Biology subjects

Lew, D. J.

Publications and source records attributed to Lew, D. J..

18 recordsLinked to original sources

bioRxiv2026

Regulation of polarity during morphological transitions in Aureobasidium pullulans

Many fungi are able to switch between different morphologies by regulating the behavior of the cytoskeleton. In turn, the cytoskeleton is regulated by Rho-family GTPases, whose differing spatiotemporal patterns of activity lead to different modes of growth. The polymorphic fungus Aureobasidium pullulans alternates between multiple growth modes, including multibudding, isotropic, and hyphal growth. Here, we describe hyphal growth and the yeast-to-hyphal transition in this organism, with a focus on cell polarity. Yeast cells switched between cycles of budding and isotropic expansion in a variable manner prior to initiating hyphal extension. Initial hyphal extension proceeded in a stop-and-start manner with frequent pauses, before switching to continuous growth. Hyphal branching occurred from the sides of hyphae, and septation at multiple sites occurred in more mature hyphae. Previous work showed that either of two Rho-family GTPases, Rac1 and Cdc42, were sufficient promote budding. However, while Rac1 was also able to drive hyphal extension without Cdc42, Cdc42 could not promote hyphal growth without Rac1. Our findings provide the basis for an investigation of how polarity components are re-purposed for different fungal growth modalities.

cell biology↗

bioRxiv2026

Organelle scaling over a 100-fold cell size range

Cell size in a proliferating cell population generally varies over a limited range ([~]2-4-fold). Within such populations, organelle content increases with cell size maintaining a relatively constant organelle density (amount per cell volume). However, cells of different types can differ greatly in cell size as well as in organelle composition. In such cases, it is often unclear to what degree, if any, the differences in organelle composition are due to the difference in cell size. In principle, this issue could be resolved by examining situations where a proliferating population of cells of the same cell type exhibit much greater size variation. Here we characterize how organelle content scales with cell volume in the polymorphic fungus, A. pullulans, whose proliferating cells span a [~]100-fold size range. We find that mitochondria and ER content increases in proportion to cell volume, while this is not the case for vacuoles and peroxisomes. Thus, organelle composition is affected by cell size in this system.

cell biology↗

bioRxiv2026

Organelle partitioning in the multi-budding yeast Aureobasidium pullulans

Cellular organelle content is fairly constant within a given cell type. This is accomplished in part by ensuring equitable organelle partitioning during division. Much of our understanding of organelle inheritance has come from investigating cells that divide in half producing two daughter cells. However, more elaborate division strategies that give rise to multiple daughters are not uncommon in nature. Here, we present the first characterization of organelle inheritance in a fungus that grows by multi-budding, producing several (2-20) daughter cells in a single cell cycle. We find that some organelles (mitochondria and ER) are evenly delivered to all growing buds, while others (vacuole and peroxisomes) are more variably inherited. We discuss the implications of even and uneven inheritance for this polyextremotolerant fungus capable of growing in dynamic, and diverse, environments.

cell biology↗

bioRxiv2026

Septins and cytokinesis in the polymorphic fungus Aureobasidium pullulans

During cytokinesis of animals and fungi, a contractile actomyosin ring (CAR) assembles at target locations and constricts to drive cell separation. In animal cells, the position of the CAR is determined by the mitotic spindle, so that the cleavage plane is perpendicular to the mitotic axis. However, in budding yeasts, the location of CAR assembly is specified by a cortical septin cytoskeleton that recruits CAR components to the neck. In the polymorphic fungus Aureobasidium pullulans, we show that septins assemble at mother-bud necks and predict the site of CAR assembly. Cells lacking septins stochastically failed to assemble CARs at a subset of bud necks. However, even cells lacking all four core septins were able to assemble CARs at 75% of bud necks. Our findings suggest the existence of a novel CAR positioning strategy that requires neither septin scaffolds nor nuclear/spindle cues to enable CAR assembly and constriction at bud necks. eTOC SUMMARYBudding yeasts are thought to use septins to mark mother-bud necks as sites for cytokinesis. Here, we find that the multibudding yeast Aureobasidium pullulans can position cytokinetic machinery at most bud necks even in the absence of septins, revealing a novel pathway to mark cytokinesis sites.

cell biology↗

bioRxiv2025

Regulation of the cell wall integrity pathway at the contact site between mating partners in yeast

