In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Structural investigations on native collagen type I fibrils using AFM

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.     

Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure–function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure–function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.     

Nanoscale structure of type I collagen fibrils: Quantitative measurement of D-spacing

This article details a quantitative method to measure the D-periodic spacing of type I collagen fibrils using atomic force microscopy coupled with analysis using a two-dimensional fast fourier transform approach. Instrument calibration, data sampling and data analysis are discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of type I collagen morphology in disease models of estrogen depletion and osteogenesis imperfecta (OI) are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage osteoporosis and OI. The ability to quantitatively characterize nanoscale morphological features of type I collagen fibrils will provide important structural information regarding type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knockout studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments.     

Mechanical properties of mineralized collagen fibrils as influenced by demineralization

Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.     

Investigations into the polymorphism of rat tail tendon fibrils using atomic force microscopy

Collagen type I displays a typical banding periodicity of 67 nm when visualized by atomic force or transmission electron microscopy imaging. We have investigated collagen fibers extracted from rat tail tendons using atomic force microscopy, under different ionic and pH conditions. The majority of the fibers reproduce the typical wavy structure with 67 nm spacing and a height difference between the peak and the grooves of at least 5 nm. However, we were also able to individuate two other banding patterns with 23+/-2 nm and 210+/-15 nm periodicities. The small pattern showed height differences of about 2 nm, whereas the large pattern seems to be a superposition of the 67 nm periodicity showing height differences of about 20 nm. Furthermore, we could show that at pH values of 3 and below the fibril structure gets dissolved whereas high concentrations of NaCl and CaCl(2) could prevent this effect.     

Role of cartilage collagen fibrils networks in knee joint biomechanics under compression

Collagen fibrils networks in knee cartilage and menisci change in content and structure from a region to another. While resisting tension, they influence global joint response as well as local strains particularly at short-term periods. To investigate the role of fibrils networks in knee joint mechanics and in particular cartilage response, a novel model of the knee joint is developed that incorporates the cartilage and meniscus fibrils networks as well as depth-dependent properties in cartilage. The joint response under up to 2000 N compression is investigated for conditions simulating the absence in cartilage of deep fibrils normal to subchondral bone or superficial fibrils parallel to surface as well as localized split of cartilage at subchondral junction or localized damage to superficial fibrils at loaded areas. Deep vertical fibrils network in cartilage play a crucial role in stiffening (by 10%) global response and protecting cartilage by reducing large strains (from maximum of 102% to 38%), in particular at subchondral junction. Superficial horizontal fibrils protect the tissue mainly from excessive strains at superficial layers (from 27% to 8%). Local cartilage split at base disrupts the normal function of vertical fibrils at the affected areas resulting in higher strains.Deep fibrils, and to a lesser extent superficial fibrils, play dominant mechanical roles in cartilage response under transient compression. Any treatment modality attempting to repair or regenerate cartilage defects involving partial or full thickness osteochondral grafts should account for the crucial role of collagen fibrils networks and the demanding mechanical environment of the tissue.     

Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.     

Assembly of alpha-synuclein fibrils in nanoscale studied by peptide truncation and AFM

α-Synuclein (α-Syn) fibrils are the major component of Lewy bodies that are closely associated with the pathogenesis of Parkinson’s disease, but the mechanism for the fibril assembly remains poorly understood. Here we report using a combination of peptide truncation and atomic force microscopy (AFM) to elucidate the self-assembly and morphology of the α-Syn fibrils. The results show that protease K significantly slims the fibrils from the mean height of ∼6.6 to ∼4.7 nm, whereas chaotropic denaturant urea completely breaks down the fibrils into small particles. The in situ enzymatic digestion also results in thinning of the fibrils, giving rise to some nicks on the fibrils. Moreover, N- or C-terminally truncated α-Syn fragments assemble into thinner filaments with the heights depending on the peptide lengths. A nine-residue peptide corresponding to the homologous GAV-motif sequence can form very thin (∼2.2 nm) but long (>1 μm) filaments. Thus, the central sequence of α-Syn forms a fibrillar core by cross-β-structure that is flanked by two flexible termini, and the orientation of the fibril growth is perpendicular to the β-sheet structures.     

