Browsing by Subject "Polarity"

Establishing an axis of cell polarity is central to cell motility, tissue morphogenesis, and cell proliferation. A highly conserved group of polarity regulators is responsible for organizing a wide variety of polarized morphologies. One of the most widely expressed polarity regulators is the Rho-type GTPase Cdc42. In response to cell cycle cues the budding yeast Saccharomyces cerevisiae polarizes Cdc42p to a discrete site on the cell periphery. GTP-Cdc42p recruits a number of effectors that aid in the organization of a polarized actin cytoskeleton. The polarized actin cytoskeleton acts as tracks to facilitate the delivery of the secretory vesicles that will grow the bud, an essential process for an organism that proliferates by budding. We have employed treatment with the actin depolymerizing drugs Latrunculin A and B as well as high-speed timelapse microscopy of fluorescently labeled polarity proteins to characterize the assembly of the incipient bud site.

Often, ensuring that only a single axis of polarity is established is as important as generating asymmetry in the cell. Even in the absence of positional cues dictating the direction of polarization, many cells are still able to self-organize and establish one, and only one, polarity axis through a process termed symmetry breaking. Symmetry breaking is thought to employ positive feedback to amplify stochastic fluctuations in protein concentration into a larger asymmetry. To test whether singularity could be guaranteed by the amplification mechanism we re-wired yeast to employ a synthetic positive feedback mechanism. The re-wired cells could establish polarity, however they occasionally made two buds simultaneously, suggesting that singularity is guaranteed by the amplification mechanism.

How do single cells—like neutrophils, amoebae, neurons, yeast, etc.—grow or move in a directed fashion in response to spatial chemical gradients? To address this question, we used the mating response in the budding yeast, Saccharomyces cerevisiae, as a biological model. To mate, pairs of yeast cells orient their cell fronts toward each other and fuse. Each cell relies on a pheromone gradient established by its partner to orient correctly. The ability for cells to resolve gradients is striking, because each cell is only ~5 μm wide and is thought to be operating in complex and noisy environments. Interestingly, mating pairs of cells often start out not facing each other. When this happens, the front of each cell—defined by a patch of cortical polarity proteins—undergoes a series of erratic and random movements along the cell cortex till it ‘finds’ the mating partner’s patch. We sought to understand how polarity patches in misaligned cells find each other. To this end, we first characterized patch movement in cells by the distribution of their step-lengths and turning angles and analyzed a bottom-up model of the polarity patch’s dynamics. The final version of our model combines 11 reaction-diffusion equations representing polarity protein dynamics with a stochastic module representing vesicle trafficking on a plane with periodic boundary conditions. We found that the model could not quantitatively reproduce step-length and turning angle distributions, which suggested that some mechanisms driving patch movement may not be present in the model. Incorpo-rating biologically inspired features into the model—such as focused vesicle delivery, sudden fluctuations in vesicle delivery rates, and the presence of polarity inhibitors on vesicles—allowed us to quantitatively match the in vivo polarity patch’s behavior. We then introduced a pathway, which connects pheromone sensing to polarity, to see how the model behaved when exposed to pheromone gradients. Concurrently, we analyzed the behavior of fluores-cently labeled polarity patches in mating pairs of cells. We discovered that the ~1 μm wide patch could (remarkably) sense and bias its movement up pheromone gradients, a result corroborated by our model. Further analysis of the model revealed that while the polarity patch tends to bias the location of a cluster of pheromone-sensing-receptors, the receptors can transform an external pheromone distribution into a peaked non-linear “polarity-activation” profile that “pulls” the patch. Stochastic perturbations cause the patch to “ping-pong” around the activation-profile. In a gradient of pheromone, this ping-ponging be-comes biased, leading to net patch movement up the gradient. We speculate that such a mechanism could be used by single cells with mobile fronts to track chemical gradients.

Bud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.

Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.

Extracellular chemical gradients provide signals that guide a broad spectrum of different cellular processes. By accurately sensing and responding to chemical gradients, immune cells can chase down invading pathogens, sperm cells can locate a distant egg, and growing axons can form connections in the developing nervous system. Haploid cells of the budding yeast Saccharomyces cerevisiae grow up gradients of pheromone in order to locate and fuse with nearby mating partners. Gradient sensing should be challenging for yeast, because they must detect a minute difference in concentration across their small cell bodies. Nevertheless, yeast cells can orient with remarkable accuracy in shallow pheromone gradients. Several mechanisms have been proposed to explain how yeast cells locate their partners, but it remains unclear whether, and to what degree each of the proposed mechanisms contributes.

We imaged fluorescent polarity probes in real time during mating events and found that cells located their partners in a multi-step process. First, cells placed a weak cluster of polarity proteins in approximately the correct location, despite a previously unappreciated challenge posed by asymmetrically distributed pheromone receptors. Cells were able to overcome receptor asymmetry by sensing the ratio, rather than the number, of active pheromone receptors. Second, the polarity cluster proceeded to move erratically around the cortex during an “indecisive phase.” We found evidence that cells switched to a local sensing mechanism during the indecisive phase, wherein cells used the mobile polarity cluster like a nose to search for areas where the local pheromone concentration was high. Third, the polarity cluster stabilized adjacent to a partner cell’s cluster and remained stationary until the partners met in the middle and fused.

Many cells are remarkably proficient at tracking even shallow chemical gradients, despite tiny differences in receptor occupancy across the cell. Stochastic receptor-ligand interactions introduce considerable noise in instantaneous receptor occupancy, so it is thought that spatial information must be integrated over time to allow noise filtering. The mechanism of temporal integration is unknown. We used the mating response of the budding yeast, Saccharomyces cerevisiae, as a model to study eukaryotic gradient tracking.

During mating, yeast cells polarize and grow up a gradient of pheromone to find and fuse with opposite-sex partners. Exposure to pheromone causes polarity regulators to cluster into a tight "patch" at the cortex, directing growth toward that site. Timelapse microscopy of fluorescently-labeled polarity proteins revealed that the patch wandered around the cortex during gradient tracking. Mathematical modeling and genetic analysis suggested that fusion of vesicles near the polarization site could perturb the polarity patch and promote wandering. Wandering is decreased due to global effects from pheromone signaling as well as interactions between receptor-activated Gβ and the exchange factor for the polarity regulator Cdc42. We found that artificially stabilizing patch wandering impaired accurate gradient tracking.

We suggest that ongoing polarized vesicle traffic causes patch wandering, which is locally reduced by pheromone-bound receptors. Thus, over time, spatial information from the pheromone gradient biases the random wandering of the polarity patch so that growth occurs predominantly up-gradient. Such temporal integration may enable sorting the low signal from stochastic noise when tracking shallow gradients.

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.

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for unipolar or multipolar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by two-component reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We investigate the behavior of a simple class of models and show that while the timescale of competition varies enormously depending on model parameters, a single factor explains a large majority of this variation. The dominant factor concerns the degree to which the maximal active GTPase concentration in a cluster approaches a “saturation point” determined by model parameters. We further show that the Rho-GTPase polarity machinery in the budding yeast S. cerevisiae, which normally generates only one bud through competition, can be manipulated to generate multiple buds

in ways consistent with this theoretical framework. We suggest that both saturation and the effect of saturation on competition reflect fundamental properties of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism, which predict whether the system will generate unipolar or multipolar outcomes.