Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

New insight into the shortening of the collagen fibril D-period in human cornea

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066–.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

In vitro Synthesis of Native,Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.     

Fibrous long spacing type collagen fibrils have a hierarchical internal structure

Nanodissection of single fibrous long spacing (FLS) type collagen fibrils by atomic force microscopy (AFM) reveals hierarchical internal structure: Fibrillar subcomponents with diameters of approximately 10 to 20 nm were observed to be running parallel to the long axis of the fibril in which they are found. The fibrillar subcomponent displayed protrusions with characteristic approximately 270 nm periodicity, such that protrusions on neighboring subfibrils were aligned in register. Hence, the banding pattern of mature FLS-type collagen fibrils arises from the in-register alignment of these fibrillar subcomponents. This hierarchical organization observed in FLS-type collagen fibrils is different from that previously reported for native-type collagen fibrils, displaying no supercoiling at the level of organization observed.     

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.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.     

Neutron diffraction studies of collagen in fully mineralized bone

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.     

Electron microscopic study on the gel of the central part of the corpus vitreum in the ox

Summary The central part of the corpus vitreum in the ox possesses a relative firmness. Electron microscopically it has been shown to consist of collagen fibrils with interfibrillar spaces containing 8 nm thick granules. The granules are made up of chains of macromolecules (hyaluronic acid in an oligomer state) 130–200 nm in length and of an oval shape. The collagen fibrils are tightly covered with 8.5 nm thick macromolecules which represent highly polymerized hyaluronic acid. These macromolecules can be stained with ruthenium-red.Dedicated to Prof. Dr. med. Drs. h.c. W. Bargmann, Kiel, on the occasion of his 70th birthday     

A model for the ultrastructure of bone based on electron microscopy of ion-milled sections

The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures ("mineral structures") are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates.     

Estimation of the binding force of the collagen molecule-decorin core protein complex in collagen fibril

Decorin belongs to the small leucine proteoglycans family and is considered to play an important role in extracellular matrix organization. Experimental studies suggest that decorin is required for the assembly of collagen fibrils, as well as for the development of proper tissue mechanical properties. In tendons, decorins tie adjoining collagen fibrils together and probably guarantee the mechanical coupling of fibrils. The decorin molecule consists of one core protein and one glycosaminoglycan chain covalently linked to a serine residue of the core protein. Several studies have indicated that each core protein binds to the surface of collagen fibrils every 67 nm, by interacting non-covalently to one collagen molecule of the fibril surface, while the decorin glycosaminoglycans extend from the core protein to connect to another decorin core protein laying on adjacent fibril surface. The present paper investigates the complex composed of one decorin core protein and one collagen molecule in order to obtain their binding force. For this purpose, molecular models of collagen molecules type I and decorin core protein were developed and their interaction energies were evaluated by means of the molecular mechanics approach. Results show that the complex is characterized by a maximum binding force of about 12.4 x 10(3) nN and a binding stiffness of 8.33 x 10(-8) N/nm; the attained binding force is greater than the glycosaminoglycan chain's ultimate strength, thus indicating that overloads are likely to damage the collagen fibre's mechanical integrity by disrupting the glycosaminoglycan chains rather than by causing decorin core protein detachment from the collagen fibril.     

Assembly of collagen fibril meshes using gold nanoparticles functionalized with tris(hydroxymethyl)phosphine-alanine as multivalent cross-linking agents

We report on the use of tris(hydroxymethyl)phosphine-alanine (THPAL) functionalized gold nanoparticles as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure. Atomic force microscopy (AFM) was used for characterization of the structure after adsorption onto an atomically flat mica substrate, revealing a mesh-like construct in which the collagen fibrils and the gold nanoparticles interact to form interconnected nodes measuring from 100 to 500 nm. As expected, the density of the collagen mesh can be increased with a higher initial concentration of gold nanoparticles. The maximum thickness of the meshes (～ 20 nm) obtained through cross-sectional height measurements confirms that the adsorbed structure consists of a single layer of collagen fibrils/gold nanoparticles assembled in two-dimensions. We propose that the capability of gold nanoparticles functionalized with the THPAL to bind to several collagen fibrils combined with the large persistence length of the fibrils, which was reported to be in the hundreds of nanometer range, are determinant factors for the preferential 2D growth of the mesh in solution.     

