A fundamental problem in cell biology is to understand how spatial information is recognized and integrated into morphogenetic responses. Budding yeast undergoes differentiation to filamentous growth, which involves changes in cell polarity through mechanisms that remain obscure. Here we define a regulatory input where spatial landmarks (bud-site–selection proteins) regulate the MAPK pathway that controls filamentous growth (fMAPK pathway). The bud-site GTPase Rsr1p regulated the fMAPK pathway through Cdc24p, the guanine nucleotide exchange factor for the polarity establishment GTPase Cdc42p. Positional landmarks that direct Rsr1p to bud sites conditionally regulated the fMAPK pathway, corresponding to their roles in regulating bud-site selection. Therefore, cell differentiation is achieved in part by the reorganization of polarity at bud sites. In line with this conclusion, dynamic changes in budding pattern during filamentous growth induced corresponding changes in fMAPK activity. Intrinsic compromise of bud-site selection also impacted fMAPK activity. Therefore, a surveillance mechanism monitors spatial position in response to extrinsic and intrinsic stress and modulates the response through a differentiation MAPK pathway.

The opportunistic fungal pathogen Candida albicans is capable of adhering to the oral mucosa despite forces created by salivary flow. Although many fungal adhesion proteins have been identified, less is known about the temporal development of cell adhesion and biofilm growth in a flow environment. In this study, we use a flow system with real-time imaging of C. albicans cells as they adhere and grow. Rates of cell attachment and dispersion of C. albicans knockout strains of putative adhesins, transcription factors, and deletions with a hyperfilamentous phenotype were quantified during 18 h of biofilm development. Cell adhesion under flow is a multi-phase process initiated with cell rolling, then an initial firm attachment to the substrate occurs. After attachment, cells enter a growth phase where cells either commit to adherence or disperse. C. albicans Δ eap1 , Δ hwp2 , Δ hyr1 , and Δ ihd1 cells had significantly reduced initial attachment and subsequent adhesion, while Δ als1 /Δ als3 had no change in initial attachment but reduced adhesion maintenance. WT cells had increased adhesion during the late growth phase when hyphae were more highly expressed. Hyperfilamentous strains had 10-fold higher total biofilm growth, a result of significantly reduced detachment rates, showing that hyphal morphogenesis is important for adhesion maintenance in the developing biofilm. The rate of C. albicans biomass dispersion was most important for determining the density of the mature biomass. Adhesion maintenance was mediated in part by Ywp1, a protein previously thought to regulate dispersion, thus it functions as an adhesion maintenance protein in C. albicans .

All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in the yeast Saccharomyces cerevisiae, which is controlled by the Rho GTPase Cdc42p. The prevailing view of bud emergence does not account for regulation by extrinsic cues. Here, we show that the filamentous growth mitogen activated protein kinase (fMAPK) pathway regulates bud emergence under nutrient-limiting conditions. The fMAPK pathway regulated the expression of polarity targets including the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and active during the period of the cell cycle leading up to bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

Many fungal species, including pathogens, undergo a morphogenetic response called filamentous growth, where cells differentiate into a specialized cell type to promote nutrient foraging and surface colonization. Despite the fact that filamentous growth is required for virulence in some plant and animal pathogens, certain aspects of this behavior remain poorly understood. By examining filamentous growth in the budding yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans, we identify responses where cells undergo filamentous growth in groups of cells or aggregates. In S. cerevisiae, aggregate invasive growth was regulated by signaling pathways that control normal filamentous growth. These pathways promoted aggregation in part by fostering aspects of microbial cooperation. For example, aggregate invasive growth required cellular contacts mediated by the flocculin Flo11p, which was produced at higher levels in aggregates than cells undergoing regular invasive growth. Aggregate invasive growth was also stimulated by secreted enzymes, like invertase, which produce metabolites that are shared among cells. Aggregate invasive growth was also induced by alcohols that promote density-dependent filamentous growth in yeast. Aggregate invasive growth also required highly polarized cell morphologies, which may affect the packing or organization of cells. A directed selection experiment for aggregating phenotypes uncovered roles for the fMAPK and RAS pathways, which indicates that these pathways play a general role in regulating aggregate-based responses in yeast. Our study extends the range of responses controlled by filamentation regulatory pathways and has implications in understanding aspects of fungal biology that may be relevant to fungal pathogenesis. IMPORTANCE Filamentous growth is a fungal morphogenetic response that is critical for virulence in some fungal species. Many aspects of filamentous growth remain poorly understood. We have identified an aspect of filamentous growth in the budding yeast Saccharomyces cerevisiae and the human pathogen Candida albicans where cells behave collectively to invade surfaces in aggregates. These responses may reflect an extension of normal filamentous growth, as they share the same signaling pathways and effector processes. Aggregate responses may involve cooperation among individual cells, because aggregation was stimulated by cell adhesion molecules, secreted enzymes, and diffusible molecules that promote quorum sensing. Our study may provide insights into the genetic basis of collective cellular responses in fungi. The study may have ramifications in fungal pathogenesis, in situations where collective responses occur to promote virulence.

The fungal cell wall is an extracellular organelle that provides structure and protection to cells. The cell wall also influences the interactions of cells with each other and surfaces. The cell wall can be reorganized in response to changing environmental conditions and different types of stress. Signaling pathways control the remodeling of the cell wall through target proteins that are in many cases not well defined. The Mitogen Activated Protein Kinase pathway that controls filamentous growth in yeast (fMAPK) was required for normal growth in media containing the cell wall perturbing agent Calcofluor White (CFW). A mass spectrometry (MASS-SPEC) approach and analysis of expression profiling data identified cell wall proteins and modifying enzymes whose levels were influenced by the fMAPK pathway. These include Flo11p, Flo10p, Tip1p, Pry2p and the mannosyltransferase, Och1p. Cells lacking Flo11p or Och1p were sensitive to CFW. The identification of cell wall proteins controlled by a MAPK pathway may provide insights into how signaling pathways regulate the cell wall.