Yeast Polarity Research Articles

Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization.

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.

Abstract 535: Paving the path to metastasis/invasion through GIV: Identification of a polarized ex(o)citing machinery for cancer invasion

Abstract Polarized exocytosis is a fundamental process involving the delivery of membrane and cargo proteins to target sites the plasma membrane (PM) and it is essential for a wide range of biological functions such as cell growth, morphogenesis, and polarized cell migration. Polarized exocytosis requires precise spatial control of vesicle tethering to target sites at the PM. This is mediated by the octameric exocyst complex that is conserved from yeast to man. How the exocyst complex imparts polarity during exocytosis remained a mystery until recent insights emerged from studies on budding yeast. A functional interaction between exocyst component Exo70p and the yeast polarity determinant scaffold protein Bem1p was reported, and it was shown that Bem1p regulates the targeting of Exo70p to polarized exocytic sites. Despite these insights, the counterpart of Bem1p in higher species remained elusive. Here we demonstrate that a novel polarity-determinant protein called GIV (Gα-interacting vesicle associated protein/Girdin) fulfills the key criteria and functions of its yeast counterpart Bem1p. GIV provides an evolutionary upgrade to the process of polarized exocytosis by making it responsive to growth factors and other external cues and by modulating it through G protein signaling that is unique to mammalian cells. GIV was originally identified in a yeast-2 hybrid screen as a binding partner of EXOC1, and subsequently validated by us as a PI4P-binding protein that is a direct binding partner of mammalian Exo70. Biochemical assays have confirmed that the mechanism of GIV:Exo70 interaction bears close semblance to the Bem1p:Exo70p interaction. Sequence alignment studies led to the identification of single point mutants in Bem1p and GIV that are selectively defective in binding to Exo70. Using genetic manipulation of cancer cells we have observed that GIV is essential for polarized tethering of Exo70-coated vesicles at the cell periphery. Consistent with the previously described prometastatic role of GIV, we found that GIV is required for EGF-stimulated exocytosis of matrix metalloproteases that aid in cancer invasion in MDA-MB-231 breast cancer cells and that specific point mutants of GIV that interrupt the interaction with Exo70 cause a reduction in the ability of these breast cancer cells to invade and degrade components of the extracellular matrix. Furthermore, GIV interacts specifically with TC-10•GTP[active], a monomeric GTPase that regulates fusion of exocytic vesicles at the plasma membrane. Insights gained continue to provide an in-depth understanding of how GIV affects a fundamental process of exocytosis, and what impact that may have on cancer invasion and metastasis. Citation Format: Cristina C. Rohena, Nina N. Sun, Nicolas Aznar, Pradipta Ghosh. Paving the path to metastasis/invasion through GIV: Identification of a polarized ex(o)citing machinery for cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2017-535

Intrinsic and Extrinsic Determinants Linking Spindle Pole Fate, Spindle Polarity, and Asymmetric Cell Division in the Budding Yeast S. cerevisiae.

The budding yeast S. cerevisiae is a powerful model to understand the multiple layers of control driving an asymmetric cell division. In budding yeast, asymmetric targeting of the spindle poles to the mother and bud cell compartments respectively orients the mitotic spindle along the mother-bud axis. This program exploits an intrinsic functional asymmetry arising from the age distinction between the spindle poles-one inherited from the preceding division and the other newly assembled. Extrinsic mechanisms convert this age distinction into differential fate. Execution of this program couples spindle orientation with the segregation of the older spindle pole to the bud. Remarkably, similar stereotyped patterns of inheritance occur in self-renewing stem cell divisions underscoring the general importance of studying spindle polarity and differential fate in yeast. Here, we review the mechanisms accounting for this pivotal interplay between intrinsic and extrinsic asymmetries that translate spindle pole age into differential fate.

