Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

Abstract

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 "fine-tunes" the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. Fungal hyphae are able to elongate over large distances and must allocate their cellular resources in order to maintain extremely polarized growth for extended periods of time. The multimorphic opportunistic fungal pathogen Candida albicans provides a useful model system in which to investigate the basic cell biological and genetic mechanisms that generate highly polarized cell shapes and the requirements for hyphal development. C. albicans has a true hyphal growth form, as well as pseudohyphal and yeast forms, and reversibly switches between these morphologies depending on environmental conditions (reviewed in reference 1). Yeast cells are ellipsoid, propagate by budding, and undergo cytokinesis and cell separation. Pseudohyphae are more elongated than yeast cells and do not undergo cell separation, resulting in the formation of chains of elongated daughter cells. Hyphae, in contrast, are extremely elongated, narrow cells, and their development can be thought of as two continuous stages, early growth and development of germ tubes (GTs) followed by the development and maintenance of mature hyphae. GTs enter the maintenance phase of polarized growth, transitioning to mature hyphae, after the formation of the first septum, a specialized structure that delimits cellular compartments. Whereas morphogenesis mechanisms in C. albicans yeast and pseudohyphae appear to follow those of the similar morpholo...

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“…However, Rsr1 is intimately connected to the function of Cdc42 in both S. cerevisiae and C. albicans (Hausauer et al 2005;Park and Bi 2007;Brand et al 2008;Pulver et al 2013). Rsr1 plays an important role in Cdc42-mediated processes because it focuses the localization of Cdc42 at the hyphal tip in C. albicans (Pulver et al 2013). In the absence of appropriate levels of Rsr1, hyphae formation is reduced due to decreased concentrations of active Cdc42.…”

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“…This requires, among others, the protein Cdc42 [46,47]. The dynamics of this GTPase and the local activity [48] control directional growth and promote thigmotropism, which contributes to invasion. Hyphal tip growth requires intracellular compartments, vesicles, and trafficking.…”