Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

β-Glucanases in Malt That Form Insoluble Materials

When brewing barley malt extracts were incubated with malt β-glucans, insoluble materials were formed in the reaction mixture. To investigate the cause of this, we studied various factors that may participate in the formation of these materials. The isolated malt β-glucans were similar to barley β-glucans with the β-(l→3) and (1→4)-linkages in a molar ratio of 1:2.38, and the molecular weight was 950,000. Three enzymes were detected and purified from malt by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and isoelectric focusing. One of these enzymes was β-(1→4)-d-glucanase (I) with a molecular weight of 40,000 and an optimum pH of 5.0. The other enzyme was β-(l→3), (l→4)-d-glucan 4-glucanohydrolase, with a molecular weight of 33,000 and an optimum pH 5.0. The third enzyme was β-(1→4)-d-glucanase (II), with a molecular weight of 49,000 and an optimum pH of 4.5. Among these three β-glucanases, β(1→4)-d-glucanases (I) and (II) had not been identified before in malt, and β-(l→4)-d-glucanase (II) was most stable on heat treatment and formed most of the precipitates in the reaction mixture.     

Loading a ring: structure of the Bacillus subtilis DnaB protein, a co-loader of the replicative helicase

Loading of the ring-shaped replicative helicase is a critical step in the initiation of DNA replication. Bacillus subtilis has adopted a two-protein strategy to load its hexameric replicative helicase: DnaB and DnaI interact with the helicase and mediate its delivery onto DNA. We present here the 3D electron microscopy structure of the DnaB protein, along with a detailed analysis of both its oligomeric state and its domain organization. DnaB is organized as an asymmetric tetramer that is comprised of two stacked components, one arranged as a closed collar and the other as an open sigma shape. Intriguingly, the 3D map of DnaB exhibits an overall architecture similar to the structure of the Escherichia coli gamma-complex, the loader of the ring-shaped processivity factor. We propose a model whereby each DnaB monomer participates in both stacked components of the tetramer and displays a different overall shape. This asymmetric quaternary organization could be a general feature of ring loaders.     

The beta-amyloid peptide of Alzheimer's disease decreases adhesion of vascular smooth muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Abeta) builds up. In this study, the possibility that Abeta disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Abeta in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Abeta was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Abeta that was fluorescently labelled at the N-terminus (fluo-Abeta) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Abeta. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Abeta1-40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Abeta treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Abeta on cell adhesion. The studies suggest that Abeta can decrease VSMC viability by disrupting VSMC-extracellular matrix (ECM) adhesion.     

Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging

Seven P2X purinergic receptor subunits have been identified: P2X1–P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His₆-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His₆ antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

Nanoscale Observations of Extracellular Polymeric Substances Deposition on Phyllosilicates by an Ectomycorrhizal Fungus

Microorganisms colonizing surfaces can exude a wide range of substances, generally called Extracellular Polymeric Substances (EPS). While EPS has often been visualized as thick mature strata embedding microbes, the initial phases of EPS production, its structure at the micro- and nanoscale and the microbial wall areas involved in its exudation are less known. In this work we use Atomic Force Microscopy to image EPS produced by the fungus Paxillus involutus on phyllosilicate surfaces. Hyphal tips initially deposit EPS which assumes the shape of a “halo” surrounding hyphae. The fusion of adjacent EPS halos is likely responsible for the creation of EPS monolayers covering mineral surfaces. It is also proposed that a specific region of hyphae initiates the formation of mineral channels produced by fungi. The results presented here permit for the first time to propose a model for the initial stages of EPS accumulation in fungi and filamentous microorganisms in general.     

Ultrastructural and cytochemical studies on the connective tissue of chaetognaths

The hydroskeleton plays a central role in the architecture of the trunk of the Chaetognath. Its fibrous part is composed by a ‘basement membrane’ which separates the epithelial and nervous level from the locomotory muscle and other tissues which surround the general cavity. This structure corresponds to a dense connective tissue sheath; together with the aqueous phase of the general cavity it constitutes the main part of the hydroskeleton. The axes of the lateral and caudal fins are extensions of this connective tissue; they are rich in ground substance and contain several kinds of fibrils and granules.The ‘basement membrane’ is made of a network of densely packed parallel layers of collagen fibrils which form helices which wrap around the trunk. The collagen fibrils of this connective stratum are sandwiched between two basal lamina; they are embedded in a reduced extracellular matrix whose components are closely related to the architecture of the collagen fibrils. In the core of the fin, the ground substance is very abundant and classical cross-striated collagen fibrils are not to be found. A compact fibrillar transition zone is to be noted between the dense connective stratum surrounding the body and the hyaline axis of the fins. In this zone, no crossbanded collagen fibrils are to be seen.The hydroskeleton and the fins show variations within the phylum. They could be related to speciation, and the ancestral pathway of the phylum. Furthermore these variations are related to the general problem of the evolution of the extracellular matrices and collagen molecule itself.     

