Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils

Abstract

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.

Structural studies of elastic fibre and microfibrillar proteins

“…B) A bead model computed from SAXS data of a region of the fibrillin molecule (protein fragment 17 (PF17)(cbEGF16-22-TB4 cbEGF23-24-TB5-cbEGF25)) with homology models of domains docked into the density [91] . C) A negative stain structure of an extracted ciliary zonule fibrillin microfibril [109] . D) A 3D segmentation of an electron tomogram of a ciliary zonule fibre next to the ciliary body basement membrane (BM) which is coloured in green [109] .…”

“…C) A negative stain structure of an extracted ciliary zonule fibrillin microfibril [109] . D) A 3D segmentation of an electron tomogram of a ciliary zonule fibre next to the ciliary body basement membrane (BM) which is coloured in green [109] . The ciliary body epithelial cell is shown in gold with its plasma membrane (PM) segmented in blue.…”

“…A ciliary zonule fibre (highlighted in orange) can be seen wrapping around a bundle of fibres (shown in blue). A single ciliary zonule fibre has been highlighted with a red circle [109] . …”

“…Microfibrils have a distinct asymmetric banding pattern and can be sub-divided into regions which have been named the bead, arm, interbead and shoulder regions [20] . Structural studies to determine the nanostructure of fibrillin microfibrils using negative-stain TEM and single particle analysis [109] , revealed greater detail of how fibrillin is organised in mature microfibrils. The dense bead region has an outer ring with a complex interwoven core which is connected to four separate arm regions.…”

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis

“…Serial block‐face scanning electron microscopy (SBF‐SEM) imaging and thick‐section electron tomography showed that FMF contain a central cavity in the interbead region, where eight symmetrically arranged fibrillin monomers can be accommodated. [ 22 ] Interestingly, AFM analysis in combination with mass spectrometry revealed that the bead morphology of skin FMF significantly differs from that of eye FMF due to their tissue‐specific molecular composition. [ 23 ]…”