Cross-talk-dependent cortical patterning of Rho GTPases during cell repair

Rho GTPases such as Rho, Rac, and Cdc42 are important regulators of the cortical cytoskeleton in processes including cell division, locomotion, and repair. In these processes, Rho GTPases assume characteristic patterns wherein the active GTPases occupy mutually exclusive “zones” in the cell cortex. During cell wound repair, for example, a Rho zone encircles the wound edge and is in turn encircled by a Cdc42 zone. Here we evaluated the contributions of cross-talk between Rho and Cdc42 to the patterning of their respective zones in wounded Xenopus oocytes using experimental manipulations in combination with mathematical modeling. The results show that the position of the Cdc42 zone relative to the Rho zone and relative to the wound edge is controlled by the level of Rho activity. In contrast, the outer boundary of the Rho zone is limited by the level of Cdc42 activity. Models based on positive feedback within zones and negative feedback from Rho to the GEF-GAP Abr to Cdc42 capture some, but not all, of the observed behaviors. We conclude that GTPase zone positioning is controlled at the level of Rho activity and we speculate that the Cdc42 zone or something associated with it limits the spread of Rho activity.     

Modeling the roles of protein kinase Cβ and η in single-cell wound repair

Wounded cells such as Xenopus oocytes respond to damage by assembly and closure of an array of actin filaments and myosin-2 controlled by Rho GTPases, including Rho and Cdc42. Rho and Cdc42 are patterned around wounds in a characteristic manner, with active Rho concentrating in a ring-like zone inside a larger, ring-like zone of active Cdc42. How this patterning is achieved is unknown, but Rho and Cdc42 at wounds are subject to regulation by other proteins, including the protein kinases C. Specifically, Cdc42 and Rho activity are enhanced by PKCβ and inhibited by PKCη. We adapt a mathematical model of Simon and coworkers to probe the possible roles of these kinases. We show that PKCβ likely affects the magnitude of positive Rho–Abr feedback, whereas PKCη acts on Cdc42 inactivation. The model explains both qualitative and some overall quantitative features of PKC–Rho GTPase regulation. It also accounts for the previous, peculiar observation that ∼20% of cells overexpressing PKCη display zone inversions—that is, displacement of active Rho to the outside of the active Cdc42.     

A Highlights from MBoC Selection: Lipid domain–dependent regulation of single-cell wound repair

After damage, cells reseal their plasma membrane and repair the underlying cortical cytoskeleton. Although many different proteins have been implicated in cell repair, the potential role of specific lipids has not been explored. Here we report that cell damage elicits rapid formation of spatially organized lipid domains around the damage site, with different lipids concentrated in different domains as a result of both de novo synthesis and transport. One of these lipids—diacylglycerol (DAG)—rapidly accumulates in a broad domain that overlaps the zones of active Rho and Cdc42, GTPases that regulate repair of the cortical cytoskeleton. Formation of the DAG domain is required for Cdc42 and Rho activation and healing. Two DAG targets, protein kinase C (PKC) β and η, are recruited to cell wounds and play mutually antagonistic roles in the healing process: PKCβ participates in Rho and Cdc42 activation, whereas PKCη inhibits Rho and Cdc42 activation. The results reveal an unexpected diversity in subcellular lipid domains and the importance of such domains for a basic cellular process.     

Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

A Rho GTPase Signal Treadmill Backs a Contractile Array

Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying "signal treadmill" in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2-powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays, and proper functioning of these gradients is dependent on both contraction and F-actin turnover. VIDEO ABSTRACT:     

Dendritic spine geometry can localize GTPase signaling in neurons

Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia.     

Abr and Bcr, two homologous Rac GTPase-activating proteins, control multiple cellular functions of murine macrophages

Small GTPases of the Rho family are key regulators of phagocytic leukocyte function. Abr and Bcr are homologous, multidomain proteins. Their C-terminal domain has GTPase-activating protein (GAP) activity that, in vitro, is specific for Rac and Cdc42. To address the in vivo relevance of these entire proteins, of which little is known, the current study examined the effect of the genetic ablation of Abr and Bcr in murine macrophages. The concomitant loss of Abr and Bcr induced multiple alterations of macrophage cellular behavior known to be under the control of Rac. Macrophages lacking both Abr and Bcr exhibited an atypical, elongated morphology that was reproduced by the ectopic expression of GAP domain mutant Abr and Bcr in a macrophage cell line and of constitutively active Rac in primary macrophages. A robust increase in colony-stimulating factor 1 (CSF-1)-directed motility was observed in macrophages deficient for both proteins and, in response to CSF-1 stimulation, Abr and Bcr transiently translocated to the plasma membrane. Phagocytosis of opsonized particles was also increased in macrophages lacking both proteins and correlated with sustained Rac activation. Bcr and Abr GAP mutant proteins localized around phagosomes and induced distinct phagocytic cup formation. These results identify Abr and Bcr as the only GAPs to date that specifically negatively regulate Rac function in vivo in primary macrophages.     

