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Maintaining Specificity in the Yeast Filamentous Growth Pathway Jessica Jerrit George Sprague Lab Institute of Molecular Biology MAP Kinase Pathways P MAPKKK P •MAP Kinase pathways are present in many organisms including humans, mice and yeast. MAPKK P MAPK Downstream components •They regulate many essential cellular functions. MAP kinase pathways in yeast Mating/Pheromone Response Ste2 3&4 Filamentous Growth (FG) Msb2 Sho1 High Osmolarity Glycerol (HOG) Sho1 Ste20 Ste20 Ste20 MAPKKK Ste11 Ste11 Ste11 MAPKK Ste7 Ste7 Pbs2 MAPK Fus3 Kss1 Hog1 Fus1 Tec1 Stl1 Figure “borrowed” from Claire How do cells regulate crosstalk when pathway components are shared? Mating/Pheromone Response Ste2 3&4 Filamentous Growth (FG) Msb2 Sho1 High Osmolarity Glycerol (HOG) Sho1 Ste20 Ste20 Ste20 Ste11 Ste11 Ste11 Ste7 Ste7 Pbs2 Fus3 Kss1 Hog1 Fus1 Tec1 Stl1 How do cells regulate crosstalk when pathway components are shared? Mating/Pheromone Response Ste2 3&4 Filamentous Growth (FG) Msb2 Sho1 High Osmolarity Glycerol (HOG) Sho1 Ste20 Ste20 Ste20 Ste11 Ste11 Ste11 Ste7 Ste7 Pbs2 Fus3 Kss1 Hog1 Fus1 Tec1 Stl1 How do cells regulate crosstalk when pathway components are shared? Mating/Pheromone Response Ste2 3&4 Filamentous Growth (FG) Msb2 Sho1 High Osmolarity Glycerol (HOG) Sho1 Ste20 Ste20 Ste20 Ste11 Ste11 Ste11 Ste7 Ste7 Pbs2 Fus3 Kss1 Hog1 Fus1 Tec1 Stl1 HOG and mating pathways use scaffolding proteins to maintain specificity Mating Pathway Ste2 3&4 HOG pathway Sho1 Ste20 Ste20 Ste11 Ste11 Ste5 Ste7 Pbs2 Fus3 Hog1 Fus1 Stl1 The mating pathway also negatively regulates the FG pathway FG Pathway Mating Pathway Ste2 3&4 Sho1 Msb2 Ste20 Ste20 Ste11 Ste11 Ste7 Ste7 Fus3 Fus1 Degradation Kss1 Tec1 Possible methods of regulation of FG pathway • Another scaffolding protein • Organelle localization • Cross pathway inhibition Filamentous Growth Conditions Msb2 Sho1 Sho1 Ste20 Ste20 Ste11 Ste11 Specificity Factor Ste7 Pbs2 Kss1 Hog1 Tec1 Filamentous Growth Conditions Msb2 Sho1 Ste20 Sho1 Mutagenesis Ste11 Ste20 Ste11 Specificity Factor Ste7 Pbs2 Kss1 Hog1 Tec1 Filamentous Growth Conditions Msb2 Sho1 Sho1 Ste20 Ste20 Ste11 Ste11 Ste7 Pbs2 Kss1 Hog1 Tec1 Stl1 Identification of Crosstalk Mutants HOG pathway activation Transcription STL1 promoter HIS3 Chromosome Transformation Reporter strains: Can only grow on -HIS media when Stl1 gene is activated Identification of Crosstalk Mutants Results after growing mutagenized cells up on -His media: 337 a mating type mutants 264 alpha mating type mutants So how do we deal with such a large number of mutants? False Positive Test • We wanted to eliminate mutants activating the HOG pathway in ways other than through crosstalk with the FG pathway. Nonselective media Non-FG conditions, -His Mutant Classification • Complementation • Degree of HOG pathway activation • Morphology – Cell growth patterns – Invasive Growth Complementation Assays Haploid Cells Gene A Gene B Gene A Gene B MATING A B Diploid Cell Complementation! Complementation Assays Haploid Cells Gene A Gene B Gene A Gene B MATING A B Diploid Cell Failure to Complement Complementation Assays Diploid Cells Unknown Specificity Factor Genes -His media Failure to complement Mutations are in the same gene -His media Complementation Mutations are in different genes Complementation Assay Overview Haploid Mutant Mating Diploid Diploid Selection Haploid Mutant His Phenotype Test Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100 Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100 Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100 Complementation Groups 1 large A mutants 7-6 7-8 8-8 8-3 8-4 Alpha mutants 4-7 5-14 12-2 12-3 15-2 1 small A mutants 2-1 Alpha mutants 10-8 29-1 Quantification of HOG pathway activation STL1 lacZ • Used beta galactosidase assays of STL1 transformants • We want to group mutants based on how strongly the HOG pathway is activated Filamentous Growth Conditions Msb2 Sho1 Sho1 Ste20 Ste20 Ste11 Ste11 Ste7 Pbs2 Kss1 Hog1 Tec1 Stl1 Sample results of HOG pathway assay STL1 B gal Activity 0.8 0.7 Activity Units 0.6 0.5 YEPD 0.4 YEPD 1M NaC l 0.3 0.2 0.1 0 Alpha strain 30min a strain 30min Alpha strain 45min a strain 45min B-gal troubleshooting • So far, two wild type strains have run with – Time course of incubation times – New transformations of reporter plasmids – Different substrates (CPRG and ONPG) Morphology Assays • Took 5 pictures of each mutant from 12 and 24 hour cultures in FG media and non-FG media. Axial budding, round cells (WT in non-FG conditions) Polar bud, elongated cell (WT in FG conditions) Polar bud, round cells Morphology Assignments • Assigned morphologies to each clump • Looked at 50-150 clumps per mutant • Grouped mutants based on % clump assignments Morphology Results for 38 Mutants Morphology >70% Axial Round (AR) >70% Polar Round (PR) >70% Polar Elongated (PE) Intermediate AR/PR Intermediate PR/PE Intermediate AR/PE Intermediate AR/PR/PE # mutants 6 2 15 9 1 0 5 Invasive Growth: Another piece of the morphology puzzle •WT yeast can invade media when growing filamentously Cross-section of invasive growth •Plate washing can reveal scars left by this growth Pre-wash Post-wash Invasive Growth: Another piece of the morphology puzzle •WT yeast can invade media when growing filamentously Cross-section of invasive growth •Plate washing can reveal scars left by this growth Pre-wash Post-wash Complementation Groups and Invasive Growth 1 large A mutants 7-6 7-8 8-8 8-3 8-4 Alpha mutants 4-7 5-14 12-2 12-3 15-2 Mostly show weak or no invasive growth 1 small A mutants 2-1 Alpha mutants 10-8 29-1 Small sample, but seem to show stronger invasive growth Future Work • Finish morphology and B Gal assays for all mutants. • Finish complementation assays to sort all mutants into complementation groups • Mate representatives of each complementation group with yeast deletion collection to determine gene(s) involved in filamentous growth specificity • Determine mechanism by which these genes promote specificity Thank yous! George Sprague Claire Romelfanger The Sprague Lab UO SPUR program
