Rod-like Domain Research Articles

A Broad Spectral Photodetector Using Organic Bisindolo Quinoxaline on ZnO Nanorods

Inorganic/organic hybrids of ZnO nanorods (NRs)/bisindolo quinoxaline (BIQ) were fabricated for broadband photosensing applications. Multiple material characterizations revealed the BIQ was self-assembled in a regular form of rod-like domain and an irregular form of amorphous aggregation that were distributed on the ZnO NRs. Optical measurements showed that BIQ can absorb visible light with a wavelength up to 630 nm and effectively generate photoelectrons. Moreover, clustering of BIQ can be observed via the 3D optical microscopy. ZnO/BIQ hybrids were promising for future UV and visible light environmental monitoring applications.

Vimentin Tail Segments Are Differentially Exposed at Distinct Cellular Locations and in Response to Stress.

The intermediate filament protein vimentin plays a key role in cell signaling and stress sensing, as well as an integrator of cytoskeletal dynamics. The vimentin monomer consists of a central rod-like domain and intrinsically disordered head and tail domains. Although the organization of vimentin oligomers in filaments is beginning to be understood, the precise disposition of the tail region remains to be elucidated. Here we observed that electrophilic stress-induced condensation shielded vimentin from recognition by antibodies against specific segments of the tail domain. A detailed characterization revealed that vimentin tail segments are differentially exposed at distinct subcellular locations, both in basal and stress conditions. The 411–423 segment appeared accessible in all cell areas, correlating with vimentin abundance. In contrast, the 419–438 segment was more scantily recognized in perinuclear vimentin and lipoxidative stress-induced bundles, and better detected in peripheral filaments, where it appeared to protrude further from the filament core. These differences persisted in mitotic cells. Interestingly, both tail segments showed closer accessibility in calyculin A-treated cells and phosphomimetic mutants of the C-terminal region. Our results lead us to hypothesize the presence of at least two distinct arrangements of vimentin tail in cells: an “extended” conformation (accessible 419–438 segment), preferentially detected in peripheral areas with looser filaments, and a “packed” conformation (shielded 419–438 segment), preferentially detected at the cell center in robust filaments and lipoxidative stress-induced bundles. These different arrangements could be putatively interconverted by posttranslational modifications, contributing to the versatility of vimentin functions and/or interactions.

726. Prevention of Muscular Dystrophy by an AAV Vector Encoding a Nonimmunogenic Protein Based on the Evolution of Utrophin and Dystrophin

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions in the dystrophin gene. Expression of recombinant dystrophin in DMD therefore risks chronic immune recognition of the “non-self” protein. Early developmental expression of the paralogous protein utrophin in the thymus may confer central immunological tolerance to its peptide sequence. Here we address previously unanswered questions about the therapeutic efficacy and immunogenicity of “µutrophins” encoded by synthetic minigenes suitable for use in AAV. First we used a comparative phylogenomic approach to address a deceptively simple question with critical implications for this field: “which came first-dystrophin or the sarcomere”. This analysis provided evidence that the long rod-like domain of utrophin and dystrophin was “coopted” from a higher molecular weight protein in dynein-propelled animals lacking striated muscle. When considered in light of recent crystalographic studies, these results suggest that the full functionality of native dystrophin may be conferred by recombinant mini-utrophins with internally deleted rod domains that preserve inter-repeat folding and hence maximal tensile strength. We maximized recombinant µUtrophin (µU) expression from synthetic open reading frames by using the codon bias of striated myosin. Our blinded studies of mdx mice injected with a µU transgene are the first to show COMPLETE recovery of peripheral myofiber nucleation following a single systemic injection of an AAV vector. Sarcoglycan expression, as measured by western blot in the µU-treated mice, are restored to control levels throughout growth to skeletal maturity. To investigate the correlation between our histological findings and functionality, we validated a novel open field cage and attached running wheel system. Our blinded data show remarkable functional rescue in µU-treated mdx mice using this non-invasive test with relevance to the clinical assessment of disease progression in DMD. These data also strongly correlate with those derived from in vivo force grip and ex vivo force transducer measurements. Remarkably, a majority of mdx mice show complete normalization of serum creatine kinase (CK), a first for single-dose treatment by any clinically translatable modality. In dystrophic puppies, intravenous injection of a 30-fold lower relative dose of AAV9U fully restored sarcoglycan levels and normalized the myofiber size-distribution following a threefold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings suggest a rationale for neonatal gene therapy using utrophin as an internally deleted “self” protein in DMD to minimize the risk of chronic immunotoxicity. We outline a rigorous translational approach using escalating vector doses in GSHPMD dogs harboring a newly defined 5 megabase deletion encompassing the dystrophin ORF.

