title: Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.
abstract: The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology.
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
authors.forename: Kristina
authors.initials: K
authors.lastname: Sliogeryte
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom. Electronic address: s.thorpe@qmul.ac.uk.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Thorpe
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
authors.forename: Clare L
authors.initials: CL
authors.lastname: Thompson
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
authors.forename: Nuria
authors.initials: N
authors.lastname: Gavara
authors.affiliation: Institute of Bioengineering and School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom.
authors.forename: Martin M
authors.initials: MM
authors.lastname: Knight
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000096:biosynthesis; D000818:Animals; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D000199:Actins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: Actin cytoskeleton; Bleb; Cell mechanics; Mesenchymal stem cell; Micropipette aspiration