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.

Actin visualization at a glance

“…Using the example of LifeAct, it has been proposed to be the most suitable actin probe to visualize the dynamic rearrangement of the actin cytoskeleton in Dictyostelium, as it labels a more complete subset of actin structures than other actin-binding probes (Lemieux et al, 2014). The application of LifeAct was also favored over actin fused to fluorophore in the context of visualizing actin rearrangements during cellular mechanotransduction events (Sliogeryte et al, 2016;Deibler et al, 2011). In contrast, LifeAct should be used with caution for actin visualization in fission yeast, as it can cause severe concentration-dependent defects in endocytosis and cytokinesis (Courtemanche et al, 2016).…”

“…In particular, GFP-actin is suitable for fluorescence recovery after photobleaching (FRAP) approaches to study actin dynamics and turnover within a given cellular F-actin structure, including, for instance, the actin cortex (Clark et al, 2013) or protrusions such as lamellipodia (Koestler et al, 2009) (see poster). However, the relatively large size (∼28 kDa) of GFP-like proteins (Sliogeryte et al, 2016) can give rise to problems in terms of incorporation of GFP-tagged actin monomers into filaments. Thus, the GFP-actin-derived localization pattern might only reflect a part of the total F-actin network in cells.…”

Phosphorylation of GMFγ by c-Abl coordinates lamellipodial and focal adhesion dynamics

“…To assess lamellipodial dynamics, smooth muscle cells were co-transfected with plasmids encoding EGFP-tagged recombinant GMF γ and LifeAct-RFP plasmid, which generates a 17 amino-acid peptide to visualize F-actin in live cells 52 . Live-cell confocal and TIRF microscopy of cells expressing WT-GMF γ revealed that the majority localized to both the near membrane region of the ventral side of the cell, and within lamellipodia and filopodia (Supplementary Fig.…”

Selective adhesive cell capture without molecular specificity: new surfaces exploiting nanoscopic polycationic features as discrete adhesive units

“…While studies have revealed differing stiffnesses in immobilized cells 52–55 and differing deformabilities in suspended cells, 56–58 for instance comparing healthy and cancerous cells or different cancer cells, the particular length and time scales of the deformation is often not clear and is critical. Specific separations strategies distinguish only a particular range in cell properties: The deformations relevant to our method could be as small as a few nanometers, evidenced by the sensitivity to ionic strength.…”