Up-regulation of the Cdc42 GTPase limits the replicative life span of budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.     

Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL/6 mice

Cdc42 is a small RhoGTPase regulating multiple functions in eukaryotic cells. The activity of Cdc42 is significantly elevated in several tissues of aged mice, while the Cdc42 gain‐of‐activity mouse model presents with a premature aging‐like phenotype and with decreased lifespan. These data suggest a causal connection between elevated activity of Cdc42, aging, and reduced lifespan. Here, we demonstrate that systemic treatment of aged (75‐week‐old) female C57BL/6 mice with a Cdc42 activity‐specific inhibitor (CASIN) for 4 consecutive days significantly extends average and maximum lifespan. Moreover, aged CASIN‐treated animals displayed a youthful level of the aging‐associated cytokines IL‐1β, IL‐1α, and INFγ in serum and a significantly younger epigenetic clock as based on DNA methylation levels in blood cells. Overall, our data show that systemic administration of CASIN to reduce Cdc42 activity in aged mice extends murine lifespan.     

GDI-Mediated Cell Polarization in Yeast Provides Precise Spatial and Temporal Control of Cdc42 Signaling

Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.     

A flip-flop switch in polarity signaling

The Rho GTPase Cdc42 is essential for polarized growth of budding yeast. Temporal control of Cdc42 depends partly on the activity of its GTPase-activating proteins (GAPs). In this issue of Developmental Cell, Saito et al. report that Cdc42 GAP activity is regulated by the phospholipid composition of the bud-tip membrane, under control of the phospholipid flippases Lem3-Dnf1 and Lem3-Dnf2.     

Cdc42 localization and cell polarity depend on membrane traffic

Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6-aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.     

Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity

Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.     

Dissociation mechanism of GDP from Cdc42 via DOCK9 revealed by molecular dynamics simulations

Cell division control protein 42 homolog (Cdc42) influences a variety of cellular responses such as cell migration and polarity. Deregulation of Cdc42 has been associated with several human diseases and developmental disorders. Over-activation of Cdc42 through guanine nucleotide exchange factor (GEF) is a critical event for Cdc42 involved cancer metastasis. Members of DOCK family of GEF are important activators of Cdc42. However, this activation mechanism is still unknown. Molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations were employed to investigate the central step of the activation of Cdc42: the dissociation mechanism of GDP from Cdc42 via DOCK9. Simulation results show that Mg²⁺ ion has a remarkable influence on the conformational change of switch I of Cdc42 through residue Pro34 which functions as a “clasp” to control the flexibility of switch I. In the GDP dissociation process, the Mg²⁺ ion leave first to result in a suitable conformation of Cdc42 for following DOCK9 binding to. When DOCK9 binds to Cdc42, it changes the orientations of residues Lys16, Thr17, Cys18 and Phe28 of Cdc42 to weaken the interactions between Cdc42 and GDP to release GDP. This study first elucidates the dissociation mechanism of GDP from Cdc42 via DOCK9 and identifies the essential residues of Cdc42 in this process. These simulation results are consistent with the recent findings of biochemical and amino acid mutational studies, and the observations are beneficial to understand the activation mechanism of Cdc42 and to provide insights for designing compounds targeting on Cdc42 related cancer metastasis.     

A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation

BACKGROUND: Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS: We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS: These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.     

Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

IQGAP1 promotes cell motility and invasion

The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42- and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1DeltaGRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1DeltaGRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.     

Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis

Cell rearrangements require dynamic changes in cell–cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.     

Regulation of WASP: PIP2 Pipped by Toca-1?

Cdc42 is a key regulator of cell polarity and actin dynamics. One of its effectors, WASP, initiates the assembly of new actin filaments. In this issue of Cell, show that a previously unknown regulator named Toca-1 is required for Cdc42 to activate WASP. This discovery changes our picture of how small GTPases and multiple other signals converge to stimulate actin polymerization and cell motility.     

Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins.     

Association of Cdc42/N-WASP/Arp2/3 signaling pathway with Golgi membranes

Recent findings indicate that Cdc42 regulates Golgi-to-ER (endoplasmic reticulum) protein transport through N-WASP and Arp2/3 (Luna et al. 2002, Mol. Biol. Cell, 13:866-879). To analyse the components of the Cdc42-governed signaling pathway in the secretory pathway, we localized Cdc42, N-WASP and Arp2/3 in the Golgi complex by cryoimmunoelectron microscopy. Cdc42 is found throughout the Golgi stack, particularly in cis/middle cisternae, whereas N-WASP and Arp3 (a component of the Arp2/3 complex) are restricted to cis cisternae. Arp3 also colocalized in peri-Golgi tubulovesicular structures with either KDEL receptor or GM130. Even though Arp3 is not found in TGN46-positive cisternal elements, a small fraction of Arp3-labeled tubulo-vesicular elements showed TGN46 labeling. Active Cdc42 (GTP-bound form) induced relocation of N-WASP and Arp3 to the lateral rims of Golgi cisternae. These results show that the actin nucleation and polymerization signaling pathway governed by Cdc42/N-WASP/Arp operates in the Golgi complex of mammalian cells, further implicating actin dynamics in Golgi-associated membrane trafficking.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.     

Cdc42 activity regulates hematopoietic stem cell aging and rejuvenation

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Highlights? Constitutively increasing Cdc42 activity causes aging of young HSCs ? Elevated Cdc42 activity correlates with a loss of cell polarity in aged HSCs ? Inhibition of Cdc42 activity rejuvenates aged LT-HSC function ? Cdc42 inhibition restores the level and spatial distribution of AcH4K16     

Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spread

The bacterial pathogen Listeria monocytogenes uses actin‐based motility to spread from infected human cells to surrounding healthy cells. Cell–cell spread involves the formation of thin extensions of the host plasma membrane (‘protrusions’) containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco‐2 BBE1 induced a decrease in the level of Cdc42‐GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen‐induced downregulation of host Cdc42.     

CLN3 Deficient Cells Display Defects in the ARF1-Cdc42 Pathway and Actin-Dependent Events

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development.