft-tissue data on the basis of three-dimensional imaging with a view to an improved three-dimensional prediction of the operative outcome of orthognathic surgery.     

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.     

Protein dynamics: Rotational diffusion of rigid and fluctuating three dimensional structures

The optimized Rouse-Zimm approximation to the local dynamics (ORZLD theory) is extended to treat three-dimensional structures. Rigid model chains of different dimensionality are considered. The local dynamics of random peptides are compared to the rotational correlation times of rigid three-dimensional protein structures. The treatment of these rigid limits is a necessary step in a more advanced ORZLD theory of the dynamics of proteins including fluctuations relative to the three-dimensional structure. © 1995 John Wiley & Sons, Inc.