SParSE++: improved event-based stochastic parameter search

BackgroundDespite the increasing availability of high performance computing capabilities, analysis and characterization of stochastic biochemical systems remain a computational challenge. To address this challenge, the Stochastic Parameter Search for Events (SParSE) was developed to automatically identify reaction rates that yield a probabilistic user-specified event. SParSE consists of three main components: the multi-level cross-entropy method, which identifies biasing parameters to push the system toward the event of interest, the related inverse biasing method, and an optional interpolation of identified parameters. While effective for many examples, SParSE depends on the existence of a sufficient amount of intrinsic stochasticity in the system of interest. In the absence of this stochasticity, SParSE can either converge slowly or not at all.ResultsWe have developed SParSE++, a substantially improved algorithm for characterizing target events in terms of system parameters. SParSE++ makes use of a series of novel parameter leaping methods that accelerate the convergence rate to the target event, particularly in low stochasticity cases. In addition, the interpolation stage is modified to compute multiple interpolants and to choose the optimal one in a statistically rigorous manner. We demonstrate the performance of SParSE++ on four example systems: a birth-death process, a reversible isomerization model, SIRS disease dynamics, and a yeast polarization model. In all four cases, SParSE++ shows significantly improved computational efficiency over SParSE, with the largest improvements resulting from analyses with the strictest error tolerances.ConclusionsAs researchers continue to model realistic biochemical systems, the need for efficient methods to characterize target events will grow. The algorithmic advancements provided by SParSE++ fulfill this need, enabling characterization of computationally intensive biochemical events that are currently resistant to analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0367-z) contains supplementary material, which is available to authorized users.

Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast.

Surface charges at the inner leaflet of the plasma membrane may contribute to regulate the surface recruitment of key signaling factors. Phosphatidylserine (PS) is an abundant charged lipid that may regulate charge distribution in different cell types. Here we characterize the subcellular distribution and function of PS in the rod-shaped, polarized fission yeast. We find that PS preferably accumulates at cell tips and defines a gradient of negative charges along the cell surface. This polarization depends on actin-mediated endocytosis and contributes to the subcellular partitioning of charged polarity-regulating Rho GTPases like Rho1 or Cdc42 in a protein charge-dependent manner. Cells depleted of PS have altered cell dimensions and fail to properly regulate growth from the second end, suggesting a role for PS and membrane charge in polarized cell growth.

A Predictive Model for Yeast Cell Polarization in Pheromone Gradients.

Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.

Fuzzy Stochastic Petri Nets for Modeling Biological Systems with Uncertain Kinetic Parameters.

Stochastic Petri nets (SPNs) have been widely used to model randomness which is an inherent feature of biological systems. However, for many biological systems, some kinetic parameters may be uncertain due to incomplete, vague or missing kinetic data (often called fuzzy uncertainty), or naturally vary, e.g., between different individuals, experimental conditions, etc. (often called variability), which has prevented a wider application of SPNs that require accurate parameters. Considering the strength of fuzzy sets to deal with uncertain information, we apply a specific type of stochastic Petri nets, fuzzy stochastic Petri nets (FSPNs), to model and analyze biological systems with uncertain kinetic parameters. FSPNs combine SPNs and fuzzy sets, thereby taking into account both randomness and fuzziness of biological systems. For a biological system, SPNs model the randomness, while fuzzy sets model kinetic parameters with fuzzy uncertainty or variability by associating each parameter with a fuzzy number instead of a crisp real value. We introduce a simulation-based analysis method for FSPNs to explore the uncertainties of outputs resulting from the uncertainties associated with input parameters, which works equally well for bounded and unbounded models. We illustrate our approach using a yeast polarization model having an infinite state space, which shows the appropriateness of FSPNs in combination with simulation-based analysis for modeling and analyzing biological systems with uncertain information.

