David F Holmes

University of Colorado at Boulder, Boulder, Colorado, United States

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Publications (71)228.88 Total impact

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    [Show abstract] [Hide abstract] ABSTRACT: Type I collagen-containing fibrils are major structural components of the extracellular matrix of vertebrate tissues, especially tendon, but how they are formed is not fully understood. MMP14 is a potent pericellular collagenase that can cleave type I collagen in vitro. Here we show that tendon development is arrested in Scleraxis-Cre::Mmp14 lox/lox mice that are unable to release collagen fibrils from plasma membrane fibripositors. In contrast to its role in collagen turnover in adult tissue, MMP14 promotes embryonic tissue formation by releasing collagen fibrils from the cell surface. Notably, tendons grow to normal size and collagen fibril release from fibripositors occurs in Col-r/r mice that have a mutated collagen-I that is uncleavable by MMPs. Furthermore, fibronectin (not collagen-I) accumulates in tendons of Mmp14-null mice. We propose a model for cell-regulated collagen fibril assembly during tendon development in which MMP14 cleaves a molecular bridge tethering collagen fibrils to the plasma membrane of fibripositors.
    Full-text · Article · Sep 2015 · eLife Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: eLife digest Young animals are able to grow in a way that allows them to maintain roughly the same shape until they reach their adult size. The growth of embryos is driven by increases in cell size and number, but it is less clear how the body grows after birth. By this point, many of the cells in the body are part of tendons and other fibrous tissues, where they are surrounded by a mesh of fibres made of collagen and other proteins. These fibres provide strength to the tissue, but may also restrict its ability to grow. Tendons connect muscles to bones. They contain fibres of collagen that run along their length, which enables them to cope with very strong pulling forces. Kalson et al. used electron microscopy to generate highly detailed three-dimensional models of mouse tendons at three stages: in the embryo, at birth and six weeks later. The experiments identified two stages in tendon development. During the first stage, the number of cells and fibres across the tendon is determined in the embryo. The fibres also slightly expand in diameter and form regular waves called crimps that are important for the structural strength of the tendon. The second stage happens after birth, during which the number of cells and fibres remains constant, but the tendons continue to grow because the fibres increase in diameter and length. The cells also move to form towers of cells running along the tendon. From these observations, Kalson et al. propose that the numbers and locations of the cells and collagen fibres that determine the shape and size of tendons are established in the embryo. The collagen fibres create a framework for the continued growth of the tendon after birth. Future challenges are to understand how the number and the arrangement of cells in the tendon is determined before the collagen fibres are made, and how these cells control the number of collagen fibres that form. DOI: http://dx.doi.org/10.7554/eLife.05958.002
    Preview · Article · May 2015 · eLife Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: Cell morphology data.DOI: http://dx.doi.org/10.7554/eLife.05958.009
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Cell-contact data.DOI: http://dx.doi.org/10.7554/eLife.05958.014
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Crimp structure data.DOI: http://dx.doi.org/10.7554/eLife.05958.017
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Fibril diameter and bundle data.DOI: http://dx.doi.org/10.7554/eLife.05958.006
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: This file contains all the source data reported in the manuscript. DOI: http://dx.doi.org/10.7554/eLife.05958.021
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Cell number data.DOI: http://dx.doi.org/10.7554/eLife.05958.012
