Chemotropism, the ability to orient growth toward external chemical cues, is a fundamental process in diverse eukaryotic systems. During mating, budding yeast cells detect pheromone gradients from potential partners, locating them by assembling a gradient-tracking machine (GTM) at the plasma membrane that redistributes upgradient prior to polarized growth. Although membrane lipids are known to influence pheromone signaling and morphogenesis, their roles in pre-morphogenic gradient tracking have remained unclear. Here, we show that phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate, and ergosterol exhibit GTM-like dynamics, polarizing to the default polarity site, redistributing upgradient, and stabilizing at the chemotropic site. Blocking PS synthesis causes a severe and specific gradient-tracking defect, whereas disruption of Bem1 binding to anionic lipids slows but does not abolish tracking. Analysis of polarity, Cdc42 activity, and exocyst dynamics indicates that Bem1 membrane binding contributes to spatial focusing of Cdc42 activation during tracking but cannot account for the pronounced defects caused by PS loss, indicating that PS influences gradient tracking through multiple GTM components. In contrast, ergosterol is dispensable for tracking but required for proper receptor organization and partner alignment after GTM stabilization. Together, these findings establish membrane lipids as integral GTM components and highlight PS as a key regulator of chemotropic gradient sensing through multivalent protein-lipid interactions.

Directional chemosensing is ubiquitous in cell biology, but some cells such as mating yeast paradoxically degrade the signal they aim to detect. While the data processing inequality suggests that such signal modification cannot increase the sensory information, we show using a reaction-diffusion model and an exactly solvable discrete-state reduction that it can. We identify a non-Markovian step in the information chain allowing the system to evade the data processing inequality, reflecting the nonlocal nature of diffusion. Our results apply to any sensory system in which degradation couples to diffusion. Experimental data suggest that mating yeast operate in the beneficial regime where degradation improves sensing.

ABSTRACT Cell polarization in response to chemical gradients is important in development and homeostasis across eukaryota. Chemosensing cells orient toward or away from gradient sources by polarizing along a front–rear axis. Using the mating response of budding yeast as a model of chemotropic cell polarization, we found that Dcv1, a member of the claudin superfamily, influences front–rear polarity. Although Dcv1 localized uniformly on the plasma membrane (PM) of vegetative cells, it was confined to the rear of cells responding to pheromone, away from the pheromone receptor. dcv1Δ conferred mislocalization of sensory, polarity and trafficking proteins, as well as PM lipids. These phenotypes correlated with defects in pheromone-gradient tracking and cell fusion. We propose that Dcv1 helps demarcate the mating-specific front domain primarily by restricting PM lipid distribution.

This paper presents the results of an archaeobotanical analysis of plant macro-remains recovered during excavations of a rural tepe site at Qaratepe, Azerbaijan, occupied during the Sasanian and Islamic periods between the 2nd and 13th centuries ad . The material derives from a 4 year Oxford University expedition which occurred between 2015 and 2018, ‘The Archaeological Exploration of Barda Project (AEB)’, established to investigate the provincial structure of the eastern Caucasus region in the Late Antique and early Islamic periods. Traditionally, archaeological practice in Azerbaijan has not embraced environmental archaeological techniques and despite the region’s importance to the understanding of early agriculture and the diffusion of crop species during the Islamic period, little archaeobotanical research has been conducted there to date. This assemblage therefore forms a rare and unique contribution to the field of archaeobotany in the Late Antique and Islamic periods in Azerbaijan and provides the first archaeobotanical evidence of crop husbandry at a rural settlement during these periods. In total, 8,676 carbonised plant remains representing a minimum of 60 species were recorded from 80 samples analysed, providing important insights into plant utilisation in Azerbaijan (Full taxonomic list available in on-line supplementary material (ESM)). Archaeobotanical evidence has revealed the range of crops cultivated and consumed at the site between the 2nd and 13th centuries. Results demonstrate that naked wheat ( Triticum aestivum / durum / turgidum ), barley ( Hordeum vulgare ), and broomcorn millet ( Panicum miliaceum ) were the primarily cultivated crops between the 2nd and 6th centuries, key crops that have been present in the Southern Caucasus for several millennia. The study has also identified the cultivation of rice ( Oryza sativa ), watermelon ( Citrullus lanatus ), and melon ( Melo sativa ) in the 13th century, indicating a change in agricultural production in the Islamic period and the introduction of several new cultivars and agricultural adaptions.

