Spatiotemporal control of pathway sensors and cross-pathway feedback regulate a differentiation MAPK pathway in yeast

Mitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the pathway that regulates the response to osmotic stress (HOG). Here, we show that the two pathways exhibit different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding mucin sensors that regulate the pathways. Cross-pathway regulation from the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. We also show that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway regulation, control a MAPK pathway that regulates cell differentiation in yeast.

Bil2 Is a Novel Inhibitor of the Yeast Formin Bnr1 Required for Proper Actin Cable Organization and Polarized Secretion

Cell growth in budding yeast depends on rapid and on-going assembly and turnover of polarized actin cables, which direct intracellular transport of post-Golgi vesicles to the bud tip. Saccharomyces cerevisiae actin cables are polymerized by two formins, Bni1 and Bnr1. Bni1 assembles cables in the bud, while Bnr1 is anchored to the bud neck and assembles cables that specifically extend filling the mother cell. Here, we report a formin regulatory role for YGL015c, a previously uncharacterized open reading frame, which we have named Bud6 Interacting Ligand 2 (BIL2). bil2Δ cells display defects in actin cable architecture and partially-impaired secretory vesicle transport. Bil2 inhibits Bnr1-mediated actin filament nucleation in vitro, yet has no effect on the rate of Bnr1-mediated filament elongation. This activity profile for Bil2 resembles that of another yeast formin regulator, the F-BAR protein Hof1, and we find that bil2Δ with hof1Δ are synthetic lethal. Unlike Hof1, which localizes exclusively to the bud neck, GFP-Bil2 localizes to the cytosol, secretory vesicles, and sites of polarized cell growth. Further, we provide evidence that Hof1 and Bil2 inhibitory effects on Bnr1 are overcome by distinct mechanisms. Together, our results suggest that Bil2 and Hof1 perform distinct yet genetically complementary roles in inhibiting the actin nucleation activity of Bnr1 to control actin cable assembly and polarized secretion.

Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

ABSTRACTMitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. MAPK pathways can share components with other pathways yet induce specific responses through mechanisms that remain unclear. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the MAPK pathway that regulates the response to osmotic stress (HOG). By exploring temporal regulation of MAPK signaling, we show here that the two pathways exhibited different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding the mucin sensors (MSB2 for fMAPK and HKR1 for HOG). We also show that positive feedback through the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. By exploring spatial regulation of MAPK signaling, we found that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway feedback, regulate a MAPK pathway that controls a cell differentiation response in yeast.

Registered report protocol: Quantitative analysis of septin Cdc10-associated proteome in Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. C. neoformans can thrive during infections due to its three main virulence-related characteristics: the ability to grow at host temperature (37°C), formation of carbohydrate capsule, and its ability to produce melanin. C. neoformans strains lacking septin proteins Cdc3 or Cdc12 are viable at 25°C; however, they fail to proliferate at 37°C and are avirulent in the murine model of infection. The basis of septin contribution to growth at host temperature remains unknown. Septins are a family of conserved filament-forming GTPases with roles in cytokinesis and morphogenesis. In the model organism Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher order complex at the mother-bud neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother-bud neck but the septin interacting proteome in this species remains largely unknown. Moreover, it remains possible that septins play other roles important for high temperature stress that are independent of their established role in cytokinesis. Therefore, we propose to perform a global analysis of septin Cdc10 binding partners in C. neoformans, including those that are specific to high temperature stress. This analysis will shed light on the underlying mechanism of survival of this pathogenic yeast during infection and can potentially lead to the discovery of novel drug targets.

A septin-Hof1 scaffold at the yeast bud neck binds and organizes actin cables

Septins and Hof1 form evenly spaced pillars at the yeast bud neck, which align, orient, and bundle actin cables entering the mother cell compartment to facilitate polarized vesicle transport.

Dynein collective behavior in mitotic nuclear positioning of Saccharomyces cerevisiae

1AbstractPositioning the nucleus at the bud-neck prior during Saccharomyces cerevisiae mitosis during anaphase involves pulling forces of cytoplasmic dynein localized in the daughter cell. While genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and dyneins numbers driving the process has remained difficult. In order to better understand the role of motor-microtubule mechanics during nuclear positioning and the role of dynein, we have used a computational model of nuclear mobility in S. cerevisiae and reconciled it to the mobility of labelled spindle pole bodies (SPBs) measured by quantifying fluorescence microscopy time-series. We model the apparent random-walk mobility of SPBs by combining diffusion of the nucleus and active pushing of MTs at the cell membrane. By minimizing the deviation between tracks of fluorescently tagged SPBs and simulations, we estimate the effective cytoplasmic viscosity to be 0.5 Pa s. The directed transport of nuclei during the budding process is similarly quantified by tracking the daughter SPB (SPB-D) in experiment. Using force-balance, we find 2 to 8 motors are required to pull the nucleus to the bud-neck. Simulations of the cytoplasmic MT (cMT) ‘search and capture’ by dynein suggest single motor binding is followed by a rapid saturation of number of bound motors. The short time and length of MT interactions with the cortex and minimal collective dynein force required, predict a functional role for dynein clustering in nuclear positioning.

Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator

Formins are essential actin assembly factors whose activities are controlled by a diverse array of binding partners. Until now, most formin ligands have been studied on an individual basis, leaving open the question of how multiple inputs are integrated to regulate formins in vivo. Here, we show that the F-BAR domain of Saccharomyces cerevisiae Hof1 interacts with the FH2 domain of the formin Bnr1 and blocks actin nucleation. Electron microscopy of the Hof1–Bnr1 complex reveals a novel dumbbell-shaped structure, with the tips of the F-BAR holding two FH2 dimers apart. Deletion of Hof1’s F-BAR domain in vivo results in disorganized actin cables and secretory defects. The formin-binding protein Bud6 strongly alleviates Hof1 inhibition in vitro, and bud6Δ suppresses hof1Δ defects in vivo. Whereas Hof1 stably resides at the bud neck, we show that Bud6 is delivered to the neck on secretory vesicles. We propose that Hof1 and Bud6 functions are intertwined as a stationary inhibitor and a mobile activator, respectively.