Single-Molecule Atomic Force Microscopy Reveals Clustering of the Yeast Plasma-Membrane Sensor Wsc1

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches

Characterization of the essential role of Ynl152/Inn1 in cell division in Saccharomyces cerevisiae

The essential open reading frame YNL152w (now called INN1) of Saccharomyces cerevisiae was previously identified in a screen for negative regulators of cell integrity signaling. Subsequent studies and data from genome-wide functional analyses suggested, that the encoded protein plays a role in cell division. This was further addressed in the thesis presented here. Functional Inn1-GFP fusions were shown to co-localize with the contractile actomyosin ring component Myo1 during cytokinesis. Mutants depleted for Inn1 failed to form a primary septum, but did not affect the dynamics of the cytokinetic actin ring (CAR). This has been attributed to the inability to couple plasma membrane ingression (hence Inn1) to CAR constriction, a phenomenon also found by Sanchez-Diaz et al. (2008). Further investigations focused on the question of how Inn1 is recruited to the bud neck and identified the cytokinetic regulators Hof1 and Cyk3, which act in concert for this purpose. Each of them contains a SH3 domain, which interacts with the proline-rich carboxy-terminal part of Inn1. Localization studies and genetic analyses indicate that Inn1 acts downstream of Hof1 and Cyk3. Either the simultaneous repression of HOF1 and CYK3 gene expression or the deletion of their SH3 domains was lethal, with a concomitant failure to localize Inn1-GFP to the division site. Overproduction of either, Hof1 or Cyk3 perturbed the dynamics of Inn1-GFP distribution, which followed that of the overproduced proteins. This atypical CAR-independent localization of Inn1 supports a presumed role of Hof1 and Cyk3 in an alternative cytokinesis pathway to form a primary septum. Since INN1 is also a multicopy suppressor of a myo1 deletion, this further supports the notion that Inn1 may be required for plasma membrane ingression, also in CAR-independent cytokinesis. Preliminary data suggest, that the protein Vrp1 is responsible for the required removal of Inn1 from the bud neck after completion of cytokinesis. The essential amino-terminal C2 domain of Inn1 may mediate plasma membrane ingression by interaction with the membrane lipid phosphatidic acid, observed in biochemical studies. Alternatively, the C2 domain has been suggested to modulate chitin synthesis in the primary septum by modulating Chs2 activity (Nishihama et al., 2009)

The Small Yeast GTPase Rho5 and Its Dimeric GEF Dck1/Lmo1 Respond to Glucose Starvation

Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways

Verplicht vrouwen in de top? Een onderzoek naar het beleidseffect van de Nederlandse inspanningsverplichting van evenwichtige verdeling tussen mannen en vrouwen op de top van grote beursgenoteerde ondernemingen in Nederland

<p>A) Phase contrast images of tip-branching in wild-type (top row) and <i>ΔAgrho2</i> strains. The images show 3 time-points of tip-branching events taken from time-lapse movies. The time-points in minutes are indicated in the top right corner of each image. The growth speed of the hyphae directly prior to tip-branching was determined from the time-lapse movie and is presented on the right side of the figure. Scale bar, 20 µm. B) Actin stained with rhodamine-phalloidin of hyphae from wild-type and <i>ΔAgrho2</i> strains. Scale bar, 5 µm.</p

Localization of mCherry-<i>Ag</i>Rho5.

<p>A) DIC and fluorescence image of a <i>ΔAgrho5</i> strain expressing a fusion of mCherry to <i>Ag</i>Rho5 from a vector. The scale bar represents 5 µm. B) Mycelia of wild-type, <i>ΔAgrho5</i> and the <i>ΔAgrho5</i> strain expressing the mCherry-<i>AgRHO5</i> construct. C) DIC images of single hyphae and tip-branching hyphae of a <i>ΔAgrho5</i> strain carrying expressing wild-type <i>AgRHO5</i> from a plasmid. D) Mycelia of wild-type, <i>ΔAgrho5</i> and the <i>ΔAgrho5</i> strain expressing wild-type <i>AgRHO5</i> from a plasmid. The scale bar is 1 cm.</p

Growth of <i>Agrho2</i> mutant strains.

<p>A) Measurement of the radial growth speed on solid medium at 30° for the wild type, <i>Agrho2</i> deletion and a strain carrying a GTP-locked allele of <i>Agrho2</i>. Shown are the arithmetic mean and standard deviation (n> = 3). The diameter of the initial inoculum was subtracted from each time-point, such that all measurements began at 0 mm. B) Example images of mycelia from the measurements in A) that display the difference in diameter taken at day 6 of the measurements. C) Measurement of the radial growth speed on solid medium at 30° for the wild type and <i>Agrho2</i> overexpression. Shown are the arithmetic mean and standard deviation (n = 2). The diameter of the initial inoculum was subtracted from each time-point, such that all measurements began at 0 mm. D) Example images of mycelia from the measurements in C) that display the difference in diameter taken at day 6 of the measurements.</p

The Small GTP-Binding Proteins <i>Ag</i>Rho2 and <i>Ag</i>Rho5 Regulate Tip-Branching, Maintenance of the Growth Axis and Actin-Ring-Integrity in the Filamentous Fungus <i>Ashbya gossypii</i>

<div><p>GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from <i>Saccharomyces cerevisiae</i> and of their homologs in other species is poorly understood. Here, we report on the analysis of the <i>Ag</i>Rho2 and <i>Ag</i>Rho5 proteins of the filamentous fungus <i>Ashbya gossypii</i>. In contrast to <i>S. cerevisiae</i> mutants of both encoding genes displayed a strong morphological phenotype. The <i>Agrho2</i> mutants showed defects in tip-branching, while <i>Agrho5</i> mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the <i>Agrho5</i> mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the <i>Agrho5</i> mutant, suggesting that both proteins act in the same pathway, but the <i>Ag</i>Rho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.</p></div

Actin instability at the hyphal tip of a <i>Δ</i><i>Agrho5</i> mutant.

<p>DIC and fluorescence images of a hypha of a <i>ΔAgrho5</i> strain carrying an actin-binding domain fused to GFP. The growth of the strain was followed by time-lapse microscopy for 150 minutes. Images were taken every 5 minutes. For each time-point, pixel intensities were measured over the first 2 µm of the hypha. The difference between the pixel intensities of the upper half of pixels versus the lower half of the hypha is plotted for each time-point in the graph below the images. In addition, for each time-point, the corresponding hyphal tip is shown in heat map coloring, with cold colors representing low-pixel intensities and warm colors represent high-pixel intensities. The scale bar represents 10 µm.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Actin ring defects in <i>Agrho5</i> and <i>Agdck1</i> mutant strains.

<p>A) Wild-type hyphae stained with rhodamine-phalloidin. The insert shows the actin-ring in the bottom right corner that was reconstructed from a z-series of images and tilted 35° towards the observer. The scale bar represents 5 µm. B) <i>ΔAgrho5</i> strain transformed with an actin-binding domain fused to GFP. The inserts show 3D-reconstructions of a z-series turned at a given angle of the aberrant actin rings in the top right and bottom left corners of the image. The scale bar represents 5 µm. C) The <i>ΔAgdck1</i> strain stained with rhodamine-phalloidin. The images on the right are 3D-reconstructions taken from z-series of the regions marked on the DIC image on the left side and rotated by the angle given in the figure. The scale bar represents 5 µm. D) DIC and fluorescence images from septa stained with Calcofluor White to visualize chitin. Strains are from left to right: wild-type, <i>ΔAgrho5</i> and <i>ΔAdck1</i>. The scale bar represents 5 µm.</p