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.
Highlights
The actin cytoskeleton plays a key role in many cellular processes such as mechanotransduction (Janmey and Weitz, 2004), motility (Pollard and Cooper, 2009) and differentiation (Titushkin and Cho, 2011)
Cell biomechanical properties are associated with actin structure and dynamics and membrane bleb formation, and play a role in dictating the cellular response to the extracellular mechanical environment (Ingber, 2006; Zhelev et al, 1994)
Live cell imaging of actin remodelling and dynamics has been widely reported through the transfection of cells with a plasmid expressing actin coupled to a fluorescent protein such as GFP (Endlich et al, 2007)
Summary
The actin cytoskeleton plays a key role in many cellular processes such as mechanotransduction (Janmey and Weitz, 2004), motility (Pollard and Cooper, 2009) and differentiation (Titushkin and Cho, 2011). Live cell imaging of actin remodelling and dynamics has been widely reported through the transfection of cells with a plasmid expressing actin coupled to a fluorescent protein such as GFP (Endlich et al, 2007). This approach labels both F-and G-actin, which can be useful for assessing the relative dynamics (Engelke et al, 2010) and reduces the signal to noise ratio when visualising F-actin structures (Lee et al, 2013). Studies have reported that actin-GFP expression directly influences actin dynamics during cell cytokinesis and migration (Aizawa et al, 1997), cell-matrix adhesion (Feng et al, 2005), and mechanically induced cell deformation (Deibler et al, 2011; Pravincumar et al, 2012)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