    Preview · Dataset · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Desmosomes and adherens junctions are intercellular adhesive structures essential for the development and integrity of vertebrate tissue, including the epidermis and heart. Their cell adhesion molecules are cadherins: type 1 cadherins in adherens junctions and desmosomal cadherins in desmosomes. A fundamental difference is that desmosomes have a highly ordered structure in their extracellular region and exhibit calcium-independent hyperadhesion, whereas adherens junctions appear to lack such ordered arrays, and their adhesion is always calcium-dependent. We present here the structure of the entire ectodomain of desmosomal cadherin desmoglein 2 (Dsg2), using a combination of small-angle X-ray scattering, electron microscopy, and solution-based biophysical techniques. This structure reveals that the ectodomain of Dsg2 is flexible even in the calcium-bound state and, on average, is shorter than the type 1 cadherin crystal structures. The Dsg2 structure has an excellent fit with the electron tomography reconstructions of human desmosomes. This fit suggests an arrangement in which desmosomal cadherins form trans interactions but are too far apart to interact in cis, in agreement with previously reported observations. Cadherin flexibility may be key to explaining the plasticity of desmosomes that maintain tissue integrity in their hyperadhesive form, but can adopt a weaker, calcium-dependent adhesion during wound healing and early development.
    Full-text · Article · Apr 2015 · Proceedings of the National Academy of Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: The small GTPase RhoA is a major regulator of actin reorganization during the formation of stress fibers; thus identifying molecules that regulate Rho activity is necessary for a complete understanding of the mechanisms that determine cell contractility. Here, we have identified Arhgap28 as a Rho GTPase activating protein (RhoGAP) that switches RhoA to its inactive form. We generated an Arhgap28-LacZ reporter mouse that revealed gene expression in soft tissues at E12.5, pre-bone structures of the limb at E15.5, and prominent expression restricted mostly to ribs and limb long bones at E18.5 days of development. Expression of recombinant Arhgap28-V5 in human osteosarcoma SaOS-2 cells caused a reduction in the basal level of RhoA activation and disruption of actin stress fibers. Extracellular matrix assembly studies using a 3-dimensional cell culture system showed that Arhgap28 was upregulated during Rho-dependent assembly of the ECM. Taken together, these observations led to the hypothesis that an Arhgap28 knockout mouse model would show a connective tissue phenotype, perhaps affecting bone. Arhgap28-null mice were viable and appeared normal, suggesting that there could be compensation from other RhoGAPs. Indeed, we showed that expression of Arhgap6 (a closely related RhoGAP) was upregulated in Arhgap28-null bone tissue. An upregulation in RhoA expression was also detected suggesting that Arhgap28 may be able to additionally regulate Rho signaling at a transcriptional level. Microarray analyses revealed that Col2a1, Col9a1, Matn3, and Comp that encode extracellular matrix proteins were downregulated in Arhgap28-null bone. Although mutations in these genes cause bone dysplasias no bone phenotype was detected in the Arhgap-28 null mice. Together, these data suggest that the regulation of Rho by RhoGAPs, including Arhgap28, during the assembly and development of mechanically strong tissues is complex and may involve multiple RhoGAPs.
    Full-text · Article · Sep 2014 · PLoS ONE
  • [Show abstract] [Hide abstract] ABSTRACT: Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1-10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures.
    No preview · Article · Nov 2013 · Proceedings of the National Academy of Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ∼8 d from isolating the tissue to having a 3D image stack.
    Full-text · Article · Jun 2013 · Nature Protocol
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    [Show abstract] [Hide abstract] ABSTRACT: The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.
    Full-text · Article · Jun 2012 · PLoS ONE
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    Dataset: Figure S4
