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.
Sethi
K K
KK
University College London, Tissue Repair and Engineering Centre (TREC), Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore, Middlesex, United Kingdom.
Mudera
V
V
Sutterlin
R
R
Baschong
W
W
Brown
R A
RA
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