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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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Negatively stained transmission electron microscope images showing primary antibody binding sites on isolated untensioned microfibrils. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position by colored arrows or lines. In higher magnification images, the bead and arm positions are indicated in green. (A) Antibody 2502 (red) binds human zonular microfibrils close to the bead on the opposite side to the arms. (B) Antibodies 11C1.3 or 12A5.18 (blue) binds bovine zonular microfibrils at the interbead striation where the arms terminate. (C) Antibody PF2 (purple) binds bovine zonular microfibrils near the middle of the interbead. (D) Antibody 2499 (orange) binds bovine zonular microfibrils on the arms close to the bead. (E) Immunofluorescence of fibrillin-1 microfibrils assembled from human dermal fibroblasts (HDFs). Cultured slides were immunolabeled with the monoclonal antibody 11C1.3 or the polyclonal antibody PF2. Fluorescence was detected using a secondary antibody conjugated to a CY3 label, and nuclei were stained with DAPI. PF2 detected microfibril structures at 3 d, whereas 11C1.3 only detected microfibrils from 14 d onwards. (a) 3-d HDF culture labeled with 1:100 dilution 11C1.3. (b) 3-d culture with 1:100 dilution PF2 (same exposure time as in a). (c) 3-d HDF with 1:20 dilution 11C1.3. (d) 3-d HDF with PF2 (same exposure as in c). (e) 14-d HDF labeled with 11C1.3. (f) 14-d HDF labeled with PF2. (g) 21-d HDF stained with DAPI and labeled with 11C1.3. (h) 21-d HDF with 11C1.3 only. (i) 21-d HDF stained with DAPI and labeled with PF2. (j) 21-d HDF labeled with PF2 only.
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Figure 2: Negatively stained transmission electron microscope images showing primary antibody binding sites on isolated untensioned microfibrils. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position by colored arrows or lines. In higher magnification images, the bead and arm positions are indicated in green. (A) Antibody 2502 (red) binds human zonular microfibrils close to the bead on the opposite side to the arms. (B) Antibodies 11C1.3 or 12A5.18 (blue) binds bovine zonular microfibrils at the interbead striation where the arms terminate. (C) Antibody PF2 (purple) binds bovine zonular microfibrils near the middle of the interbead. (D) Antibody 2499 (orange) binds bovine zonular microfibrils on the arms close to the bead. (E) Immunofluorescence of fibrillin-1 microfibrils assembled from human dermal fibroblasts (HDFs). Cultured slides were immunolabeled with the monoclonal antibody 11C1.3 or the polyclonal antibody PF2. Fluorescence was detected using a secondary antibody conjugated to a CY3 label, and nuclei were stained with DAPI. PF2 detected microfibril structures at 3 d, whereas 11C1.3 only detected microfibrils from 14 d onwards. (a) 3-d HDF culture labeled with 1:100 dilution 11C1.3. (b) 3-d culture with 1:100 dilution PF2 (same exposure time as in a). (c) 3-d HDF with 1:20 dilution 11C1.3. (d) 3-d HDF with PF2 (same exposure as in c). (e) 14-d HDF labeled with 11C1.3. (f) 14-d HDF labeled with PF2. (g) 21-d HDF stained with DAPI and labeled with 11C1.3. (h) 21-d HDF with 11C1.3 only. (i) 21-d HDF stained with DAPI and labeled with PF2. (j) 21-d HDF labeled with PF2 only.

Mentions: Fibrillin-1 antibody epitopes accessible on isolated untensioned microfibrils were mapped after incubation, centrifugation, and ultrastructural examination (Fig. 2 and Table ). Antibody 2502, which recognizes an NH2-terminal fibrillin-1 sequence within residues 45–450 (referred to as antibody 26 in Reinhardt et al. 1996), bound human zonular microfibrils (Fig. 2 A), but not bovine zonular microfibrils (not shown). This antibody generated both parallel microfibril arrays with beads in-register and antiparallel microfibrils with beads offset. The binding site was close to the bead on the side opposite the arms [15.9% (2.5% SD) of bead-to-bead distance]. Antibody 11C1.3 or 12A5.18 [epitope(s) within fibrillin-1 residues 654–909] bound to bovine zonular microfibrils (Fig. 2 B) as well as human and murine microfibrils (not shown) at the interbead striation where the arms terminate. Incubation with either of these antibodies generated numerous extensive parallel double-banded microfibril arrays with beads in-register and the antibody binding at 41.1% of bead-to-bead distance. The PF2 antibody, which recognizes pepsin fragment PF2 (sequences encoded by exons 41–45; Maddox et al. 1989; Maslen et al. 1991), bound close to the centre of the interbead (either 47.1 or 52.9% of bead-to-bead distance, microfibril orientation was difficult to establish because of the interbead binding position; Fig. 2 C). A few microfibril arrays were detected, but these preparations mainly contained single antibody-associated microfibrils. Antibody 2499, which recognizes a COOH-terminal fibrillin-1 sequence within residues 2093–2732 (assuming furin cleavage; referred to as antibody 69 in Reinhardt et al. 1996), generated bovine zonular microfibril arrays (Fig. 2 D) and human zonular microfibril arrays (not shown) that were either parallel with beads-in-register or antiparallel with beads offset. The binding site was on the arms close to the bead [20.2% of the bead-to-bead distance (2.1% SD)], on the same side as antibody 12A5.18.


