Contraction-mediated pinocytosis of RGD-peptide by dermal fibroblasts: inhibition of matrix attachment blocks contraction and disrupts microfilament organisation.
Force generation in collagen and matrix contraction are basic functions of fibroblasts and important elements of tissue repair. Cell-matrix attachment is critical to this contraction, involving RGD-binding integrins. We have investigated how this process operates, in terms of force generation (in the Culture Force Monitor) and cytoskeletal structure, using a synthetic RGD-decapeptide. The RGD-peptide blocked force generation over the first 6 h, followed by near complete recovery by 20 h. However, dose response was complex indicating multiple processes were operating. Analysis of cytoskeletal structure after treatment with RGD-peptide indicated major disruption with condensed aggregates of actin and microtubular fragmentation. Fluorescent labeling and tracking of the RGD-peptide demonstrated intracellular uptake into discrete cytoplasmic aggregates. Critically, these RGD-peptide pools co-localised with the condensed actin microfilament aggregates. It is concluded that RGD-peptide uptake was by a form of contraction-mediated pinocytosis, resulting from mechanical tension applied to the untethered RGD-peptide-integrin, as contractile microfilament were assembled. These findings emphasize the importance of sound mechanical attachment of ligand-occupied integrins (e.g., to extracellular matrix) for normal cytoskeletal function. Conversely, this aspect of unrestrained cytoskeletal contraction may have important pathogenic and therapeutic applications.
University College London, Tissue Repair and Engineering Centre (TREC), Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore, Middlesex, United Kingdom.
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Sethi
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Mudera
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Sutterlin
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Baschong
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Brown
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D001672:Biocompatible Materials / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D020405:Dermis / Q000166:cytology*; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010873:Pinocytosis / Q000502:physiology*
D000199:Actins; D001672:Biocompatible Materials; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D003094:Collagen; D016650:Fluorescein-5-isothiocyanate