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Since positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking. What prevents the GTM from triggering polarized growth at the DS? Here, we describe two mechanisms that are essential for tracking: inactivation of the Ras GTPase Bud1 and positioning of actin-independent vesicle delivery upgradient.

Cell polarization in response to chemical gradients is important in development and homeostasis across eukaryota. Chemosensing cells orient toward or away from gradient sources by polarizing along a front-rear axis. Using the chemotropic mating response of the budding yeast S. cerevisiae as a model of environmentally-induced cell polarization, we found that Dcv1, a claudin homolog, is a determinant of front-rear polarity. Although Dcv1 localized uniformly on the plasma membrane (PM) † of vegetative cells, it was confined to the rear of cells responding to pheromone, away from the pheromone receptor and mating projection. Deletion of DCV1 conferred mislocalization of sensory, polarity, and trafficking proteins, as well as the PM lipids ergosterol, phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylserine (PS). These phenotypes correlated with defects in pheromone-gradient tracking and cell fusion. We propose that the novel claudin-like and rear-domain protein, Dcv1, demarcates the mating-specific front domain primarily by restricting PM lipid distribution. Consistent with this hypothesis, a mutation that blocks ergosterol biosynthesis partially phenocopies dcv1 Δ. Summary statement The yeast claudin Dcv1 facilitates the proper localization of plasma membrane lipids and proteins that are required for front-rear polarity, efficient chemotropism, and cell fusion.

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gβ subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gβ and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gβ and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gβ and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gβ correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gβ contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gβ exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through the secretion of mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Because strong positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking after its assembly is complete. What prevents the GTM from triggering polarized growth at the DS? Here we describe two mechanisms that enable tracking. First, the Ras GTPase Bud1 must be inactivated to release the GTM. Second, actin-independent – but not actin-dependent – vesicle delivery must be targeted upgradient to effect GTM redistribution.

The mating-specific yeast G alpha controls pheromone signaling by sequestering G beta gamma and by regulating the Fus3 MAP kinase. Disrupting G alpha-Fus3 interaction leads to severe defects in chemotropism. Because Ga concentrates at the chemotropic growth site where Fus3 is required for the phosphorylation of two known targets, we screened for additional proteins whose phosphorylation depends on pheromone stimulation and G alpha-Fus3 interaction. Using a mutant form of G alpha severely defective in Fus3-binding, G alpha(DSD), and quantitative mass spectrometry, fourteen proteins were identified as potential targets of G alpha-recruited Fus3, ten of which were previously implicated in cell polarity and morphogenesis. To explore the biological relevance of these findings, we focused on the Spa2 polarisome protein, which was hypophosphorylated on multiple serine residues in pheromone-treated G alpha(DSD) cells. Six sites were mutagenized to create the Spa2(6xsA) mutant protein. Spa2(6xsA) exhibited increased affinity for Fus3, consistent with a kinase-substrate interaction, and Spa2(6xsA) cells exhibited dramatic defects in gradient sensing and zygote formation. These results suggest that G alpha promotes the phosphorylation of Spa2 by Fus3 at the cortex of pheromone-stimulated cells, and that this mechanism plays a role in chemotropism. How the G alpha-Fus3 signaling hub affects the other putative targets identified here has yet to be determined. Significance: Previously, interaction between the G alpha protein, Gpa1, and the MAPK of the pheromone response pathway, Fus3, was shown to be important for efficient sensing of the pheromone gradient and for the maintenance of cell polarity during mating. Here we show that the underlying molecular mechanisms involve the phosphorylation of specific cortical targets of Gpa1/Fus3. These have been identified by quantitative phosphoproteomics using a mutant of Gpa1, which is defective in interacting with Fus3. One of these targets is the polarisome protein Spa2. Alanine substitution of the Spa2 phosphorylation sites targeted by Gpa1/Fus3 lead to a dramatic defect in pheromone gradient sensing and zygote formation. These results reveal how the G alpha protein and the MAPK control cell polarity in a prototypical model system. Our results have wider significance as similar mechanisms exist in higher eukaryotes and are involved in important biological such as neuron development, immunity, and cancer cell metastasis.

