The 10+4 microfibril structure of thin cartilage fibrils

Abstract

Determining the structure of cartilage collagen fibrils will provide insights into how mutations in collagen genes affect cartilage formation during skeletal morphogenesis and understanding the mechanism of fibril growth. The fibrils are indeterminate in size, heteropolymeric, and highly cross-linked, which make them refractory to analysis by conventional high-resolution structure determination techniques. Electron microscopy has been limited to making simple measurements of fibril diameter and immunolocalizing certain molecules at the fibril surface. Consequently, structural information on the fibrils is limited. In this study we have used scanning transmission electron microscopic mass mapping, analysis of axial stain exclusion pattern, and r-weighted back-projection techniques to determine the intermediate resolution (to Ϸ4 nm) structure of thin collagen fibrils from embryonic cartilage. The analyses show that the fibrils are constructed from a 10؉4 microfibrillar arrangement in which a core of four microfibrils is surrounded by a ring of 10 microfibrils. Accurate mass measurements predict that each microfibril contains five collagen molecules in cross-section. Based on the proportion of collagen II, IX, and XI in the fibrils, the fibril core comprises two microfibrils each of collagen II and collagen XI. Single molecules of collagen IX presumably occur at the fibril surface between the extended N-terminal domains of collagen XI. The 10؉4 microfibril structure explains the mechanism of diameter limitation in the narrow fibrils and the absence of narrow collagen fibrils in cartilage lacking collagen XI.chondrodysplasia ͉ collagen ͉ electron microscopy ͉ mass mapping ͉ reconstruction T he ability of cartilage to withstand cycles of compression and relaxation relies on a felt-like extracellular matrix (ECM) of insoluble collagen fibrils within a concentrated solution of proteoglycans and glycoconjugates. The collagen fibrils withstand the swelling pressure exerted on the ECM by the hydrated glycosaminoglycan side chains of the proteoglycans. However, this apparently simple role of the collagen fibrils belies enormous effort over many years to understand fibril structure and function as a means of explaining how mutations in cartilage fibril genes produce developmental defects. In particular, it is perplexing why cartilage has two distinct populations of collagen fibrils; one thin (Ϸ20-nm diameter) and the other thick (Ϸ40-nm diameter). Also, it is a quandary why cartilage fibrils of diameter between 20 and 40 nm do not exist in cartilage; also whereas the thin fibrils are all Ϸ20 nm in diameter, the thick fibrils have a much broader diameter distribution. In this respect, the thick fibrils are more like fibrils in noncartilagenous tissues, which can range from 30 to 500 nm in diameter (1). We reasoned that determination of the structure of the thin fibrils was an essential first step in understanding cartilage fibril structure and function.Cartilage fibrils are D-periodic (where D Ϸ 67 nm), indeterminate in leng...

Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse

“…To speculate, the α1(XI) collagen chain and in particular its N-propeptide domain may play a regulatory role in restricting the diameter of type II collagen fibrils assembled in developing cartilages. Exactly how the N-propeptide domain may affect fibril diameter is unclear, but recent structural analysis (Fallahi et al, 2005) and microfibril modeling predictions suggest that the N-propeptide globular domains are excluded from the microfibril interior, encircle the fibril and prevent further lateral aggregation of collagen molecules (Holmes and Kadler, 2006). The absence of α1(XI) chains from type V/XI hybrid molecules in cho/cho cartilage (Figure 2, tables 1, 2) may be responsible at least in part for an impaired regulation of type II collagen fibril diameters.…”

“…Type XI collagen molecules are also cross-linked to type II collagen presumably as co-components of heterofibrils (Wu and Eyre, 1995). Since type XI collagen is proposed to be within the core of type II collagen fibrils (Mendler et al, 1989), it is becoming clear that type XI collagen may act as both the template for type II collagen fibrillogenensis and as a regulator of fibril diameter in cartilage (Blaschke et al, 2000;Holmes and Kadler, 2006;Wu and Eyre, 1995).…”

The binding capacity of α1β1-, α2β1- and α10β1-integrins depends on non-collagenous surface macromolecules rather than the collagens in cartilage fibrils

“…According to these predictions, binding of A-domains as well as entire integrins could occur also to collagen fibrils. However, this notion is difficult to reconcile with the structural model of thin cartilage fibrils as proposed by Holmes and Kadler [46] in which, both, collagen II and XI molecules bearing potential integrin-binding sites are tightly packed into fibrils composed of 10 and 4 microfibrils located at the surface and the core of the fibrils, respectively. Thus, the accessibility of collagen molecules to integrins would be severely limited.…”

Nano-Morphology of Cartilage in Hydrated and Dehydrated Conditions Revealed by Atomic Force Microscopy

“…Although no D-banding patterns were found along these fibrils, we suspect they are also type II collagen fibrils. Distinctively thin fibrils were noted before and it was suggested that thin fibrils may serve as a precursor for fibrilogenesis [13]. The sub-fibrillar structure of ~20 nm thin-fibrils was described by Holmes et al as a "10+4" arrangement of microfibrils, which contains a core of four microfibrils and a ring of ten surrounding microfibrils [13].…”

“…Distinctively thin fibrils were noted before and it was suggested that thin fibrils may serve as a precursor for fibrilogenesis [13]. The sub-fibrillar structure of ~20 nm thin-fibrils was described by Holmes et al as a "10+4" arrangement of microfibrils, which contains a core of four microfibrils and a ring of ten surrounding microfibrils [13]. These 4 nm thin microfibril are constructed by five tropocollagens in the equatorial plane [13,14].…”