| Popis: |
Polarity refers to the asymmetric organization of shape, structure, or function of cells. Cell polarization is critical for the function of nearly every cell type and underlies essential processes such as cell growth and division. Indeed, cell polarity is critical for proper cellular function — maintenance of cell polarity has been proposed to contribute to tumor suppression, while loss of cell polarity is commonly observed in tumors and correlates with invasion and the formation of metastases. The Cdc42 GTPase plays a central role in polarity development in species ranging from yeast to humans. Cdc42 polarization is directed by spatial cues in most organisms, yet the link between spatial landmarks and Cdc42 machinery has not been clear. Due to the conservation of polarity signaling across species, Saccharomyces cerevisiae, commonly known as budding yeast, has proven to be a tractable model system to study cell polarity. In budding yeast, selection of a growth site (i.e. bud site) determines the axis of Cdc42 polarization and subsequently the plane of cell division. The Rsr1 GTPase module composed of a Ras family GTPase Rsr1 and its regulators is required for selecting the proper bud site. These proteins interact with Cdc42 and its regulators to direct organization of the actin cytoskeleton and septin filaments for polarized growth at the selected site. While a number of studies in yeast have revealed an intricate crosstalk of polarity components underlying spatial cue-directed polarization, many questions remain. Budding yeast cells always select a new non-overlapping division site in each cell cycle. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the current division site by inhibiting Cdc42 repolarization. While a protein complex including Nba1 and Nis1 had been proposed to prevent rebudding at old division sites, here we show that Rga1 is the core of negative polarity signaling at any division site. Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. Remarkably, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and the axis of polarity is established prior to delocalization of Rga1. These findings highlight that coordination of spatial distribution of a Cdc42 GAP with cell cycle progression in G1 may be critical for fine-tuning the orientation of the polarity axis in yeast.Previous studies have suggested that Cdc42 polarization occurs in two steps in the G1 phase and that the Ras GTPase Rsr1 is necessary for Cdc42 polarization in the first step. While Rsr1-GTP interacts with Cdc42 and Cdc24, a guanine nucleotide exchange factor (GEF) for Cdc42, Rsr1-GDP preferentially interacts with Bem1, a scaffold protein, in vitro. It had been unclear why and how Rsr1-GDP interacts with Bem1, especially since both Cdc24 and Bem1 function together for Cdc42 polarization. In this work, we find that the interaction between GDP-bound Rsr1 and Bem1 is important to ensure the proper timing of events leading to bud emergence. Rsr1 associates with Bem1 preferentially in its GDP-bound state in early G1. This interaction involves a part of the Bem1 Phox homology (PX) domain, which overlaps with a region previously shown to interact with Exo70, an exocyst component. Furthermore, overexpression of the constitutively GDP-bound Rsr1 interferes with Bem1-dependent Exo70 polarization leading to delayed polarized secretion. Thus, Rsr1 coordinates spatial and temporal regulation of polarity establishment via its GTP- and GDP-bound states. Overall, this work provides key insights into the mechanisms that regulate polarity establishment in yeast and reveals underlying concepts of cell polarization that may be applicable to higher eukaryotes. This study highlights that Ras and Rho GTPases may not simply act as on and off switches, where the GTP bound form is only functional. Additionally, this work reveals how negative polarity cues are established and maintained through each cell division cycle for proper regulation of Cdc42 polarization and normal development. |
