IFT88 influences chondrocyte actin organization and biomechanics.
OBJECTIVE
Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes.


METHODS
The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP.


RESULTS
IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation.


CONCLUSIONS
This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology.
Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
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Wang
Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom.
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Wann
Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom. Electronic address: clare.l.thompson@qmul.ac.uk.
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Thompson
Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
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Hassen
Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
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Wang
Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
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Knight
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D048430:Cell Shape / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002923:Cilia / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D004548:Elasticity; D008817:Mice, Mutant Strains; D009154:Mutation; D024501:Nonmuscle Myosin Type IIB / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000502:physiology*; D014783:Viscosity
D000199:Actins; C490898:Tg737Rpw protein, mouse; D025521:Tumor Suppressor Proteins; D015638:Cytochalasin D; D024501:Nonmuscle Myosin Type IIB
Cell mechanics; F-actin; ITF88; Micropipette aspiration; Myosin IIB