Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions.

While Rho GTPases are indispensible regulators of cellular polarity, the mechanisms underlying their anisotropic activation at membranes have been elusive. Using the budding yeast Cdc42 GTPase module, which includes a guanine nucleotide exchange factor (GEF) Cdc24 and the scaffold Bem1, we find that avidity generated via multivalent anionic lipid interactions is a critical mechanistic constituent of polarity establishment. We identify basic cluster (BC) motifs in Bem1 that drive the interaction of the scaffold-GEF complex with anionic lipids at the cell pole. This interaction appears to influence lipid acyl chain ordering, thus regulating membrane rigidity and feedback between Cdc42 and the membrane environment. Sequential mutation of the Bem1 BC motifs, PX domain, and the PH domain of Cdc24 lead to a progressive loss of cellular polarity stemming from defective Cdc42 nanoclustering on the plasma membrane and perturbed signaling. Our work demonstrates the importance of avidity via multivalent anionic lipid interactions in the spatial control of GTPase activation.

Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity.

Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.

Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk1(11A)), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.

Plasma membrane aminoglycerolipid flippase function is required for signaling competence in the yeast mating pheromone response pathway.

The class 4 P-type ATPases ("flippases") maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MAT A: cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection ("shmoo") tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MAT A: dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MAT A: dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.

Membrane-protein binding measured with solution-phase plasmonic nanocube sensors.

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed in a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrated the LSPR peak shift due to protein binding to the membrane surface and then characterized the lipid-binding specificity of a pleckstrin homology domain protein.

The distinct interaction between cell cycle regulation and the widely conserved morphogenesis-related (MOR) pathway in the fungus Ustilago maydis determines morphology.

The morphogenesis-related NDR kinase (MOR) pathway regulates morphogenesis in fungi. In spite of the high conservation of its components, impairing their functions results in highly divergent cellular responses depending on the fungal species. The reasons for such differences are unclear. Here we propose that the species-specific connections between cell cycle regulation and the MOR pathway could be partly responsible for these divergences. We based our conclusion on the characterization of the MOR pathway in the fungus Ustilago maydis. Each gene that encodes proteins of this pathway in U. maydis was deleted. All mutants exhibited a constitutive hyperpolarized growth, contrasting with the loss of polarity observed in other fungi. Using a conditional allele of the central NDR kinase Ukc1, we found that impairing MOR function resulted in a prolonged G2 phase. This cell cycle delay appears to be the consequence of an increase in Cdk1 inhibitory phosphorylation. Strikingly, prevention of the inhibitory Cdk1 phosphorylation abolished the hyperpolarized growth associated with MOR pathway depletion. We found that the prolonged G2 phase resulted in higher levels of expression of crk1, a conserved kinase that promotes polar growth in U. maydis. Deletion of crk1 also abolished the dramatic activation of polar growth in cells lacking the MOR pathway. Taken together, our results suggest that Cdk1 inhibitory phosphorylation may act as an integrator of signaling cascades regulating fungal morphogenesis and that the distinct morphological response observed in U. maydis upon impairment of the MOR pathway could be due to a cell cycle deregulation.

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula.

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.

Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants.

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitin-tagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin/proteasome pathway to promote disease.