Glycation changes the charge distribution of type I collagen fibrils

In aging and diabetes, glycation of collagen molecules leads to the formation of cross-links that could alter the surface charge on collagen fibrils, and hence affect the properties and correct functioning of a number of tissues. The electron-optical stain phosphotungstic acid (PTA) binds to positively charged amino acid side-chains and leads to the characteristic banding pattern of collagen seen in the electron microscope; any change in the charge on these side-chains brought about by glycation will affect the uptake of PTA. We found that, upon glycation, a decrease in stain uptake was observed at up to five regions along the collagen D-period; the greatest decrease in stain uptake was apparent at the c1 band. This reduction in PTA uptake indicates that the binding of fructose leads to an alteration in the surface charge at several sites along the D-period. Not all lysine and arginine residues are involved; there appear to be specific residues that suffer a loss of positive charge.     

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.The present study was supported in part by Grant-in-Aid for Developmental Scientific Research (07558249), by The Japan Society for the Promotion of Science, Research for the Future Program (JSPS-RFTF96I00201), by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR), by Grant-in-Aid for the Creation of Innovations through Business-Academic-Public Sector Cooperation to T.H., and by Grant-in-Aid for Scientific Research (B) to Y.I.     

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell–cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm² areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.     

Structural impact of cations on lipid bilayer models: Nanomechanical properties by AFM-force spectroscopy

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules – DNA or proteins – to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.     

Interactions between a luminescent conjugated polyelectrolyte and amyloid fibrils investigated with flow linear dichroism spectroscopy

Luminescent conjugated polyelectrolytes (LCPs) have emerged as novel stains to detect and distinguish between various amyloidogenic species, including prefibrillar aggregates and mature fibril deposits, both in vitro and in histological tissue samples, offering advantages over traditional amyloid stains. We here use linear dichroism (LD) spectroscopy under shear alignment to characterize interactions between the LCP poly(3-thiophene acetic acid) (PTAA) and amyloid fibrils. The positive signature in the LD spectrum of amyloid-bound PTAA suggests that it binds in the grooves between adjacent protein side-chains in the amyloid fibril core, parallel to the fibril axis, similar to thioflavin-T and congo red. Moreover, using LD we record the absorption spectrum of amyloid-bound PTAA in isolation from free dye showing a red-shift by ca 30 nm compared to in solution. This has important implications for the use of PTAA as an amyloid probe in situ and in vitro and we demonstrate how to obtain optimal amyloid-specific fluorescence read-outs using PTAA. We use the shift in maximum absorption to estimate the fraction of bound PTAA at a given concentration. PTAA binding reaches saturation when added in 36 times excess and at this concentration the PTAA density is 4–5 monomer units per insulin monomer in the fibril. Finally, we demonstrate that changes in LD intensity can be related to alterations in persistence length of amyloid fibrils resulting from changes in solution conditions, showing that this technique is useful to assess macroscopic properties of these biopolymers.     

Study of interaction between Smad7 and DNA by single-molecule force spectroscopy

Smad7 is an antagonist of TGF-β signaling pathway and the mechanism of its inhibitory effect is of great interest. We recently found that Smad7 could function in the nucleus by binding to the DNA elements containing the minimal Smad binding element CAGA box. In this work, we further applied single-molecule force spectroscopy to study the DNA-binding property of Smad7. Smad7 showed similar binding strength to the oligonucleotides corresponding to the CAGA-containing activin responsive element (ARE) and the PAI-1 promoter, as that of Smad4. However, Smad7 also exhibited a binding activity to the mutant ARE with the CAGA sequence substituted, indicating its DNA-binding specificity is different from other Smads. Moreover, we demonstrated that the MH2 domain of Smad7 had a higher binding affinity to the DNA elements than the full-length Smad7, while the N-terminal domain exhibited an inhibitory effect.     

Structural changes of human RNase L upon homodimerization investigated by Raman spectroscopy

RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-β structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.     

Energy landscape investigation by wavelet transform analysis of atomic force spectroscopy data in a biorecognition experiment

Force fluctuations recorded in an atomic force spectroscopy experiment, during the approach of a tip functionalized with biotin towards a substrate charged with avidin, have been analyzed by a wavelet transform. The observation of strong transient changes only when a specific biorecognition process between the partners takes place suggests a drastic modulation of the force fluctuations when biomolecules recognize each other. Such an analysis allows to investigate the peculiar features of a biorecognition process. These results are discussed in connection with the possible role of energy minima explored by biomolecules during the biorecognition process.