Aspects of Mineral Structure in Normally Calcifying Avian Tendon

Structural characteristics of normally calcifying leg tendons of the domestic turkey Meleagris gallopavo have been observed for the first time by tapping mode atomic force microscopy (TMAFM), and phase as well as corresponding topographic images were acquired to gain insight into the features of mineralizing collagen fibrils and fibers. Analysis of different regions of the tendon has yielded new information concerning the structural interrelationships in vivo between collagen fibrils and fibers and mineral crystals appearing in the form of plates and plate aggregates. TMAFM images show numerous mineralized collagen structures exhibiting characteristic periodicity (54-70 nm), organized with their respective long axes parallel to each other. In some instances, mineral plates (30-40 nm thick) are found interspersed between and in intimate contact with the mineralized collagen. The edges of such plates lie parallel to the neighboring collagen. Many of these plates appear to be aligned to form larger aggregates (475-600 nm long x 75-90 nm thick) that also retain collagen periodicity along their exposed edges. Intrinsic structural properties of the mineralizing avian tendon have not previously been described on the scale reported in this study. These data provide the first visual evidence supporting the concept that larger plates form from parallel association of smaller ones, and the data fill a gap in knowledge between macromolecular- and anatomic-scale studies of the mineralization of avian tendon and connective tissues in general. The observed organization of mineralized collagen, plates, and plate aggregates maintaining a consistently parallel nature demonstrates the means by which increasing structural complexity may be achieved in a calcified tissue over greater levels of hierarchical order.     

Telomeric nucleosomes are intrinsically mobile

Nucleosomes are no longer considered only static basic units that package eukaryotic DNA but they emerge as dynamic players in all chromosomal processes. Regulatory proteins can gain access to recognition sequences hidden by the histone octamer through the action of ATP-dependent chromatin remodeling complexes that cause nucleosome sliding. In addition, it is known that nucleosomes are able to spontaneously reposition along the DNA due to intrinsic dynamic properties, but it is not clear yet to what extent sequence-dependent dynamic properties contribute to nucleosome repositioning. Here, we study mobility of nucleosomes formed on telomeric sequences as a function of temperature and ionic strength. We find that telomeric nucleosomes are highly intrinsically mobile under physiological conditions, whereas nucleosomes formed on an average DNA sequence mostly remain in the initial position. This indicates that DNA sequence affects not only the thermodynamic stability and the positioning of nucleosomes but also their dynamic properties. Moreover, our findings suggest that the high mobility of telomeric nucleosomes may be relevant to the dynamics of telomeric chromatin.     

Configurational entropy modulates the mechanical stability of protein GB1

Configurational entropy plays important roles in defining the thermodynamic stability as well as the folding/unfolding kinetics of proteins. Here we combine single-molecule atomic force microscopy and protein engineering techniques to directly examine the role of configurational entropy in the mechanical unfolding kinetics and mechanical stability of proteins. We used a small protein, GB1, as a model system and constructed four mutants that elongate loop 2 of GB1 by 2, 5, 24 and 46 flexible residues, respectively. These loop elongation mutants fold properly as determined by far-UV circular dichroism spectroscopy, suggesting that loop 2 is well tolerant of loop insertions without affecting GB1′s native structure. Our single-molecule atomic force microscopy results reveal that loop elongation decreases the mechanical stability of GB1 and accelerates the mechanical unfolding kinetics. These results can be explained by the loss of configurational entropy upon closing an unstructured flexible loop using classical polymer theory, highlighting the important role of loop regions in the mechanical unfolding of proteins. This study not only demonstrates a general approach to investigating the structural deformation of the loop regions in mechanical unfolding transition state, but also provides the foundation to use configurational entropy as an effective means to modulate the mechanical stability of proteins, which is of critical importance towards engineering artificial elastomeric proteins with tailored nanomechanical properties.     

Periodically arranged interactions within the myosin filament backbone revealed by mechanical unzipping

Numerous types of biological motion are driven by myosin thick filaments. Although the exact structure of the filament backbone is not known, it has long been hypothesized that periodically arranged charged regions along the myosin tail are the main contributors to filament stability. Here we provide a direct experimental test of this model by mechanically pulling apart synthetic myosin thick filaments. We find that unzipping is accompanied by broad force peaks periodically spaced at 4-, 14- and 43-nm intervals. This spacing correlates with the repeat distance of highly charged regions along the myosin tail. Lowering ionic strength does not change force-peak periodicity but increases the forces necessary for unzipping. The force peaks are partially reversible, indicating that the interactions are rapidly re-established upon mechanical relaxation. Thus, the zipping together of myosin tails via consecutive formation of periodically spaced bonds may be the underlying mechanism of spontaneous thick filament formation.     

Kinetics of different processes in human insulin amyloid formation

Human insulin has long been known to form amyloid fibrils under given conditions. The molecular basis of insulin aggregation is relevant for modeling the amyloidogenesis process, which is involved in many pathologies, as well as for improving delivery systems, used for diabetes treatments. Insulin aggregation displays a wide variety of morphologies, from small oligomeric filaments to huge floccules, and therefore different specific processes are likely to be intertwined in the overall aggregation. In the present work, we studied the aggregation kinetics of human insulin at low pH and different temperatures and concentrations. The structure and the morphogenesis of aggregates on a wide range of length scales (from monomeric proteins to elongated fibrils and larger aggregates networks) have been monitored by using different experimental techniques: time-lapse atomic force microscopy (AFM), quasi-elastic light-scattering (QLS), small and large angle static light-scattering, thioflavin T fluorescence, and optical microscopy. Our experiments, along with the analysis of scattered intensity distribution, show that fibrillar aggregates grow following a thermally activated heterogeneous coagulation mechanism, which includes both tip-to-tip elongation and lateral thickening. Also, the association of fibrils into bundles and larger clusters (up to tens of microns) occurs simultaneously and is responsible for an effective lag-time.     

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young''s modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging.