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The supramolecular organization of fibrillin-rich microfibrils.

Baldock C, Koster AJ, Ziese U, Rock MJ, Sherratt MJ, Kadler KE, Shuttleworth CA, Kielty CM - J. Cell Biol. (2001)

Bottom Line: Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare.Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section.We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Schools of Biological Sciences and Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom. clair.baldock@man.ac.uk

ABSTRACT
We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

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Antibody and gold binding to microfibrils extended by surface tension. (A) Fibrillin-rich microfibrils were extended by surface tension, and then labeled with 5-nm colloidal gold and negatively stained. (B) Negatively stained images of microfibrils labeled with antibody12A5.18, either untensioned or extended by surface tension. Only extension up to ∼10 nm is tolerated before the antibody banding pattern is lost. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position is represented by blue arrows.
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Figure 3: Antibody and gold binding to microfibrils extended by surface tension. (A) Fibrillin-rich microfibrils were extended by surface tension, and then labeled with 5-nm colloidal gold and negatively stained. (B) Negatively stained images of microfibrils labeled with antibody12A5.18, either untensioned or extended by surface tension. Only extension up to ∼10 nm is tolerated before the antibody banding pattern is lost. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position is represented by blue arrows.

Mentions: Several approaches were investigated to generate extended isolated microfibrils. When microfibril preparations were centrifuged at 60,000 g for 1 h, a small proportion of microfibrils appeared stretched in the range 70–110 nm, but most retained untensioned periodicity. When microfibril preparations were repeatedly drawn through narrow bore needles, the majority of microfibrils retained untensioned (∼56 nm) periodicity, although a few were extended to ∼70 nm. Interbead morphology of many of these microfibrils appeared diffuse, suggesting conformation change (not shown). However, a significant number of microfibrils in extended state (70–110-nm range) were captured from sample drop–air interfaces directly onto carbon-coated grids (Fig. 3). Interbead morphology of these microfibrils was diffuse, indicating major conformational changes. By contrast, canine zonules associated with dislocated lenses contained numerous stable highly extended microfibrils (see Fig. 4).


The supramolecular organization of fibrillin-rich microfibrils.

Baldock C, Koster AJ, Ziese U, Rock MJ, Sherratt MJ, Kadler KE, Shuttleworth CA, Kielty CM - J. Cell Biol. (2001)

Antibody and gold binding to microfibrils extended by surface tension. (A) Fibrillin-rich microfibrils were extended by surface tension, and then labeled with 5-nm colloidal gold and negatively stained. (B) Negatively stained images of microfibrils labeled with antibody12A5.18, either untensioned or extended by surface tension. Only extension up to ∼10 nm is tolerated before the antibody banding pattern is lost. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position is represented by blue arrows.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198817&req=5

Figure 3: Antibody and gold binding to microfibrils extended by surface tension. (A) Fibrillin-rich microfibrils were extended by surface tension, and then labeled with 5-nm colloidal gold and negatively stained. (B) Negatively stained images of microfibrils labeled with antibody12A5.18, either untensioned or extended by surface tension. Only extension up to ∼10 nm is tolerated before the antibody banding pattern is lost. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position is represented by blue arrows.
Mentions: Several approaches were investigated to generate extended isolated microfibrils. When microfibril preparations were centrifuged at 60,000 g for 1 h, a small proportion of microfibrils appeared stretched in the range 70–110 nm, but most retained untensioned periodicity. When microfibril preparations were repeatedly drawn through narrow bore needles, the majority of microfibrils retained untensioned (∼56 nm) periodicity, although a few were extended to ∼70 nm. Interbead morphology of many of these microfibrils appeared diffuse, suggesting conformation change (not shown). However, a significant number of microfibrils in extended state (70–110-nm range) were captured from sample drop–air interfaces directly onto carbon-coated grids (Fig. 3). Interbead morphology of these microfibrils was diffuse, indicating major conformational changes. By contrast, canine zonules associated with dislocated lenses contained numerous stable highly extended microfibrils (see Fig. 4).

Bottom Line: Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare.Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section.We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Schools of Biological Sciences and Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom. clair.baldock@man.ac.uk

ABSTRACT
We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Show MeSH
Related in: MedlinePlus