Electrochemical Regulation of Budding Yeast Polarity

Citation: Haupt A, Campetelli A, Bonazzi D, Piel M, Chang F, et al. ( Electrochemical Regulation of Budding Yeast Polarity Armin Haupt 0 Alexis Campetelli 0 Daria Bonazzi 0 Matthieu Piel 0 Fred Chang 0 Nicolas Minc 0 Mark D. Rose, Princeton University, United States of America 0 1 Institut Jacques Monod, UMR7592 CNRS, Paris, France, 2 Institut Curie, UMR 144 CNRS/IC, Paris, France, 3 Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons , New York, New York , United States of America Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization. - Funding: This work was supported by funds from the National Institutes of Health (http://www.nih.gov/, GM056836) to FC and the CNRS, ANR (http://www. agence-nationale-recherche.fr/, grant 10PDOC00301), FRM (http://www.frm.org/, grant AJE20130426890), the FP7 CIG and ITN FungiBrain (http://ec.europa.eu/ research/mariecurieactions/) and the Mairie de Paris emergence program (http://www.paris.fr/, LS100805) to NM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: EF, electric field; LatA, latrunculin A; PS, phosphatidylserine; TMP, transmembrane potential; WT, wild-type. . These authors contributed equally to this work. Cell polarization arises from the asymmetric accumulation of cellular components near a region of the plasma membrane. Although the roles of polarity proteins such as small GTPases and cytoskeletal elements have been studied extensively [1], much less is known about the possible contribution of electrochemical elements. Recent studies identifying certain ion transporters in regulating processes such as cell migration and polarized cell growth indicate potential roles of local pH, ion fluxes, and membrane potentials at the plasma membrane [28]. How these elements interface with established modules of polarity networks remains to be defined. The importance of electricity in cell polarization is illustrated by the ability of electric fields (EFs) to direct cell polarization. It has been appreciated for decades that most cellsranging from bacteria, fungi, and amoebas to animal cellsare electrotactic, and robustly orient polarity, migration, or division to applied exogenous EFs [914]. EFs of similar intensities as those used in these experiments naturally surround cells in tissues, and even individual cells such as fungal cells [10,15,16]. The physiological relevance of endogenous EFs has been demonstrated in fungal infection [17], immune cell response [18], wound healing, regeneration, and development [6,10,19,20]. These findings have led to the proposal that in addition to responding to chemical and mechanical signals, cells may also be responding to endogenous electrotactic signals to guide cell polarization [20]. The response of cells to exogenous EFs provides a powerful tool to study electrochemical elements in cell polarization. The molecular mechanisms of cell polarity are currently best understood in the budding yeast, Saccharomyces cerevisiae. Polarized cell growth in these cells is tightly controlled by intrinsic and extrinsic spatial cues. Haploid budding yeast cells display an axial budding pattern, in which new buds form adjacent to previous bud sites, while diploid cells exhibit a bipolar pattern, in which buds emerge at sites of previous division or growth [21,22]. During mating, cells of opposite mating type polarize towards each other in response to gradients of secreted pheromones; exogenous application of the pheromone a-factor causes cells to grow a mating projection, forming a pear-shaped shmoo. The core polarity machinery required for both bud and shmoo formation is organized around the small GTPase Cdc42p, which coordinates actin assembly and exocytosis [2325]. Bud site selection is specified by a Ras-like protein Rsr1p and its regulators [23]. During mating, these spatial cues used to direct budding are turned off, so that cells can polarize towards the mating partner. This reorientation of polarity involves Far1p and its interactions with the receptor-coupled Gb protein and Cdc42 GEF [2527]. As demonstrated by mutants affected in the regulation of only shmoos or only budding [23,28,29], there are specific molecular The 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 (...truncated)