    [Show abstract] [Hide abstract] ABSTRACT: Domain architecture of the different recombinant proteins and constructs used in this study. Key to domain labels: PfN, phage fibre N-terminal domain; PCoil, phage coil domain; Col, collagen domain; PfC, phage fibre C-terminal domain; H, hexahistidine tag; Trx, thioredoxin tag. (PDF)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Analysis of rEPclA and its Col–PfC fragment by SEC/MALLS. (A) Chromatogram showing the elution of nickel-affinity purified rEPclA from a Superose 6 10/300 GL size exclusion column; the red trace corresponds to the light scattering detector and the green trace to the UV absorption detector, both in arbitrary units. Peak 1 corresponds to the void volume and contains high molecular aggregates; peak 2 corresponds to native rEPclA. (B) Molar mass distribution or native rEPclA (peak 2 in A) measured by light scattering. The blue trace corresponds to the refractive index detector (in arbitrary units) and the dashed black line shows the weight-average molecular mass for each slice, as measured by the light scattering detector. The molar mass distribution is consistent with trimeric rEPclA (Table 5). (C) Chromatogram showing the elution of a nickel-affinity purified auto-induction sample of rEPclA from a Superdex 200 10/300 GL size exclusion column (traces as in panel A). Peak 1 corresponds to the void volume and contains high molecular aggregates; peaks 2 and 3 show molar mass distributions consistent with trimeric rEPclA and trimeric Col–PfC fragment, respectively (Table 5); peak 4 is consistent with monomeric rEPclA. (D) Molar mass distribution of peak 3 from C (Col–PfC) re-chromatographed in the same Superdex 200 column. The blue trace corresponds to the refractive index detector (arbitrary units) and the dashed black line shows the weight-average molecular mass for each slice, as measured by the light scattering detector. The molar mass distribution is consistent with trimeric Col–PfC (Table 5). (PDF)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S2
    [Show abstract] [Hide abstract] ABSTRACT: Large-scale expression of rEPclA: SDS-PAGE analysis of the different fractions after purification by nickel affinity chromatography. Individual peptides identified by mass spectrometry on each protein band are shown in red against the original EPclA sequence. (A) Overexpression of rEPclA by IPTG induction. Lane 1: molecular weight markers; lane 2: flow-through; lanes 3–4: fractions eluted with 5 mM and 100 mM imidazole (washes); lanes 5–10: fractions eluted with 1 M imidazole. The overexpressed band of rEPclA, confirmed by mass spectrometry, shows an apparent molecular weight of ∼66 kDa (higher than the true molecular weight of 47 kDa). (B) Overexpression of rEPclA by auto-induction. Lane 1: molecular weight markers; lanes 2–10: fractions eluted with 500 mM imidazole. The rEPclA band runs at ∼66 kDa, also confirmed by mass spectrometry. Two additional protein bands were identified by mass spectrometry as endogenous proteolytic fragments of rEPclA fragments. The mapped peptides reveal the extent and domain composition of each fragment. The band corresponding to the Col–PfC fragment shows an apparent molecular weight of ∼30 kDa (higher than the predicted molecular weight of ∼21 kDa). Another band at ∼60 kDa seems to correspond to a fragment with a partial digestion of the PfN domain and including the PCoil–Col–PfC domains. (PDF)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S3
    [Show abstract] [Hide abstract] ABSTRACT: Analysis of PfN, PfN–PCoil and Trx–PfC fragments by SEC/MALLS. (A) Chromatogram showing the elution of nickel-affinity purified PfN–PCoil fragment (green trace) or PfN fragment (red trace), from a Superdex 200 10/300 GL size exclusion column. Both traces correspond to the UV absorption detector, in arbitrary units. The dashed green and red lines show weight-average molecular masses for each slice of peaks 1 to 4, as measured by the light scattering detector. Peaks 1 and 2 correspond to trimeric and monomeric PfN–PCoil fragment, respectively, whereas peaks 3 and 4 correspond to trimeric and monomeric PfN. Molar mass distributions on each peak are consistent with these oligomerization states (Table 5). The predominant species in the PfN–PCoil sample is the trimer (peak 1), but a small amount of monomer (peak 2) can be detected. For PfN the predominant species is the monomer (peak 4), but a small amount of trimer (peak 3) can be detected. Elution volumes appear to be non-linear between the two purifications as the PfN–PCoil monomer elutes at a lower volume than the PfN trimer. (B) Molar mass distribution or the Trx–PfC fragment, measured by light scattering. The blue trace corresponds to the refractive index detector (in arbitrary units) and the dashed black line shows the weight-average molecular mass for each slice, as measured by the light scattering detector. The molar mass distribution is consistent with trimeric Trx–PfC (Table 5). (PDF)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S5
