Fast activation cycles of Rac1 at the lamellipodium tip trigger membrane protrusion

Abstract

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss-or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.

Integrin-based adhesion compartmentalizes ALK3 of the BMPRII to control cell adhesion and migration

“…We next used single-protein tracking (SPT) coupled to photoactivation localization microscopy (sptPALM) (Rossier et al, 2012;Chazeau et al, 2014;Manley et al, 2008;Mehidi et al, 2017) to better determine how BMP2 treatment affects the molecular dynamics of ALK3 within and outside β3 integrin-containing FAs (Fig. 6).…”

“…A typical sptPALM experiment leads to a set of 16,000 images per cell, which are then analyzed to extract information about the localization and dynamics of the molecule. Singlemolecule fluorescent spots were localized and tracked over time using a combination of wavelet segmentation and simulated annealing algorithms developed in Metamorph software (Molecular Devices), as described previously (Chazeau et al, 2014;Mehidi et al, 2017;Rossier et al, 2012). For ALK3 enrichment and trajectory analysis, ROIs of FAs were identified manually from GFP-β3-integrin images.…”

“…We used a weighted fit to reduce the inaccuracy of the MSD fit due to down sampling for larger time intervals. Trajectories were sorted into three groups: immobile, confined diffusion, and free-diffusion, as described previously (Mehidi et al, 2017;Chazeau et al, 2014;Rossier et al, 2012). Immobile trajectories were defined as trajectories with D < 0.011 μm 2 .s -1 , corresponding to molecules that explored an area inferior to that defined by the image spatial resolution ~(0.05 μm)² during the time used to fit the initial slope of the MSD (Rossier et al, 2012).…”