The fungal cell wall is constantly remodeled to allow cell growth, but any holes in the cell wall would lead to catastrophic lysis. The "Cell Wall Integrity" pathway (CWI) detects cell wall defects and promotes cell wall thickening or repair to protect cell integrity. However, cell walls must be removed at contact sites between fusing cells during mating or mycelium formation. Here we show that in Saccharomyces cerevisiae, the CWI is downregulated specifically at the contact site between mating cells. A key component of the CWI, Pkc1, accumulated at polarity sites (shmoo tips) in cells exposed to mating pheromone, but not at contact sites. Pkc1 exclusion required a cell wall protein, Fig2, induced by pheromone. In mutants lacking Fig2, cell wall removal was delayed, blocked, or even reversed after transient fusion, leading to reduced mating. These results suggest that Fig2 designates the contact site as a "safe" spot to degrade the cell wall. eTOCCurtis and Lew show that the fungal "Cell Wall Integrity" repair pathway is silenced at contact sites between mating partners to allow cell wall degradation and fusion. They identify a cell wall protein needed to distinguish the contact site as a safe spot for wall removal.

cell biology↗

bioRxiv2025

Allocation of resources among multiple daughter cells

Cell division commonly produces two daughter cells, but there are many exceptions where large cells produce multiple daughters. Multiple fission of some green algae and bacteria, cellularization during embryogenesis of plants and insects, and growth of Ichthyosporeans, Chytrids, and Apicomplexans all provide variations on this theme. In some yeast species, a large multi-nucleate mother cell grows multiple buds (daughters) simultaneously. Here we address how mothers partition growth equally among their buds in the multi-budding yeast Aureobasidium pullulans. Bud growth is directed by actin cable networks that appear to be optimized for even partitioning despite complex cell geometries. Even partitioning does not rely on compensatory mechanisms to adjust bud volumes, but rather stems directly from effective equalization of polarity sites. These results reveal how conserved cell polarity and cytoskeletal networks are adapted to build complex morphologies in fungi.

cell biology↗

bioRxiv2025

A genetic strategy to allow detection of F-actin by phalloidin staining in diverse fungi

Actin is highly conserved across eukaryotes. This versatile protein builds cytoskeletal networks central to diverse cellular processes, including cell division and cell motility. The most potent and broadly used reagents to detect polymerized actin distribution in fixed cells are fluorescently conjugated derivatives of the basidiomycete-derived toxin, phalloidin. However, despite its conservation, actin in many ascomycete fungi fails to bind phalloidin. Here we trace the failure to bind phalloidin to a single amino acid change in a phalloidin-binding residue in actin. Reverting this change in the fungus Aureobasidium pullulans by introducing the point mutation act1V75I at the native ACT1 locus confers phalloidin binding without disrupting actin function. We describe a simple genetic technique to introduce this point mutation that may be effective in other fungal systems. This strategy should enable characterization of F-actin in a wider range of fungi. SUMMARYA single point mutation, act1V75I, enables phalloidin staining in ascomycete fungi

cell biology↗

bioRxiv2025

Local collective memory from ratiometric signaling outperforms cellular gradient sensing limits

Cells excel at interpreting noisy chemical gradients to guide fertilization, development, and immune responses, but the mechanisms underlying this remarkable ability remain poorly understood. Previous work showed that some G protein signaling pathways can overcome challenges from uneven receptor distribution by using a ratiometric signaling strategy. In this mechanism, G proteins receive information from both bound and unbound receptors, unlike classical signaling where only bound receptors contribute. Here, we show that ratiometric signaling also provides an unexpected ability to suppress noise from low receptor numbers. The benefit stems from each G protein remembering the last receptor state it encountered, so that at any instant, ratiometric G protein collectives reflect time-averaged receptor activity. Unlike classical signaling, this averaging remains unbiased and accurate across the varying ligand concentrations present in a spatial gradient. Using theory and simulations, we demonstrate that this averaging mechanism allows cells to surpass theoretical limits for gradient detection from instantaneous receptor information alone. Our findings reveal how ratiometric biochemical architectures enable robust temporal integration across spatially varying signals, providing cells with enhanced directional accuracy under noisy conditions.

biophysics↗

bioRxiv2025

Optimized vectors for genetic engineering of Aureobasidium pullulans

Aureobasidium pullulans is a polyextremotolerant black yeast that exhibits impressive morphological plasticity. Consequently, it shows promise as a model system for investigating mechanisms of cell adaptation to different environments and the regulation of cell shape. Here, we build upon the current toolkit for working with A. pullulans and design and test 25 vectors with seven different codon-optimized fluorophores and three selection cassettes. This includes vectors that allow for dual expression of GFP and mCherry tagged proteins at the URA3 locus and vectors that enable homology-based deletion or C-terminal tagging of endogenous genes without the need for cloning. This versatile vector series for working with A. pullulans will enable a broad range of experiments in this emerging model system. SUMMARYAn optimized plasmid toolkit for genetic engineering of the emerging model fungus, A. pullulans.