    [Show abstract] [Hide abstract] ABSTRACT: Nucleotide and amino acid sequences of rEPclA from DNA sequencing of the product amplified from a sample of genomic DNA from E. coli O157:H7 Sakai and cloned into a pET-28a(+) expression vector (see Methods). Sequence colour code: red, PfN domain; orange, PCoil domain; green, Col domain; blue, PfC domain; black, additional amino acids introduced by cloning to the protein expression vector, including N-terminal and C-terminal hexahistidine tags and a thrombin cleavage site preceding the PfN domain. Twelve nucleotide changes with respect to the most similar deposited EPclA sequence (ECs2717) are highlighted in yellow. Of those, eight are silent and four lead to changes in the amino acid sequence, also highlighted in yellow. All these amino acid changes correspond to normal sequence variability amongst EPclA sequences from different O157:H7 strains. (PDF)
    Preview · Dataset · Jun 2012
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    [Show abstract] [Hide abstract] ABSTRACT: A distinctive feature of embryonic tendon development is the steady increase in collagen fibril diameter and associated improvement of tissue mechanical properties. A potential mechanical stimulus for these changes is slow stretching of the tendon during limb growth. Testing this hypothesis in vivo is complicated by the presence of other developmental processes including muscle development and innervation. Here we used a cell culture tendon-like construct to determine if slow stretch can explain the increases in fibril diameter and mechanical properties that are observed in vivo. Non-stretched constructs had an ultrastructural appearance and mechanical properties similar to those of early embryonic tendon. However, slowly stretching during 4 days in culture increased collagen fibril diameter, fibril packing volume, and mechanical stiffness, and thereby mimicked embryonic development. 3D EM showed cells with improved longitudinal alignment and elongated nuclei, which raises the hypothesis that nuclear deformation could be a novel mechanism during tendon development.
    Full-text · Article · Nov 2011 · Developmental Dynamics
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    [Show abstract] [Hide abstract] ABSTRACT: Tendons are composed of longitudinally aligned collagen fibrils arranged in bundles with an undulating pattern, called crimp. The crimp structure is established during embryonic development and plays a vital role in the mechanical behaviour of tendon, acting as a shock-absorber during loading. However, the mechanism of crimp formation is unknown, partly because of the difficulties of studying tendon development in vivo. Here, we used a 3D cell culture system in which embryonic tendon fibroblasts synthesise a tendon-like construct comprised of collagen fibrils arranged in parallel bundles. Investigations using polarised light microscopy, scanning electron microscopy and fluorescence microscopy showed that tendon constructs contained a regular pattern of wavy collagen fibrils. Tensile testing indicated that this superstructure was a form of embryonic crimp producing a characteristic toe region in the stress-strain curves. Furthermore, contraction of tendon fibroblasts was the critical factor in the buckling of collagen fibrils during the formation of the crimp structure. Using these biological data, a finite element model was built that mimics the contraction of the tendon fibroblasts and monitors the response of the Extracellular matrix. The results show that the contraction of the fibroblasts is a sufficient mechanical impulse to build a planar wavy pattern. Furthermore, the value of crimp wavelength was determined by the mechanical properties of the collagen fibrils and inter-fibrillar matrix. Increasing fibril stiffness combined with constant matrix stiffness led to an increase in crimp wavelength. The data suggest a novel mechanism of crimp formation, and the finite element model indicates the minimum requirements to generate a crimp structure in embryonic tendon.
    Full-text · Article · Jul 2011 · Biomechanics and Modeling in Mechanobiology

Publication Stats

3k Citations
228.88 Total Impact Points

Institutions

  • 2007
    • University of Colorado at Boulder
      • Department of Molecular, Cellular, and Developmental Biology (MCDB)
      Boulder, Colorado, United States
  • 1990
    • The University of Edinburgh
      • Department of Clinical Biochemistry
      Edinburgh, Scotland, United Kingdom
  • 1985
    • The University of Manchester
      • Wellcome Trust Centre for Cell-Matrix Research
      Manchester, ENG, United Kingdom