Structure of the Fibrillin-1 N-Terminal Domains Suggests that Heparan Sulfate Regulates the Early Stages of Microfibril Assembly

SummaryThe human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly.

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.

Focus on Molecules: Collagens V and XI

The collagens are a family of 28 extracellular matrix glycoproteins. Collagens are composed of 3 polypeptides, called alpha chains. Each α chain contains at least one stiff, rod-like domain of varying length with a glycine at every third residue that can form a triple helix, i.e., collagenous domain. Collagens V and XI are members of the fibril-forming class of collagens, along with collagens I, II, III, XXIV and XXVII. Collagens V and XI comprise a subclass of regulatory fibrillar collagens that co-assemble with collagens I, II and III. All collagens are trimers composed of 3 α chains with at least one triple helical or collagen (COL) domain. Alpha chains of collagens V (P20908 and P25940) and XI (P12107 and P13942) are encoded by genes with different phylogenetic origins, belonging to different clades or `ancestral groups' (clade 2) from collagens I, II and III (clade 1) or XXIV and XXVII (clade 3). Clade 1 also contains the α2(V) gene (P05997; i.e., the α2 chain of collagen V) and the α1(II) gene, termed α3(XI) when part of a collagen XI trimer (P02458). These genes code for multi-domain polypeptide chains with features common to all fibrillar collagens: a signal peptide; an N-terminal non-collagenous region (NC3); a short N-terminal triple helical domain of interrupted Gly-X–Y repeats (COL2), where X and Y can be any amino acid, but are often proline and hydroxyproline, respectively; a short non-collagenous domain (NC2 or `hinge'); the main triple helical or collagenous domain of continuous (GlyX–Y)n repeats (COL1); and a non-collagenous C-propeptide (NC1) (Fig.1).

Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA ofAggregatibacter actinomycetemcomitansby Electron Tomography

The periodontal pathogen Aggregatibacter actinomycetemcomitans displays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140 degrees and 170 degrees at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of approximately 5 nm and a cylindrical domain ( approximately 4.4 nm by 5.8 nm) separated by a linker region with a diameter of approximately 3 nm, followed by a cylindrical domain ( approximately 4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein of A. actinomycetemcomitans.

Migfilin, a Molecular Switch in Regulation of Integrin Activation

The linkage of heterodimeric (alpha/beta) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin beta cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin beta CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin beta CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.

Making Connections: Desmoplakin as an Intermediate Filament-Binding Protein

Early work on desmosome morphology and biochemical composition laid the groundwork for the utilization of molecular biological approaches in the 1990s. Important breakthroughs included the cloning and sequencing of the desmoplakin gene, the identification of desmoplakin as an intermediate filament-binding protein and the realization that desmoplakin was part of a family of filamentbinding proteins now known as the plakin family of cytolinkers (reviewed in Kowalczyk et al., 1999; Leung et al., 2002). Collectively, these studies form an important foundation for our understanding of how intermediate filaments associate with the plasma membrane and other cytoskeletal networks. Green et al. (1988), at Northwestern University in Chicago, first isolated and analyzed the desmoplakin coding sequence, which allowed for structural analysis of the amino-acid sequence of the desmoplakin protein (Green et al., 1990). From this work, desmoplakin was predicted to harbor a central alpha-helical coiledcoil domain similar to that found in intermediate filaments. This region of desmoplakin would thus encode a rod-like domain connecting globular aminoand carboxy-terminal domains. In addition, the carboxyterminal domain harbored several interesting features, including the presence of a 38-amino-acid repeated homology domain that was also present in the 230-kDa bullous pemphigoid antigen (BPAG1). The periodicity of charged amino acids within these carboxy-terminal repeats was similar to that found in the 1B rod domain of intermediate filament proteins, leading to the prediction that the desmoplakin carboxyl terminal domain might interact with intermediate filaments. More recent studies have revealed several different means of desmoplakin–intermediate filament interactions, but this early study by Green and colleagues made two major predictions that were substantiated by further studies. First, that desmoplakin and BPAG1 were part of a gene family, and second, that the carboxy-terminal region of this family of proteins might confer intermediate filament binding activity. Using the desmoplakin cDNA and structural information from the 1990 study as a foundation, along with emerging information about BPAG1 and plectin as intermediate filamentassociated proteins, Stappenbeck and Green used molecular biological approaches to define desmoplakin domains functionally (Stappenbeck and Green, 1992; Stappenbeck et al., 1993). The most striking finding of their studies was that the desmoplakin carboxy-terminal domain aligned along intermediate filament networks when expressed transiently in cultured cells. These studies demonstrated that the desmoplakin carboxy-terminal domain was both necessary and sufficient for interacting with intermediate filaments, and, when overexpressed, could alter intermediate filament organization. Subsequent studies by the Fuchs laboratory demonstrated that the desmoplakin intermediate filament interactions were direct (Kouklis et al., 1994). These findings thereby complemented the structural studies of the desmoplakin gene and were followed by numerous additional biochemical, structural, and yeast genetic approaches (two-hybrid), confirming that the desmoplakin carboxyl terminus is an intermediate filament-binding domain (reviewed in Kowalczyk et al., 1999). Subsequent studies in the late 1990s as well as current studies continue to build upon the foundation initiated by the pioneering discoveries of desmoplakin and plakin family structure. The desmoplakin amino-terminal domain was found to target desmoplakin to desmosomes through associations with armadillo proteins such as plakoglobin and plakophilins. Collectively, these studies revealed the modular nature of desmoplakin and the mechanisms by which it couples filaments to sites of tight cell–cell adhesion. Current studies are focused on the regulation of these interactions and the dynamic properties of desmoplakin in the assembly of adhesive structures (Godsel et al., 2005).