How cells integrate complex stimuli: the effect of feedback from phosphoinositides and cell shape on cell polarization and motility

To regulate shape changes, motility and chemotaxis in eukaryotic cells, signal transduction pathways channel extracellular stimuli to the reorganization of the actin cytoskeleton. The complexity of such networks makes it difficult to understand the roles of individual components, let alone their interactions and multiple feedbacks within a given layer and between layers of signalling. Even more challenging is the question of if and how the shape of the cell affects and is affected by this internal spatiotemporal reorganization. Here we build on our previous 2D cell motility model where signalling from the Rho family GTPases (Cdc42, Rac, and Rho) was shown to organize the cell polarization, actin reorganization, shape change, and motility in simple gradients. We extend this work in two ways: First, we investigate the effects of the feedback between the phosphoinositides (PIs) , and Rho family GTPases. We show how that feedback increases heights and breadths of zones of Cdc42 activity, facilitating global communication between competing cell “fronts”. This hastens the commitment to a single lamellipodium initiated in response to multiple, complex, or rapidly changing stimuli. Second, we show how cell shape feeds back on internal distribution of GTPases. Constraints on chemical isocline curvature imposed by boundary conditions results in the fact that dynamic cell shape leads to faster biochemical redistribution when the cell is repolarized. Cells with frozen cytoskeleton, and static shapes, consequently respond more slowly to reorienting stimuli than cells with dynamic shape changes, the degree of the shape-induced effects being proportional to the extent of cell deformation. We explain these concepts in the context of several in silico experiments using our 2D computational cell model.     

The Exo70 Subunit of the Exocyst Is an Effector for Both Cdc42 and Rho3 Function in Polarized Exocytosis

The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation of exocytosis by Rho GTPases.     

Wave-pinning and cell polarity from a bistable reaction-diffusion system

Motile eukaryotic cells polarize in response to external signals. Numerous mechanisms have been suggested to account for this symmetry breaking and for the ensuing robust polarization. Implicated in this process are various proteins that are recruited to the plasma membrane and segregate at an emergent front or back of the polarizing cell. Among these are PI3K, PTEN, and members of the Rho family GTPases such as Cdc42, Rac, and Rho. Many such proteins, including the Rho GTPases, cycle between active membrane-bound forms and inactive cytosolic forms. In previous work, we have shown that this property, together with appropriate crosstalk, endows a biochemical circuit (Cdc42, Rac, and Rho) with the property of inherent polarizability. Here we show that this property is present in an even simpler system comprised of a single active/inactive protein pair with positive feedback to its own activation. The simplicity of this minimal system also allows us to explain the mechanism using insights from mathematical analysis. The basic idea resides in a well-known property of reaction-diffusion systems with bistable kinetics, namely, propagation of fronts. However, it crucially depends on exchange between active and inactive forms of the chemicals with unequal rates of diffusion, and overall conservation to pin the waves into a stable polar distribution. We refer to these dynamics as wave-pinning and we show that this phenomenon is distinct from Turing-instability-generated pattern formation that occurs in reaction-diffusion systems that appear to be very similar. We explain the mathematical basis of the phenomenon, relate it to spatial segregation of Rho GTPases, and show how it can account for spatial amplification and maintenance of polarity, as well as sensitivity to new stimuli typical in polarization of eukaryotic cells.     

Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P₂ specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.     

Concentric zones of active RhoA and Cdc42 around single cell wounds

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.     

Mathematical Model for Spatial Segregation of the Rho-Family GTPases Based on Inhibitory Crosstalk

Cdc42, Rac, and Rho are small GTPases known to play a central role in signal transduction to the actin cytoskeleton. These proteins regulate cell motility, by affecting nucleation, uncapping, and depolymerization of actin filaments, and acto-myosin contractility. Studies of crosstalk and mutual feedbacks in these three proteins have led to a number of proposals for their interaction. At the same time, observations of the spatio-temporal dynamics of Rho-family proteins give evidence of spatial polarization and mutual exclusion between Cdc42/Rac and Rho. In this paper, we formulate a mathematical model to account for such observations, based on the known underlying biology of these proteins. We first investigate which of the crosstalk schemes proposed in the literature is consistent with observed dynamics, and then derive a simple model that can correctly describe these dynamics (assuming crosstalk is mediated via Rho GEFs). We show that cooperativity is an essential ingredient in the interactions of the proteins. The co-occurrence of a stable rest state with the possibility of fast spatial segregation can be related to bistability in a set of underlying ODEs in which the inactive forms of these proteins are fixed at a constant level. We show that the fast diffusion of the inactive forms is essential for stabilizing the transition fronts in the PDE formulation of the model, leading to robust spatial polarization, rather than traveling waves.     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling

Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.     

A Highlights from MBoC Selection: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.     

Phosphoinositides and Rho proteins spatially regulate actin polymerization to initiate and maintain directed movement in a one-dimensional model of a motile cell

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive cell motility, with parameter values derived from in vitro and in vivo studies. In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that the PIs filter noise, and define the front versus back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore the importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. We also suggest a mechanism to explain the spatial exclusion of Cdc42 and PTEN and the inability of cells lacking active Cdc42 to properly detect chemoattractant gradients.