Patterning of the cell cortex by Rho GTPases

Abstract

The Rho GTPases - RHOA, RAC1 and CDC42 - are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.

Fig. 1 ∣. Basic principles of Rho GTPase regulation.

a, The Rho GTPase cycle. Activation of Rho GTPase (that is, exchange of GDP for GTP) results from interaction with a GEF (guanine nucleotide exchange factor). The active GTPase can then bind to an effector resulting in changes in the cortical cytoskeleton. GTPase inactivation results from interaction with a GAP (GTPase activating protein) and is followed by extraction of the GTPase from the plasma membrane by RhoGDI, rendering the GTPase soluble in the cytoplasm. RhoGDI then somehow returns the inactive Rho to the plasma membrane, completing the cycle. b, Activation-centric view of Rho GTPase signalling. In this view, the path from the stimulus to the response is essentially linear, with the stimulus activating a GEF, the GEF activating the GTPase and the active GTPase directing the response, whereas the contributions of GAPs to the response are considered to merely limit or terminate the response. c, Self-organizing view of Rho GTPase signalling. In this view, the path from the signal to the response is highly non-linear, with the stimulus activating both the GEF and the GAP resulting in continuous GTPase cycling and self-organization of the GTPases into patterns which then dictate the response. In the figure, Rho indicates RHOA, RAC1 or CDC42.

Fig. 2 ∣. Self-organizing Rho GTPase patterns.

a, Pulsed contractions in a Caenorhabditis elegans embryo. Top: single frame from TIRF movie showing RHOA-GTP (green) and myosin 2 (red); anterior end of the embryo on the left, posterior on the right. Bottom: kymograph derived from the embryo in the top panel; total elapsed time 200 s. Pulses are evident in the kymograph as streaks which, on average, move towards the anterior end of the embryo over time. RHOA activity rises before myosin 2 in the contractions. b, Mitotic CDC42-GTP wave in an RBL cell from a TIRF movie. Image shows a composite of three successive timepoints with each time point coloured differently to reveal movement (red, T = 0 s; blue, T = 4 s; green, T = 8 s). The image captures a target pattern wave (that is, one that forms from a spot and spreads outward from the spot) of CDC42 activity. c, Experimentally induced RHOA-GTP and F-actin waves in frog oocytes. Single frame from a timelapse light sheet movie showing travelling waves of RHOA-GTP (cyan) chased by F-actin waves (red) in frog oocyte expressing the RHOA GEF Ect2 and the RHOA GAP RGA-3/4. Both target and spiral wave patterns are evident. d, Pulsed contractions in a U2OS cell. Left: series of images from timelapse TIRF movie of a nocodazole-treated U2OS cell showing RHOA-GTP (green) and myosin 2 (magenta); images taken 30 s apart. Right: kymograph corresponding to the white arrow on leftmost image; elapsed time 770 s. RHOA activity rises ahead of myosin 2 recruitment in the pulsed contractions. e, Travelling waves of Rho GTPase activity in a wounded frog oocyte. Top: single frame from a confocal movie of wounded frog oocyte showing a CDC42-GTP wave (red) and a RHOA-GTP wave (cyan); the CDC42-GTP wave encircles the RHOA-GTP wave. Bottom: kymograph from the cell depicted in the top panel; elapsed time 240 s. Single waves of RHOA-GTP and CDC42-GTP converge on the wound. f, Cytokinetic RHOA and F-actin waves in starfish blastomere. Left: single frame from a confocal movie of dividing starfish blastomere showing RHOA-GTP (green) and F-actin (orange). The cell is undergoing cytokinesis and the RHOA-GTP and F-actin waves are confined to the equatorial cortex. Right: kymograph taken from the area indicated by a box in the central region of the dividing cell on the left; elapsed time 960 s. Furrow waves of RHOA-GTP and chasing F-actin waves are evident as angled lines. D, distance; T, time. Source of images: panel a courtesy of John Michaux and Ed Munro, University of Chicago; panel b courtesy of Cheesan Tong and Min Wu, Yale University; panel c courtesy of Ani Michaud, Promega Corp.; panel d courtesy of Melanie Graessl, Perihan Nalbant, and Leif Dehmelt, University of Duisburg and Technical University of Dortmund; panel e courtesy of Lila Hoachlander-Hobby, University of Wisconsin–Madison; panel f provided by the authors.