A PDE-DDE Model for Cell Polarization in Fission Yeast

We consider a one-dimensional model of cell polarization in fission yeast consisting of a hybrid partial differential equation--delay differential equation system. The model describes bulk diffusion of the signaling molecule Rho GTPase Cdc42 in the cytoplasm, which is coupled to a pair of delay differential equations at the ends of the cell via boundary conditions. The latter represent the binding of Cdc42 to the cell membrane and rerelease into the cytoplasm via unbinding. The nontrivial nature of the dynamics arises from the fact that both the binding and unbinding rates at each end are taken to depend on the local membrane concentration of Cdc42. In particular, the association rate is regulated by positive feedback and the dissociation rate is regulated by delayed negative feedback. We use linear stability analysis and numerical simulations to investigate the onset of limit cycle oscillations at the end compartments for a cell of fixed length, distinguishing between symmetric solutions in which the mean concentration is identical at both ends and asymmetric solutions where the mean concentration at one end dominates. We find that the critical time delay for the onset of oscillations via a Hopf bifurcation increases as the diffusion coefficient $D$ decreases. We then solve the diffusion equation on a growing domain under the additional assumption that the total amount $C_{\rm tot}$ of the signaling molecule increases as the cell length increases. We show that the system undergoes a transition from asymmetric to symmetric oscillations as the cell grows, consistent with experimental findings of “new-end-take-off” in fission yeast. (The latter refers to the switch from monopolar to bipolar growth as the cell grows.) The critical length where the switch occurs depends on $D$ and the growth rate.

Imaging Polarization in Budding Yeast.

We describe methods for live-cell imaging of yeast cells that we have exploited to image yeast polarity establishment. As a rare event occurring on a fast time-scale, imaging polarization involves a trade-off between spatiotemporal resolution and long-term imaging without excessive phototoxicity. By synchronizing cells in a way that increases resistance to photodamage, we discovered unexpected aspects of polarization including transient intermediates with more than one polarity cluster, oscillatory clustering of polarity factors, and mobile "wandering" polarity sites.

A stable microtubule array drives fission yeast polarity reestablishment upon quiescence exit.

Cells perpetually face the decision to proliferate or to stay quiescent. Here we show that upon quiescence establishment, Schizosaccharomyces pombe cells drastically rearrange both their actin and microtubule (MT) cytoskeletons and lose their polarity. Indeed, while polarity markers are lost from cell extremities, actin patches and cables are reorganized into actin bodies, which are stable actin filament-containing structures. Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle. We have identified proteins involved in this process and propose a molecular model for Q-MT bundle formation. Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state. Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation.

Probing Yeast Polarity with Acute, Reversible, Optogenetic Inhibition of Protein Function.

We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud. By taking advantage of the reversibility of Bem1 inactivation, we show that pole size scales with cell size, and that this scaling is dependent on the actin cytoskeleton. Our experiments reveal how rapid reversible inactivation of protein function complements traditional genetic approaches. This strategy should be widely applicable to other biological contexts.

Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast.

Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42-GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback.

In vitro systems for the study of microtubule-based cell polarity in fission yeast.

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries.

Electrochemical regulation of budding yeast polarity.

Author SummaryThe ability of cells to orient towards spatial cues is critical for processes such as migration, wound healing, and development. Although the role of electrochemical signals is well characterized in processes such as neuronal signaling, their function in cell polarity is much less understood or appreciated. Application of exogenous electric fields can direct cell polarization in many cell types, and electric fields of similar magnitude surround cells and tissues naturally. However, the significance and mechanism of these responses remain poorly understood. Here, we introduce budding yeast (Saccharomyces cerevisiae) as a powerful model system to study electrochemical regulation of cell polarity. We show that application of electric fields causes budding yeast to polarize in particular directions. We begin to identify key proteins involved in this response, which implicate an electrochemical pathway involving membrane potential, membrane charge, and an ion channel, which ultimately regulate the central polarity factor Cdc42p. These key proteins are not only needed for response to electric fields, but also contribute to cell polarity more generally. To test whether a change in membrane potential is sufficient to control cell polarization, we introduce a light-sensitive ion channel into yeast and show that we can now control the site of polarization simply by using a focused laser beam. Thus, our study shows that electrochemical regulation is an integral component of cell polarity pathways.

Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast.

Aip1p cooperates with actin-depolymerizing factor (ADF)/cofilin to disassemble actin filaments in vitro and in vivo, and is proposed to cap actin filament barbed ends. We address the synergies between Aip1p and the capping protein heterodimer Acp1p/Acp2p during clathrin-mediated endocytosis in fission yeast. Using quantitative microscopy and new methods we have developed for data alignment and analysis, we show that heterodimeric capping protein can replace Aip1p, but Aip1p cannot replace capping protein in endocytic patches. Our quantitative analysis reveals that the actin meshwork is organized radially and is compacted by the cross-linker fimbrin before the endocytic vesicle is released from the plasma membrane. Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking. Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity. We identified two independent pathways that control polarization of endocytic sites, one depending on acp2(+) and aip1(+) during interphase and the other independent of acp1(+), acp2(+), and aip1(+) during mitosis.

