Effects of Fibrillin-1 Degradation on Microfibril Ultrastructure

Abstract

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.Microfibrils have been extracted from a variety of tissues and visualized by electron microscopy as distinctive beaded string structures (1, 2). The molecular composition of these structures is assumed to be complex. However, based upon immunolocalization of fibrillin to these structures (2, 3) and the shape of fibrillin monomers (4, 5), fibrillins are thought to be the major backbone components of these extracted microfibrils. Fibrillincontaining microfibrils are ubiquitous in the connective tissue space (6), providing architectural support as well as information essential for appropriate signaling during morphogenesis (7-9). In human disorders associated with fibrillins, the structural integrity of fibrillin microfibrils is required for the proper function of certain connective tissues (10). Therefore, determination of the organization of fibrillin molecules within the microfibril is important basic information.The organization of fibrillin molecules within microfibrils has been controversial. Both parallel (head-to-tail) (3, 4) and antiparallel (11) arrangements of fibrillin molecules within microfibrils have been...

Biogenesis of extracellular microfibrils: Multimerization of the fibrillin-1 C terminus into bead-like structures enables self-assembly

“…Although many publications over the last years address the static spatial organization of fibrillins in microfibrils (11)(12)(13)(14)(15)(16)(17)(18), very little information is available about the dynamic processes of microfibril formation. Revealing these mechanisms is critical for the understanding of the pathobiology of fibrillinopathies.…”

“…To advance the understanding of disease pathogenesis, detailed information on microfibril assembly mechanisms is urgently needed. Although current models for the static organization of fibrillins in microfibrils agree on a head-to-tail alignment with approximately eight fibrillin monomers in cross section, it is not clear whether the molecules are arranged in an unstaggered or staggered orientation (10)(11)(12)(13)(14)(15)(16)(17)(18). The initial formation of microfibrils from monomeric fibrillins most likely occurs close to or at the cell surface and involves formation of disulfide bonds in early stages (19).…”

Fibrillin Assembly Requires Fibronectin

“…Although many publications in recent years have addressed the spatial organization of fibrillins in microfibrils Downing et al, 1996;Liu et al, 1996;Reinhardt et al, 1996;Qian and Glanville, 1997;Baldock et al, 2001;Lee et al, 2004;Baldock et al, 2006;Kuo et al, 2007), relatively little information is available about mechanisms and components involved in microfibril formation. Revealing these mechanisms is ultimately critical for the understanding of the pathobiology of microfibrillopathies associated with mutations in fibrillins.…”

“…Extracted microfibrils from cell culture or tissues display a typical bead-on-a-string ultrastructure, having a 50 -55-nm periodicity when analyzed by electron microscopy after rotary shadowing Kielty et al, 1991). Although many publications over recent years have addressed the spatial organization of fibrillins as it relates to the bead-on-a-string microfibrillar structure Reinhardt et al, 1996;Downing et al, 1996;Liu et al, 1996;Qian and Glanville, 1997;Baldock et al, 2001Baldock et al, , 2006Lee et al, 2004;Kuo et al, 2007), little information is available about the dynamic processes and components involved in microfibril formation. The pathogenetic relevance of this is highlighted by the fact that microfibril assembly is frequently disturbed in patients with fibrillinopathies (Tiedemann et al, 2004).…”

Fibrillin‐rich microfibrils: Structural determinants of morphogenetic and homeostatic events

“…Fibrillins are produced prior to tropoelastin deposition and initially give rise to structures that are visualized by rotary shadowing electron microscopy as strings with regularly-spaced beads, the periodicity of which can extend from $50 to $200 nm depending on the tissue source and conditions used for microfibril extraction (Handford et al, 2000;Kielty et al, 2002;Hubmacher et al, 2006). Molecular models of microfibril assembly remain controversial and revolve around the extent to which these extracellular elements can stretch and recoil (Lee et al, 2004;Baldock et al, 2006;Kuo et al, 2007).…”