Fig. 3 ∣. Feedback to Rho GTPase GEFs and GAPs.

a, Overview of known mechanisms of positive and negative feedback of Rho GTPases acting through their GEFs and GAPs. Feedback is considered positive if the end result is an increase in the activity of the Rho GTPase or negative if the end result is a decrease in the activity of the GTPase. Thus, stimulation of a GEF by its target GTPase is considered positive feedback whereas stimulation of a GAP by its target GTPase is considered negative feedback. Positive interactions indicated by arrows with pointed ends; negative interactions indicated by arrows with flat ends. See Table 1 for specific examples. b, Example mechanisms of the different classes of feedback. Direct feedback: an active Rho GTPase binds allosterically to its GEF via the PH domain in the GEF, thereby targeting it to the plasma membrane and exposing the active site (DH domain) which can then activate an inactive GTPase. Effector-based feedback: an active GTPase binds an effector, which binds a GEF or GAP, targeting it to the plasma membrane. Effector target-based feedback: an active GTPase stimulates an effector which promotes formation of (in this case) F-actin which, in turn, targets the GEF or GAP to the plasma membrane. Direct feedback has only been described for positive feedback; effector-based feedback and effector target-based feedback can be either positive or negative. In the figure, Rho indicates the Rho GTPase RHOA, RAC1 or CDC42. D, direct feedback (active GTPase binds GEF); E, effector-based feedback (effector binds or modifies GEF or GAP); T, effector target-based feedback (downstream target of effector binds to or modifies GEF or GAP).

Fig. 4 ∣. Proposed feedbacks for examples of self-organization.

For each example, the upstream signal is indicated at the top and the pattern produced is indicated at the bottom. a, Formation of the polarizing CDC42 cluster in budding yeast relies on at least one positive feedback loop (via CDC42-GTP to Bem1 to the GEF (guanine nucleotide exchange factor) Cdc24) and two negative feedback loops (from CDC42-GTP to septins and the GAP (GTPase activating protein) Bem2 and from CDC42-GTP to F-actin cables and secretory vesicles). b, Pulsed contractions in Caenorhabditis elegans may arise from direct positive feedback from RHOA-GTP to the GEF Ect2 and from negative feedback from RHOA-GTP to F-actin to the GAP RGA-3/4. Pulsed contractions in U2OS cells arise from direct positive feedback from RHOA-GTP to the GEF GEF-H1 and two negative feedback loops: from RHOA-GTP to myosin 2 which inhibits GEF-H1, and from RHOA-GTP to F-actin to the GAP myosin 9. c, Travelling waves during actin coating of secretory vesicles arise from negative feedback from RHOA-GTP to F-actin to the GAP C-GAP; the basis of positive feedback has yet to be identified. Travelling waves during plasma membrane repair arise from positive feedback from RHOA-GTP to the dual GEF–GAP ABR; ABR is also responsible for negative crosstalk from RHOA-GTP to CDC42-GTP and participates in positive feedback for RHOA. CDC42-GTP is responsible for negative crosstalk to RHOA-GTP by an as yet unidentified RHOA GAP (not shown). Travelling waves during embryonic cytokinesis arise from direct positive feedback from RHOA-GTP to the GEF Ect2 and from negative feedback from RHOA-GTP to F-actin that engages in negative feedback with the GAP RGA-3/4. d, Homeostasis in adherens junctions relies on positive feedback from RHOA-GTP to Rho-associated protein kinase (ROCK) and myosin 2, which negatively regulate non-canonical Rho GTPase RND3 which positively regulates the GAP p190-RhoGAP. The basis of the negative feedback has not been identified. Homeostasis in tight junctions is restored following junction stretching via positive direct positive feedback from RHOA-GTP to p115-RhoGEF. The basis of negative feedback is unknown but may be dependent on ROCK. Pointed arrows indicate positive regulatory interactions; flat-headed arrows indicate negative regulatory interactions. ? indicates players assumed but not yet identified. Circled plus signs indicate positive feedback loops; circled minus signs indicate negative feedback loops.