A RhoC Biosensor Reveals Differences in the Activation Kinetics of RhoA and RhoC in Migrating Cells

Abstract

RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

Rho GTPase isoforms in cell motility: Don't fret, we have FRET

“…53 This analysis showed that areas of RhoC activation appeared to be more diffuse and evenly distributed at the edge of the cell. 53 Furthermore, the authors showed that RhoA and RhoC activities have different kinetics during lamellipodial protrusions. These different activation dynamics suggest that the key determining factors that control subcellular GTPase activation dynamics is likely the spatial distribution of their upstream regulators.…”

“…49,53,56 At invadopodia, RhoC has a geometrically confined activation pattern, while RhoA displays a more random activation pattern. 56 At leading edge protrusions, RhoC activation is confined spatially behind RhoA activation.…”

“…49 Also, using computational approaches in fibroblasts, the activation of RhoC was quantitatively compared with that of RhoA. 53 This analysis showed that areas of RhoC activation appeared to be more diffuse and evenly distributed at the edge of the cell. 53 Furthermore, the authors showed that RhoA and RhoC activities have different kinetics during lamellipodial protrusions.…”

Rho family GTPases: Making it to the third dimension

“…In 2D migration, RhoA localizes to the rear of the cell where it is thought to promote rear retraction. At the front of the cell, Rho A is found at the border of the lamellipod and lamella, while RhoC is found at the leading edge (Zawistowski et al, 2013)(Bravo-Cordero et. al., 2014).…”

“…Both RhoA and RhoC play roles in the extension of membrane protrusions during migration on a 2D surface, with RhoC preceding protrusion formation (Pertz et al, 2006, Zawistowski, Sabouri-Ghomi, 2013). Rho A also regulates the contraction at the lateral and rear of the cell, further supporting forward migration.…”