The mating of budding yeast depends on chemotropism, a fundamental cellular process. The two yeast mating types secrete peptide pheromones that bind to GPCRs on cells of the opposite type. Cells find and contact a partner by determining the direction of the pheromone source and polarizing their growth toward it. Actin-directed secretion to the chemotropic growth site (CS) generates a mating projection. When pheromone-stimulated cells are unable to sense a gradient, they form mating projections where they would have budded in the next cell cycle, at a position called the default polarity site (DS). Numerous models have been proposed to explain yeast gradient sensing, but none address how cells reliably switch from the intrinsically determined DS to the gradient-aligned CS, despite a weak spatial signal. Here we demonstrate that, in mating cells, the initially uniform receptor and G protein first polarize to the DS, then redistribute along the plasma membrane until they reach the CS. Our data indicate that signaling, polarity, and trafficking proteins localize to the DS during assembly of what we call the gradient tracking machine (GTM). Differential activation of the receptor triggers feedback mechanisms that bias exocytosis upgradient and endocytosis downgradient, thus enabling redistribution of the GTM toward the pheromone source. The GTM stabilizes when the receptor peak centers at the CS and the endocytic machinery surrounds it. A computational model simulates GTM tracking and stabilization and correctly predicts that its assembly at a single site contributes to mating fidelity.

In classical Cell Biology, fundamental cellular processes are revealed empirically, one experiment at a time. While this approach has been enormously fruitful, our understanding of cells is far from complete. In fact, the more we know, the more keenly we perceive our ignorance of the profoundly complex and dynamic molecular systems that underlie cell structure and function. Thus, it has become apparent to many cell biologists that experimentation alone is unlikely to yield major new paradigms, and that empiricism must be combined with theory and computational approaches to yield major new discoveries. To facilitate those discoveries, three workshops will convene annually for one day in three successive summers (2017-2019) to promote the use of computational modeling by cell biologists currently unconvinced of its utility or unsure how to apply it. The first of these workshops was held at the University of Illinois, Chicago in July 2017. Organized to facilitate interactions between traditional cell biologists and computational modelers, it provided a unique educational opportunity: a primer on how cell biologists with little or no relevant experience can incorporate computational modeling into their research. Here, we report on the workshop and describe how it addressed key issues that cell biologists face when considering modeling including: (1) Is my project appropriate for modeling? (2) What kind of data do I need to model my process? (3) How do I find a modeler to help me in integrating modeling approaches into my work? And, perhaps most importantly, (4) why should I bother?

The ability of cells to direct their movement and growth in response to shallow chemical gradients is essential in the life cycles of all eukaryotic organisms. The signaling mechanisms underlying directional sensing in chemotactic cells have been well studied; however, relatively little is known about how chemotropic cells interpret chemical gradients. Recent studies of chemotropism in budding and fission yeast have revealed 2 quite different mechanisms-biased wandering of the polarity complex, and differential internalization of the receptor and G protein. Each of these mechanisms has been proposed to play a key role in decoding mating pheromone gradients. Here we explore how they may work together as 2 essential components of one gradient sensing machine.

