Browsing by Subject "Cdc42"

The Rho GTPase Cdc42 is a master regulator of cell polarity that orchestrates reorganization of the cytoskeleton. During polarity establishment, active GTP-Cdc42 accumulates at a part of the cell cortex that becomes the front of the cell. Localized GTP-Cdc42 orients the cytoskeleton through a set of “effector” proteins that bind specifically to GTP-Cdc42 and not GDP-Cdc42. A family of Cdc42 effectors, called GICs in yeast and BORGs in mammals, have been implicated in regulation of both the actin cytoskeleton and the septin cytoskeleton. Yeast cells lacking both Gic1 and Gic2 are able to polarize and grow at low temperatures, but many mutant cells fail to polarize the cytoskeleton at high temperature. This led to the conclusion that GICs communicate between Cdc42 and different cytoskeletal elements.

To better characterize the role of GIC proteins in yeast, we utilized time-lapse fluorescent microscopy to examine morphogenetic events in living single cells. Surprisingly, we found that not only the cytoskeleton but also Cdc42 itself failed to polarize in many gic1 gic2 mutant cells at high temperature. This observation indicates that GICs may act upstream of polarization rather than downstream.

Polarization of Cdc42 is triggered by cell-cycle progression, and in particular by G1 Cyclin-dependent kinase (CDK) activity. Using a live-cell reporter for G1 CDK activation, we found that cells lacking GICs were not defective in CDK activation, but showed a specific defect in polarization downstream of the CDK. Previous work had implicated the scaffold protein Bem1 in a positive feedback loop important for polarization. Cells lacking GICs failed to polarize Bem1 as well as Cdc42 at high temperature. Future work will be directed at understanding how GICs contribute to polarity establishment. Because many of the mechanisms and proteins involved in polarization are highly conserved, we anticipate our findings will help inform how this process regulated in higher eukaryotes.

Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.

This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.

Cell polarity is critical for essential functions in many types of cells. Rho-family GTPases are master regulators of cell polarity. Thus, mechanisms for controlling the activity of Rho GTPases are of both academic interest and practical concern. While much has been discovered about regulation of Rho GTPase activity by partners like GEFs, GAPs, and GDIs, there are still many things which remain unclear about how their behavior is enforced in cells of various kinds.

The budding yeast, Saccharomyces cerevisiae, has long been a model for studying cell polarity. Its Rho GTPase Cdc42 is responsible for defining a single polarity site for the purpose of either mating with a single partner or making a single bud. Other types of yeasts, however, can generate multiple polarity sites utilizing the same core polarity machinery. What are the rules which allow for such differences in behavior while using a similar set of proteins? We highlight key design principles established in mathematical models for Rho GTPase polarity machineries and show that they apply in budding yeast cells: strains featuring specific genetic perturbations which increase the total amount of polarity proteins can go from making one bud, to making multiple buds.

Bud emergence in yeast is enabled by cell cycle activity in G1. It is known that bud emergence also requires cytoskeletal changes which are orchestrated by Cdc42 and its effectors. How are these changes coordinated with cell cycle progression? It seems likely that G1 CDK activity regulates many aspects of Cdc42 polarization. We use live-cell fluorescence microscopy to reveal one such avenue whereby input from the cell cycle is required for many Cdc42 effector proteins to localize to sites with active Cdc42, thus restricting bud formation until the time is right.

Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.

Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.

While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.

Many cells spontaneously establish a polarity axis even in the absence of directional cues, a process called symmetry breaking. A central question concerns how cells polarize towards one, and only one, randomly oriented "front". The conserved Rhotype GTPase Cdc42p is an essential factor for both directed and spontaneous polarization in various organisms, whose local activation is thought to define the cell's front. We previously proposed that in yeast cells, a small stochastic cluster of GTP-Cdc42p at a random site on the cortex can grow into a large, dominating cluster via a positive feedback loop involving the scaffold protein Bem1p. As stochastic Cdc42p clusters could presumably arise at many sites, why does only one site become the dominating "front"? We speculated that competition between growing clusters for limiting factors would lead to growth of a single winning "front" at the expense of the others. Utilizing time-lapse imaging with high spatiotemporal resolution, we now document initiation of multiple polarized clusters that competed rapidly to resolve a winning cluster. Such multicluster intermediates are observed in wild-type yeast cells with functional directional cues, but the locations where they are initiated are biased by the spatial cues. In addition, we detected an unexpected oscillatory polarization in a majority of the cells breaking symmetry, in which polarity factors initially concentrated very brightly and then dimmed in an oscillatory manner, dampening down to a final intermediate level after 2-3 peaks. Dampened oscillation suggests that the polarity circuit contains an in-built negative feedback loop. Mathematical modeling predicts that negative feedback would confer robustness to the polarity circuit and make the kinetics of competition between polarity factor clusters relatively insensitive to polarity factor concentration.

We are trying to understand how competition between clusters occurs. We find that the yeast guanine-nucleotide dissociation inhibitor (GDI), Rdi1p, is needed for rapid competition between clusters. In the absence of Rdi1p the initial clustering of polarity

factors is slowed, and competition is also much slower: in some cases cells still have two clusters at the time of bud emergence and they form two buds. We suggest that in the absence of Rdi1p, the clusters compete for a limiting pool of Cdc42p, and that slow

exchange of Cdc42p on and off the membrane in the absence of Rdi1p leads to slow competition.

Cryptococcus neoformans pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of C. neoformans yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.