Functional analysis of BcBem1 and its interaction partners in Botrytis cinerea: impact on differentiation and virulence.

In phytopathogenic fungi the establishment and maintenance of polarity is not only essential for vegetative growth and differentiation, but also for penetration and colonization of host tissues. We investigated orthologs of members of the yeast polarity complex in the grey mould fungus Botrytis cinerea: the scaffold proteins Bem1 and Far1, the GEF (guanine nucleotide exchange factor) Cdc24, and the formin Bni1 (named Sep1 in B. cinerea). BcBem1 does not play an important role in regular hyphal growth, but has significant impact on spore formation and germination, on the establishment of conidial anastomosis tubes (CATs) and on virulence. As in other fungi, BcBem1 interacts with the GEF BcCdc24 and the formin BcSep1, indicating that in B. cinerea the apical complex has a similar structure as in yeast. A functional analysis of BcCdc24 suggests that it is essential for growth, since it was not possible to obtain homokaryotic deletion mutants. Heterokaryons of Δcdc24 (supposed to exhibit reduced bccdc24 transcript levels) already show a strong phenotype: an inability to penetrate the host tissue, a significantly reduced growth rate and malformation of conidia, which tend to burst as observed for Δbcbem1. Also the formin BcSep1 has significant impact on hyphal growth and development, whereas the role of the putative ortholog of the yeast scaffold protein Far1 remains open: Δbcfar1 mutants have no obvious phenotypes.

Inhibitory GEF Phosphorylation Provides Negative Feedback in the Yeast Polarity Circuit

Cell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical "front" that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop) that then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4-7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site.

Rassf5 and Ndr kinases regulate neuronal polarity through Par3 phosphorylation in a novel pathway.

The morphology and polarized growth of cells depend on pathways that control the asymmetric distribution of regulatory factors. The evolutionarily conserved Ndr kinases play important roles in cell polarity and morphogenesis in yeast and invertebrates but it is unclear whether they perform a similar function in mammalian cells. Here, we analyze the function of mammalian Ndr1 and Ndr2 (also known as STK38 or STK38L, respectively) in the establishment of polarity in neurons. We show that they act downstream of the tumor suppressor Rassf5 and upstream of the polarity protein Par3 (also known as PARD3). Rassf5 and Ndr1 or Ndr2 are required during the polarization of hippocampal neurons to prevent the formation of supernumerary axons. Mechanistically, the Ndr kinases act by phosphorylating Par3 at Ser383 to inhibit its interaction with dynein, thereby polarizing the distribution of Par3 and reinforcing axon specification. Our results identify a novel Rassf5-Ndr-Par3 signaling cascade that regulates the transport of Par3 during the establishment of neuronal polarity. Their role in neuronal polarity suggests that Ndr kinases perform a conserved function as regulators of cell polarity.

Role of Internalization of Cell Surface Receptors in Regulation of Cell Polarity

Internalization of cell surface receptors is often stimulated after ligand binding. It plays important roles in biological processes such as signal desensitization, substance uptake, and signal transduction. Antagonist-stimulated internalization may be also important for cell polarization [1]. We have developed a general model to study the role of receptor internalization in the establishment of cell polarity. Our results show that regulated antagonist-stimulated internalization can provide an important mechanism to initiate cell polarization. It also provides additional regulation of signal amplification and noise filtering, which are important for the sensitivity and the accuracy of signal sensing. We discuss applications of our model to the study of yeast polarity and compare computational results with experimental observations.[1] Polarization of the Yeast Pheromone Receptor Requires Its Internalization but Not Actin-dependent, D.V. Suchkov et al, Mol Biol Cell. 2010 May 15; 21(10): 1737-1752.

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.