Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales

Godwin, Alan R.; Starborg, Tobias; Sherratt, Michael J.; Roseman, Alan M.; Baldock, Clair

doi:10.1016/j.actbio.2016.12.023

Cited by 33 publications

(32 citation statements)

References 69 publications

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“…Splice-modulating antisense oligomers targeting the pseudoexon pre-mRNA sequence are used to promote its skipping as a therapeutic strategy. (E) Splicing reporter (minigene) constructs were prepared by subcloning various segments of COL6A1 genomic DNA (intervening sequence 11 , exons 11 to 13 [Ex- [11][12][13], or exons 10 to 13 [Ex-10-13]) into the pET01 vector (top). Minigenes were transfected into HEK293T cells, and RNA was isolated 24 hours later.…”

Section: Resultsmentioning

confidence: 99%

“…Monomers join laterally to form staggered antiparallel dimers, which then associate in parallel to form a 112-nm-long tetramer. The tetramers are subsequently secreted into the extracellular space where they form higher-order structures by end-to-end association and branching (9)(10)(11)(12)(13)(14). Functional collagen VI is a connected meshwork of tetramers, ultrastructurally appearing as beaded microfibrils (15).…”

Section: Introductionmentioning

confidence: 99%

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A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

Bolduc¹,

Foley²,

Solomon-Degefa³

et al. 2019

JCI Insight

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

Bolduc¹,

Foley²,

Solomon-Degefa³

et al. 2019

JCI Insight

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“…WT and homozygous eyes were prepared for serial block face scanning electron microscopy (SBF-SEM) as previously described ( 43 , 44 ). Eyes were oriented so the CZ to lens capsule was imaged in longitudinal cross section.…”

Section: Methodsmentioning

confidence: 99%

ADAMTS10-mediated tissue disruption in Weill–Marchesani syndrome

Mularczyk

Singh

Godwin

et al. 2018

Human Molecular Genetics

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Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-β superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill–Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

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“…The initial template was an average image of the unaligned stack of particles. An initial 3D model was constructed by creating a cylindrical average model from the sum average of the aligned particle set, using SPIDER, filtered to 20 Å; this was subsequently refined using a projection matching approach as we described for collagen VI microfibrils [49] . To evaluate the reconstructions, projections were compared non-quantitatively to class sum images created with a non-reference based classification method using IMAGIC5 [50] .…”

Section: Methodsmentioning

confidence: 99%

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils

Godwin

Starborg

Smith

et al. 2018

Journal of Molecular Biology

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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.

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Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales

Abstract: Graphical abstract

Cited by 33 publications

References 69 publications

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

ADAMTS10-mediated tissue disruption in Weill–Marchesani syndrome

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils

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