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)

Negatively stained transmission electron microscope images showing primary antibody binding sites on isolated untensioned microfibrils. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position by colored arrows or lines. In higher magnification images, the bead and arm positions are indicated in green. (A) Antibody 2502 (red) binds human zonular microfibrils close to the bead on the opposite side to the arms. (B) Antibodies 11C1.3 or 12A5.18 (blue) binds bovine zonular microfibrils at the interbead striation where the arms terminate. (C) Antibody PF2 (purple) binds bovine zonular microfibrils near the middle of the interbead. (D) Antibody 2499 (orange) binds bovine zonular microfibrils on the arms close to the bead. (E) Immunofluorescence of fibrillin-1 microfibrils assembled from human dermal fibroblasts (HDFs). Cultured slides were immunolabeled with the monoclonal antibody 11C1.3 or the polyclonal antibody PF2. Fluorescence was detected using a secondary antibody conjugated to a CY3 label, and nuclei were stained with DAPI. PF2 detected microfibril structures at 3 d, whereas 11C1.3 only detected microfibrils from 14 d onwards. (a) 3-d HDF culture labeled with 1:100 dilution 11C1.3. (b) 3-d culture with 1:100 dilution PF2 (same exposure time as in a). (c) 3-d HDF with 1:20 dilution 11C1.3. (d) 3-d HDF with PF2 (same exposure as in c). (e) 14-d HDF labeled with 11C1.3. (f) 14-d HDF labeled with PF2. (g) 21-d HDF stained with DAPI and labeled with 11C1.3. (h) 21-d HDF with 11C1.3 only. (i) 21-d HDF stained with DAPI and labeled with PF2. (j) 21-d HDF labeled with PF2 only.
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Figure 2: Negatively stained transmission electron microscope images showing primary antibody binding sites on isolated untensioned microfibrils. Microfibril direction is indicated by a white arrow, bead position by green arrows, and antibody position by colored arrows or lines. In higher magnification images, the bead and arm positions are indicated in green. (A) Antibody 2502 (red) binds human zonular microfibrils close to the bead on the opposite side to the arms. (B) Antibodies 11C1.3 or 12A5.18 (blue) binds bovine zonular microfibrils at the interbead striation where the arms terminate. (C) Antibody PF2 (purple) binds bovine zonular microfibrils near the middle of the interbead. (D) Antibody 2499 (orange) binds bovine zonular microfibrils on the arms close to the bead. (E) Immunofluorescence of fibrillin-1 microfibrils assembled from human dermal fibroblasts (HDFs). Cultured slides were immunolabeled with the monoclonal antibody 11C1.3 or the polyclonal antibody PF2. Fluorescence was detected using a secondary antibody conjugated to a CY3 label, and nuclei were stained with DAPI. PF2 detected microfibril structures at 3 d, whereas 11C1.3 only detected microfibrils from 14 d onwards. (a) 3-d HDF culture labeled with 1:100 dilution 11C1.3. (b) 3-d culture with 1:100 dilution PF2 (same exposure time as in a). (c) 3-d HDF with 1:20 dilution 11C1.3. (d) 3-d HDF with PF2 (same exposure as in c). (e) 14-d HDF labeled with 11C1.3. (f) 14-d HDF labeled with PF2. (g) 21-d HDF stained with DAPI and labeled with 11C1.3. (h) 21-d HDF with 11C1.3 only. (i) 21-d HDF stained with DAPI and labeled with PF2. (j) 21-d HDF labeled with PF2 only.
Mentions: Fibrillin-1 antibody epitopes accessible on isolated untensioned microfibrils were mapped after incubation, centrifugation, and ultrastructural examination (Fig. 2 and Table ). Antibody 2502, which recognizes an NH2-terminal fibrillin-1 sequence within residues 45–450 (referred to as antibody 26 in Reinhardt et al. 1996), bound human zonular microfibrils (Fig. 2 A), but not bovine zonular microfibrils (not shown). This antibody generated both parallel microfibril arrays with beads in-register and antiparallel microfibrils with beads offset. The binding site was close to the bead on the side opposite the arms [15.9% (2.5% SD) of bead-to-bead distance]. Antibody 11C1.3 or 12A5.18 [epitope(s) within fibrillin-1 residues 654–909] bound to bovine zonular microfibrils (Fig. 2 B) as well as human and murine microfibrils (not shown) at the interbead striation where the arms terminate. Incubation with either of these antibodies generated numerous extensive parallel double-banded microfibril arrays with beads in-register and the antibody binding at 41.1% of bead-to-bead distance. The PF2 antibody, which recognizes pepsin fragment PF2 (sequences encoded by exons 41–45; Maddox et al. 1989; Maslen et al. 1991), bound close to the centre of the interbead (either 47.1 or 52.9% of bead-to-bead distance, microfibril orientation was difficult to establish because of the interbead binding position; Fig. 2 C). A few microfibril arrays were detected, but these preparations mainly contained single antibody-associated microfibrils. Antibody 2499, which recognizes a COOH-terminal fibrillin-1 sequence within residues 2093–2732 (assuming furin cleavage; referred to as antibody 69 in Reinhardt et al. 1996), generated bovine zonular microfibril arrays (Fig. 2 D) and human zonular microfibril arrays (not shown) that were either parallel with beads-in-register or antiparallel with beads offset. The binding site was on the arms close to the bead [20.2% of the bead-to-bead distance (2.1% SD)], on the same side as antibody 12A5.18.

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