The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales

Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the 'cellular zone' of the V. carteri ECM. Pherophorin-V1 and -V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae.

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion.

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.

Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells

Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs). In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep-to-superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod-like fibers, whereas in the molecular layer, the rod-like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod-like domain was composed of glial fibrillary acidic protein (GFAP)-positive/GLAST-positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP-negative/GLAST-positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC-BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses.

Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells

Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs). In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep-to-superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod-like fibers, whereas in the molecular layer, the rod-like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod-like domain was composed of glial fibrillary acidic protein (GFAP)-positive/GLAST-positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP-negative/GLAST-positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC-BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses. J. Comp. Neurol. 418:106–120, 2000. © 2000 Wiley-Liss, Inc.

The distribution of end-to-end distances of the weakly bending rod model.

The weakly bending rod model is an approximation to a worm-like chain in the limit where the ratio L(0)/P of the contour length L(0) to the persistence length P is not too large. The range of validity of the weakly bending rod model is investigated by deriving analytical expressions for its distribution of end-to-end distances P(L) and its moments <L(m) > and numerically comparing the results with corresponding values for the worm-like chain model. No general, closed form analytical expression for either P(L) or the average length <L> of a worm-like chain exists, so those quantities are obtained by Monte Carlo simulations. Exact analytical expressions for <L(2)> and <L(4)> for the worm-like chain are employed in the comparison of the computed moments. Moments calculated for the approximate distributions of Daniels and Yamakawa and Stockmayer are also compared with the others. In addition, P(L) and its moments for the alternative model of Winkler et al. are compared with the others. The weakly bending rod model gives a reasonably good account of both P(L) and <L(m) >, m = 1,2,4, over the range L(0)/P </= 0.6, but deviates significantly for L(0)/P >/= 1.0. In contrast, the alternative model of Winkler et al. yields rather poor results in the rodlike domain.

Oscillatory shear induced anisotropic domain growth and related rheological properties of binary mixtures

Numerical simulation based on the modified time-dependent Ginzburg–Landau (TDGL) model has been performed on the domain growth and related rheological properties of binary mixtures under oscillatory shear. The simulation results reveal that the domain growth is anisotropic and depends on the quench depth. It is found that, in the deep quench case, the disclike domain with the normal parallel to the velocity gradient direction is observed, while in the shallow quench case, the rodlike domain with rod axis aligned along the flow direction is observed. The scattering functions for different light incident directions are calculated and suggest that the undulated rodlike morphology is formed in the shallow quench case. This undulated rodlike morphology shows the anomalous rheological response. A plausible interpretation for the anomalous rheological property is proposed based on the deformation of the undulated rodlike morphology under oscillatory shear.

Entactin-2: A New Member of Basement Membrane Protein with High Homology to Entactin/Nidogen

Using the new signal sequence trap (SST) method, we isolated several clones encoding secreted and transmembrane proteins from KUSA cells, a murine osteoblast-like cell line. One isolated novel clone, termed entactin-2, exhibited a high similarity to mouse entactin/nidogen, a basement membrane protein. Although deduction of the amino acid sequence of entactin-2 revealed only 27.4% homology to entactin, many structural similarities were seen between both proteins. Entactin-2 contains five EGF-like and two thyroglobulin-like motifs, which are both cysteine-rich. Comparison of both proteins clearly revealed that entactin-2 also contains related domain structures. The rod-like domain of entactin-2, containing the RGD integrin recognition sequence, fused to glutathione-Stransferase (GST), revealed a cell surface-binding activity similar to that of entactin. In addition, the tissue distribution of entactin-2 mRNA resembled that of entactin. Furthermore, mRNA expression of both genes decreased as osteoblastic differentiation progressed. These results suggest that entactin-2 is a member of the entactin gene family, may have entactin-related functions, and might act as a basement membrane component.