cell biology↗

bioRxiv2024

Negative feedback equalizes polarity sites in a multi-budding yeast

Morphogenesis in fungi and animals is directed by polarization of small GTPases Cdc42 and Rac. In the budding yeast Saccharomyces cerevisiae competition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeast Aureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1, and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.

cell biology↗

bioRxiv2023

Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are insufficient to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated a mobile polarity site with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient. Author summaryCells perform many complex tasks, including directed growth, migration, division and differentiation. To accomplish these tasks, the relevant molecular machinery is localized to specific cellular regions. The asymmetric distribution of cellular components is referred to as cell polarity. Polarity is established by localized activation of the protein Cdc42. Establishing mechanisms that regulate the spatiotemporal activity of Cdc42 is a fundamental area of cell biology. Mating yeast cells dynamically relocate a region of high Cdc42 activity, referred to as the polarity site, and grow toward each other after proper alignment of the sites. We investigated mechanisms that generate dynamic polarity sites by performing particle-based simulations of the biochemical reactions that regulate Cdc42 activity. The reactions contain two positive feedback loops that reinforce Cdc42 activity. The first involves autocatalytic activation of Cdc42 through recruitment of an activator. While necessary for polarity establishment, this feedback loop on its own created a stable polarity site that did not relocate. Incorporation of the second feedback loop, which couples the polarity machinery to extracellular mating signals, generated mobile polarity sites. This feedback loop also provides a mechanism for developing stable alignment of polarity sites. Our findings provide insight into how cells regulate polarity dynamics to accomplish complex tasks.

systems biology↗

bioRxiv2022

Mechanism of commitment to a mating partner in Saccharomyces cerevisiae

Many cells detect and follow gradients of chemical signals to perform their functions. Yeast cells use gradients of extracellular pheromones to locate mating partners, providing a tractable model to understand how cells decode the spatial information in gradients. To mate, yeast cells must orient polarity toward the mating partner. Polarity sites are mobile, exploring the cell cortex until they reach the proper position, where they stop moving and "commit" to the partner. A simple model to explain commitment posits that a high concentration of pheromone is only detected upon alignment of partner cells polarity sites, and causes polarity site movement to stop. Here we explore how yeast cells respond to partners that make different amounts of pheromone. Commitment was surprisingly robust to varying pheromone levels, ruling out the simple model. We also tested whether adaptive pathways were responsible for the robustness of commitment, but our results show that cells lacking those pathways were still able to accommodate changes in pheromone. To explain this robustness, we suggest that the steep pheromone gradients near each mating partners polarity site trap the polarity site in place. This mechanism has evolutionary implications because it enables sexual selection for cells making more pheromone.

cell biology↗

bioRxiv2020

A novel stochastic simulation approach enables exploration of mechanisms for regulating polarity site movement

Cells polarize their movement or growth toward external directional cues in many different contexts. For example, budding yeast cells grow toward potential mating partners in response to pheromone gradients. Directed growth is controlled by polarity factors that assemble into clusters at the cell membrane. The clusters assemble, disassemble, and move between different regions of the membrane before eventually forming a stable polarity site directed toward the pheromone source. Pathways that regulate clustering have been identified but the molecular mechanisms that regulate cluster mobility are not well understood. To gain insight into the contribution of chemical noise to cluster behavior we simulated clustering within the reaction-diffusion master equation (RDME) framework to account for molecular-level fluctuations. RDME simulations are a computationally efficient approximation, but their results can diverge from the underlying microscopic dynamics. We implemented novel concentration-dependent rate constants that improved the accuracy of RDME-based simulations of cluster behavior, allowing us to efficiently investigate how cluster dynamics might be regulated. Molecular noise was effective in relocating clusters when the clusters contained low numbers of limiting polarity factors, and when Cdc42, the central polarity regulator, exhibited short dwell times at the polarity site. Cluster stabilization occurred when abundances or binding rates were altered to either lengthen dwell times or increase the number of polarity molecules in the cluster. We validated key results using full 3D particle-based simulations. Understanding the mechanisms cells use to regulate the dynamics of polarity clusters should provide insights into how cells dynamically track external directional cues. Author summaryCells localize polarity molecules in a small region of the plasma membrane forming a polarity cluster that directs functions such as migration, reproduction, and growth. Guided by external signals, these clusters move across the membrane allowing cells to reorient growth or motion. The polarity molecules continuously and randomly shuttle between the cluster and the cell cytosol and, as a result, the number and distribution of molecules at the cluster constantly changes. Here we present an improved stochastic simulation algorithm to investigate how such molecular-scale fluctuations induce cluster movement across the cell membrane. Unexpectedly, cluster mobility does not correlate with variations in total molecule abundance within the cluster, but rather with changes in the spatial distribution of molecules that form the cluster. Cluster motion is faster when polarity molecules are scarce and when they shuttle rapidly between the cluster and the cytosol. Our results suggest that cells control cluster mobility by regulating the abundance of polarity molecules and biochemical reactions that affect the time molecules spend at the cluster. We provide insights into how cells harness random molecular behavior to perform functions important for survival, such as detecting the direction of external signals.