Chemotropism and chemotaxis are fundamental processes required for a broad range of biological phenomena. The mating process of budding yeast (Saccharomyces cerevisiae) is, to date, the best‐studied example of chemotropism. In mating mixtures, haploid yeast cells can interpret a shallow pheromone gradient, chemotrope toward the closest mating partner, and fuse to form a diploid zygote. The molecular machinery required for polarized growth has been well characterized. However, how yeast cells accurately position the polarity machinery towards the source of pheromone is unclear. It is well known that the pheromone receptor and its cognate G protein are uniformly distributed on the plasma membrane of vegetative cells, and that they polarize in response to pheromone. In our published model of yeast gradient sensing, inhibition of receptor phosphorylation by Gβg results in differential phosphorylation of the receptor across the cell surface, and consequently, lesser internalization of the receptor and G protein on the up‐gradient side of the cell. A key question is how the uniformly distributed surface receptor competes for a limiting amount of G protein. Here we show that in mating cells, the initially uniform receptor and G protein first localize as polarized crescents at the default polarity site. The receptor and G protein crescents then track along the plasma membrane until they reach the region of highest pheromone concentration, centered around the position at which the cell ultimately shmoos towards its partner. We also show that polarization of Gβ to the default polarity site is independent of receptor phosphorylation and polarization, whereas Gβ tracking from the default site to the eventual chemotropic site does not occur if receptor phosphorylation and redistribution are blocked. These observations suggest a new mechanism that localizes the receptor with its much less abundant G protein. In our revised model, we propose that mating cells that are arrested in G1 concentrate Gβg at the default polarity site, likely through its interaction with Far1‐Bem1‐Cdc24‐Cdc42. The polarized Gβg then protects the receptor from being phosphorylated and internalized, thereby triggering local accumulation of the receptor and G protein. Because the pheromone gradient is mirrored by a gradient of signaling activation within the receptor/G‐protein crescent, there are higher proportions of active‐unphosphorylated receptors and active G protein closer to the pheromone source. The peak of signaling activity incrementally moves up the pheromone gradient, as unprotected receptors are phosphorylated and co‐internalized with G proteins at the back, while vesicles containing nascent receptors and G proteins preferentially dock where the receptor is most abundant.Support or Funding InformationNSF

Chemotropic and chemotactic cells exhibit a remarkable ability to interpret chemical gradients and sense direction. The mating response of the budding yeast S. cerevisiae is a model chemotropic system. The two haploid mating types, MAT a and MAT α, sense the pheromone secreted by cells of the opposite type, polarize their growth to form a mating projection towards the closest mating partner, and fuse to form diploids. In MAT a cells, the Ste2 pheromone receptor is the primary gradient sensor. Ste2 is uniformly distributed on the plasma membrane (PM) in vegetative cells, but upon ligand binding, it is rapidly internalized. It then reappears as a polarized crescent that coincides with the incipient mating projection site. In mating mixtures, the newly emerged receptor crescents orient towards the closest mating partner. Although actin‐dependent directed secretion stabilizes and amplifies receptor polarity, we have shown that receptor polarization precedes the polarization of actin cables and occurs in the absence of actin‐directed secretion. In contrast, internalization of the receptor is essential for its polarization. How is receptor polarity established upstream of actin‐directed secretion and what are the key players? In a directed genetic screen, we found that deletion of the yeast claudin homolog, DCV1 , conferred a significant defect in receptor polarization without affecting actin‐directed secretion. Unlike wild type (WT) cells, dcv1Δ cells were unable to establish a receptor polarization site that could be amplified independently of f‐actin. Based on its primary structure, Dcv1 was predicted to be an integral membrane protein. Immuno‐fluorescence (IF) microscopy in cells expressing Dcv1‐HA suggested that the protein localizes uniformly to the PM in vegetative cells and away from the mating projection in pheromone‐treated cells. Studies of cells expressing fluorescently‐tagged Dcv1 confirmed the IF results. Furthermore, Dcv1 appeared to localize as a ring at the base of newly formed mating projections. Cells co‐expressing fluorescently‐tagged forms of Dcv1 and the receptor exhibited inverse localization of these proteins concurrent with morphogenesis. This result is consistent with the observation that claudins promote the formation of membrane domains and act as membrane barriers. dcv1 Δ cells also exhibited abnormal localization of various lipids in the PM. These include phosphotidylinositol, phosphotidylserine, and sterols. Preliminary lipidomic studies using mass spectrometry suggest differences in the phospholipid composition of the PM in WT and dcv1 Δ cells. Consistent with its effect on receptor polarization, dcv1 Δ conferred a defect in pheromone‐gradient sensing. Time‐lapse imaging of mating mixtures also revealed that Dcv1 is required for a variety of polarized mating functions in addition to receptor redistribution, and for efficient mating. In summary, our data implicate a gene of previously unknown function in the polarization of proteins and plasma membrane lipids that contribute to efficient chemotropism and mating. We propose that the yeast claudin Dcv1 plays a role in organizing mating‐specific membrane domains essential for the polarization of the receptor and other mating‐specific proteins. Support or Funding Information National Science Foundation