Numerical Study of Elastic Effects by Cubic-Tetragonal Symmetry Breaking on the Ordering Kinetics

Elastic effects by cubic-tetragonal symmetry breaking on the ordering kinetics are investigated. We propose a coarse-grained model characterized by a Ginzburg-Landau free energy with nonconserved order parameters coupled to an elastic field. By numerical study we obtain an oval shaped domain and a square domain in two dimensions and a plate-like domain and a rod-like domain in three dimensions. And in the coarsening process, a striped pattern is obtained. These patterns are remarkably similar to morphologies observed in real cubic-tetragonal order-disorder alloys. Moreover, we study the dynamics during the coarsening process by analyzing the change of the total domain boundaries L DB ( t ). L DB ( t ) decreases as a power of time: t -α (α>0). The exponent α becomes small with increasing the magnitude of the elastic effects.

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Influence of nidogen complexed or not with laminin on attachment, spreading, and albumin and laminin B2 mRNA levels of rat hepatocytes.

Nidogen/entactin is a Mr = 150,000 glycoprotein which is present within basement membranes in a noncovalent stable complex with laminin. We have studied the effects of nidogen/entactin complexed or not with laminin on attachment, spreading, and functions of adult rat hepatocytes in primary culture. Freshly isolated hepatocytes attached on either recombinant or EHS-derived nidogen, although to a lesser extent than on laminin/nidogen complex, laminin, and E8 and P1 fragments of laminin. Hepatocytes bound on a nidogen fragment bearing the N-terminal and rod-like domains but not on either the N-terminal globules or the rod-like domain which contains a RGD sequence. Attachment of hepatocytes on nidogen and laminin/nidogen complex was inhibited by anti-beta 1 integrin antibodies. Hepatocytes remained rounded on nidogen and laminin, whereas they rapidly spread on laminin/nidogen complex and collagen IV. Nidogen, laminin, and laminin/nidogen complex transiently maintained high steady-state albumin mRNA levels in cultured hepatocytes, but a decrease in albumin mRNA content was observed after 24 h, independently of the substrates. Actinomycin D and cycloheximide treatment indicated that the transient effect of these substrates on albumin expression was related to post-transcriptional mechanisms. Laminin B2 mRNAs were not detectable in freshly isolated hepatocytes but were expressed in 4 h hepatocyte cultures. After 24 h, a dramatic increase in the steady-state level of laminin B2 mRNA was found in hepatocytes cultured on nidogen and laminin/nidogen complex. This effect was slightly prevented in hepatocytes plated on laminin. These results show that interactions of hepatocytes with nidogen/entactin in vitro result only in a transient modulation of hepatocyte functions.

Three-dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron microscopy and difference map analysis.

The three-dimensional structure of the rotavirus spike haemagglutinin viral protein 4 (VP4) has been determined to a resolution of 26 A by cryo-electron microscopy and difference analysis of intact virions and smooth (spikeless) particles. Native and spikeless virions were mixed prior to cryo-preservation so that both structures could be determined from the same micrograph, thereby minimizing systematic errors. This mixing strategy was crucial for difference map analysis since VP4 only accounts for approximately 1% of the virion mass. The VP4 spike is multi-domained and has a radial length of approximately 200 A with approximately 110 A projecting from the surface of the virus. Interactions between VP4 and cell surface receptors are facilitated by the bi-lobed head, which allows multi-site interactions, as well as the uniform distribution of the VP4 heads at maximum radius. The bi-lobed head is attached to a square-shaped body formed by two rods that have a slight left-handed helical twist. These rods merge with an angled, rod-like domain connected to a globular base approximately 85 A in diameter. The anchoring base displays pseudo 6-fold symmetry. This surprising finding may represent a novel folding motif in which a single polypeptide of VP4 contributes similar but non-equivalent domains to form the arms of the hexameric base. The VP4 spike penetrates the virion surface approximately 90 A and interacts with both outer (VP7) and inner (VP6) capsid proteins. The extensive VP4-VP7 and VP4-VP6 interactions imply a scaffolding function in which VP4 may participate in maintaining precise geometric register between the inner and outer capsids.(ABSTRACT TRUNCATED AT 250 WORDS)