systems biology↗

bioRxiv2020

Exploratory polarization facilitates mating partner selection in Saccharomyces cerevisiae

Yeast decode pheromone gradients to locate mating partners, providing a model for chemotropism. How yeast polarize toward a single partner in crowded environments is unclear. Initially, cells often polarize in unproductive directions, but then they relocate the polarity site until two partners polarity sites align, whereupon the cells "commit" to each other by stabilizing polarity to promote fusion. Here we address the role of the early mobile polarity sites. We found that commitment by either partner failed if just one partner was defective in generating, orienting, or stabilizing its mobile polarity sites. Mobile polarity sites were enriched for pheromone receptors and G proteins, and we suggest that such sites engage in an exploratory search of the local pheromone landscape, stabilizing only when they detect elevated pheromone levels. Mobile polarity sites were also enriched for pheromone secretion factors, and simulations suggest that only focal secretion at polarity sites would produce high pheromone concentrations at the partners polarity site, triggering commitment.

cell biology↗

bioRxiv2020

How cells determine the number of polarity sites

The diversity of cell morphologies arises, in part, through regulation of cell polarity by Rho-family GTPases. A poorly understood but fundamental question concerns the regulatory mechanisms by which different cells can generate different numbers of polarity sites. Theoretical models of polarity circuits develop multiple initial polarity sites, but then those sites engage in competition, leaving a single winner. The timescale of competition slows dramatically as GTPase concentrations at polarity sites approach a "saturation point", allowing multiple sites to coexist. Here, we show that these principles hold in more complex mechanistic models of the Saccharomyces cerevisiae polarity machinery, and confirm model predictions in vivo. Further, we elucidate a novel design principle whereby cells can switch from competition to equalization among polarity sites. These findings provide insight into how cells determine the number of polarity sites.

cell biology↗

bioRxiv2018

How yeast cells find their mates

Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors during mating encounters, we found that gradient decoding involves two steps. First, cells bias orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. To achieve this, they measure the local fraction of occupied receptors, rather than absolute number. However, this process is error-prone, and subsequent exploratory behavior of the polarity factors corrects initial errors via communication between mating partners. The mobile polarity sites convert the difficult problem of spatial gradient decoding into the easier one of sensing temporal changes in local pheromone levels.

cell biology↗

bioRxiv2018

Mating in wild yeast: variable interest in sex after spore germination

Studies of lab strains of Saccharomyces cerevisiae have uncovered signaling pathways involved in mating, including information processing strategies to optimize decisions to mate or to bud. However, lab strains are heterothallic (unable to self-mate) while wild yeast are homothallic. And while mating of lab strains is studied using cycling haploid cells, mating of wild yeast is thought to involve germinating spores. Thus, it was unclear whether lab strategies would be appropriate in the wild. Here, we have investigated the behaviors of several yeast strains derived from wild isolates. Following germination, these strains displayed large differences in their propensity to mate or to enter the cell cycle. The variable interest in sex following germination was correlated with differences in pheromone production, which were due to both cis- and trans-acting factors. Our findings suggest that yeast spores germinating in the wild may often enter the cell cycle and form microcolonies prior to engaging in mating.

cell biology↗

bioRxiv2017

Principles that govern competition or co-existence in Rho-GTPase driven polarization

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 uni-polar or multi-polar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by 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 derive a \"saturation rule\" general to all relevant models that governs the timescale of competition, and thereby predicts whether the system will generate uni-polar or multi-polar outcomes. We suggest that the saturation rule is a fundamental property of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism.

cell biology↗