Spatial Organization and Mineralization of the Basal Plate of Elasmoid Scales in Osteichthyans

In Sarcopterygii (Latimeria, Neoceratodus, Protopterus, Leptdosiren)and Amiidae (Amia) collagen fibrils of the basal plate are packedin bundles whereas they remain isolated in Teleostei. The basalplate looks like plywood, a system of superimposed layers ofparallel fibers or fibrils the directions of which rotate witha regular angle in two successive layers. The double twistedplywood is constituted of two imbricate systems, the odd andthe even, where the rotation of the fibrillar directions isright-handed in Sarcopterygii and lefthanded in Amiidae andnumerous primitive Teleostei. The orthogonal plywood, with itstwo main orthogonal fibrillar directions, characterizes theevolved Teleostei and some more primitive ones. In most teleosteanspecies, as in Amia and Protopterus, mineralization of the basalplate in elasmoid scales involves Mandl's corpuscles that mineralizewithout contact with a pre-existing calcified tissue; they growand coalesce with the neighbouring ones and fuse to the mineralizingfront. Their shape is directly influenced by the local arrangementof the collagenous fibrils. In two teleostean families (Osteoglossidaeand Mormyridae) Mandl's corpuscles are completely lacking butspreading of mineralization in the basal plate has a peculiaraspect. Whatever that may be, the various characteristic organizationsof the skeletal tissues or isopedine that constitute the basalplate of osteichthyan elasmoid scales, all are varieties ofbone tissue that have undergone more or less important specializationlinked to the general regression of dermal ossifications andto functional adaptations.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

Corneal morphogenesis in the Mov13 mutant mouse is characterized by normal cellular organization but disordered and thin collagen

This paper compares corneal development in the normal and in the Mov13 mutant mouse homozygote which does not synthesize type I collagen. During the period 12-14 days of development, there is no obvious difference between cellular organization in the normal and the mutant corneas or, indeed, elsewhere in the eye. In particular, there is normal colonization of the mutant cornea by the mesenchymal cells which will form the endothelium and the fibroblasts. In the early stages of stromal deposition (less than 14 days), when relatively little collagen is normally laid down, mutant and wild-type corneas differ only in that mutant collagen fibrils are less uniform than normal ones. Later development in the Mov13 mutant cannot usually be studied because almost all mutant embryos are dead by 14 days, but we now have two homozygous embryos from a single, 16-day litter. Their stromas obviously differed from those of their normal littermates: there was markedly less collagen in the mutant cornea and the collagen that was deposited lacked orthogonal organization. Fibril morphology also differed: the diameters of fibrils in the normal corneas peaked sharply at about 20 nm, whereas the diameters of mutant fibrils were spread over the range 5-15 nm, with only a small percentage overlapping the normal distribution. These results suggest that type I collagen is of negligible importance in controlling the cellular organization of the cornea, but has a dominant role in the formation of normal 20 nm fibrils and of normal stromal organization. They also show that, as collagen production is markedly lower in the mutant than in the wild-type cornea, the production of other collagens cannot compensate in any way for the lack of type I collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D=67nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.     

Collagen remodeling during decidualization in the mouse

Summary An ultrastructural study of the features and distribution of collagen fibrils was performed in the endometrium of virgin and pregnant (2nd to 11th day) mice. Collagen-containing structures were observed in the cytoplasm of fibroblasts on the 2nd day of pregnancy. Treatment of tissues with lanthanum nitrate established that these structures were intracytoplasmic. Their association with lysosome-like bodies suggested the occurrence of intracellular digestion of collagen, probably connected with remodeling of the endometrial stroma prior to decidualization. On the 4th day of pregnancy, very few collagen fibrils were present in the intercellular space. From the 6th day of pregnancy onwards, thick collagen fibrils were observed between decidual cells. The diameter of these fibrils measured up to 300 nm whereas the fibrils present in the endometrium of virgin mice measured 40–68 nm.