Chemotaxis (directed cell movement) and chemotropism (directed cell growth) are vital mechanisms essential to a variety of biological processes, including cell development, cancer metastasis, angiogenesis, and axon guidance. Both chemotaxis and chemotropism depend on the ability of the cell to interpret shallow and complex chemical gradients. One of the best‐studied models of eukaryotic directional sensing is the mating response of Saccharomyces cerevisiae (budding yeast). Yeast exist as two haploid mating types, MATa and MATα, that can sense pheromone gradients, chemotrop toward the closest mating partner, and eventually fuse at their tips to form diploid cells. In vegetative cells, the G protein coupled receptor (GPCR) that binds pheromone and its associated heterotrimeric G protein are uniformly distributed on the plasma membrane. Upon receptor activation, Gβγ is released from Gα and Gβ is rapidly phosphorylated on multiple residues. Free Gβγ signals through a MAP kinase cascade to trigger cell‐cycle arrest and changes in gene expression. Pheromone‐activated receptors are globally internalized and then reappear as polarized crescents that mark the chemotropic growth site, where the cell ultimately forms its mating projection. Previous studies have shown that the receptor crescents are visible before detectable actin polarization and that the receptors can polarize without actin‐dependent directed secretion [1]. On the contrary, inhibiting receptor internalization confers a moderate defect in gradient sensing [2]. A key question raised by these observations is how does the yeast cell interpret the external signals and establish the chemotropic growth site? From a directed genetic screen, we found that Pcl1 is critical for the establishment of receptor polarity. Pcl1 is a cyclin required by the cyclin dependent kinase Pho85, which has been implicated in polarization during budding. It is known that Gβ phosphorylation is crucial to receptor polarization, and Gβ is predicted to be a substrate of Pho85‐Pcl1 [3]. Additionally, our data indicated a genetic interaction between Gβ and Pcl1. Here, we show that Pcl1 localizes to the mating projection during the mating response. Moreover, Pcl1 and Gβ interacted directly on the plasma membrane of pheromone‐treated cells, as indicated by a Bimolecular fluorescence complementation assay. Lastly, Pho85 inactivation also appears to affect receptor polarity. Taken together, these results suggest that Pho85‐Pcl1 regulates polarization of the receptor by phosphorylating Gβ.Support or Funding InformationNational Science Foundation

Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion.

Virtually all eukaryotic cells can grow in a polarized fashion in response to external signals. Cells can respond to gradients of chemoattractants or chemorepellents by directional growth, a process referred to as chemotropism. The budding yeast Saccharomyces cerevisiae undergoes chemotropic growth during mating, in which two haploid cells of opposite mating type grow towards one another. Mating pheromone gradients are essential for efficient mating in yeast and different yeast mutants are defective in chemotropism. Two methods of assessing the ability of yeast strains to respond to pheromone gradients are presented here.

Cell polarization, the generation of cellular asymmetries, is a fundamental biological process. Polarity of different molecules can arise through several mechanisms. Among these, internalization has been shown to play an important role in the polarization of cell surface receptors. The internalization of cell surface receptors can be upregulated upon ligand binding. Additional regulatory mechanism can downregulate the internalization process. Here we describe a general model, which incorporates these two opposing processes, to study the role of internalization in the establishment of cell polarity. We find that the competition between these two processes is sufficient to induce receptor polarization. Our results show that regulated internalization provides additional regulation on polarization as well. In addition, we discuss applications of our model to the yeast system, which shows the capability and potential of the model.