1. Maintaining Specificity in the Yeast Filamentous Growth Pathway Jessica Jerrit George Sprague Lab Institute of Molecular Biology

2. MAP Kinase Pathways MAPKKK MAPKK MAPK P P P Downstream components MAP Kinase pathways are present in many organisms including humans, mice and yeast. They regulate many essential cellular functions.

3. Ste20 Ste11 Ste7 Hog1Fus3Kss1 Sho1 Msb2 Ste2 3&4 Pbs2 Filamentous Growth (FG) High Osmolarity Glycerol (HOG) Mating/Pheromone Response MAP kinase pathways in yeast MAPKKK MAPKK MAPK Fus1 Tec1Stl1 Figure “borrowed” from Claire

4. Ste20 Ste11 Ste7 Hog1Fus3Kss1 Sho1 Msb2 Ste2 3&4 Pbs2 Filamentous Growth (FG) High Osmolarity Glycerol (HOG) Mating/Pheromone Response How do cells regulate crosstalk when pathway components are shared? Fus1 Tec1Stl1

5. Ste20 Ste11 Ste7 Hog1Fus3Kss1 Sho1 Msb2 Ste2 3&4 Pbs2 Filamentous Growth (FG) High Osmolarity Glycerol (HOG) Mating/Pheromone Response How do cells regulate crosstalk when pathway components are shared? Fus1 Tec1Stl1

6. Ste20 Ste11 Ste7 Hog1Fus3Kss1 Sho1 Msb2 Ste2 3&4 Pbs2 Filamentous Growth (FG) High Osmolarity Glycerol (HOG) Mating/Pheromone Response How do cells regulate crosstalk when pathway components are shared? Fus1 Tec1Stl1

7. HOG and mating pathways use scaffolding proteins to maintain specificity Ste20 Ste11 Ste7 Fus3 Ste2 3&4 Fus1 Mating Pathway Ste20 Ste11 Hog1 Sho1 Stl1 HOG pathway Ste5 Pbs2

8. The mating pathway also negatively regulates the FG pathway Ste20 Ste11 Ste7 Fus3Kss1 Sho1 Msb2 Ste2 3&4 Fus1 Tec1 Mating Pathway FG Pathway  Degradation

9. Possible methods of regulation of FG pathway Another scaffolding protein Organelle localization Cross pathway inhibition

10. Ste20 Ste11 Ste7Pbs2 Hog1Kss1 Sho1 Msb2 Tec1 Filamentous Growth Conditions Specificity Factor

11. Ste20 Ste11 Ste7Pbs2 Hog1Kss1 Sho1 Msb2 Tec1 Filamentous Growth Conditions Mutagenesis Specificity Factor

12. Ste20 Ste11 Ste7Pbs2 Hog1Kss1 Sho1 Msb2 Tec1 Filamentous Growth Conditions Stl1

13. Identification of Crosstalk Mutants HOG pathway activation HIS3 Transformation STL1 promoter Reporter strains: Can only grow on -HIS media when Stl1 gene is activated Chromosome Transcription

14. 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?

15. False Positive Test We wanted to eliminate mutants activating the HOG pathway in ways other than through crosstalk with the FG pathway. Nonselective mediaNon-FG conditions, -His

16. Mutant Classification Complementation Degree of HOG pathway activation Morphology –Cell growth patterns –Invasive Growth

17. Complementation Assays Haploid Cells Gene AGene BGene AGene B MATING A B Complementation! Diploid Cell

18. Complementation Assays Haploid Cells Gene AGene BGene AGene B MATING A B Failure to Complement Diploid Cell

19. Complementation Assays Diploid Cells Unknown Specificity Factor Genes -His media  Mutations are in the same gene -His media Mutations are in different genes Failure to complement Complementation

20. Haploid Mutant Mating Diploid Diploid Selection His Phenotype Test Complementation Assay Overview

21. Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100

22. Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100

23. Complementation Results Wild type controls (background growth) 1:1 1:10 1:100 1:1 1:10 1:100

24. 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

25. Quantification of HOG pathway activation Used beta galactosidase assays of STL1 transformants We want to group mutants based on how strongly the HOG pathway is activated STL1 lacZ

26. Ste20 Ste11 Ste7Pbs2 Hog1Kss1 Sho1 Msb2 Tec1 Filamentous Growth Conditions Stl1

27. Sample results of HOG pathway assay

28. 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)

29. 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, round cells Polar bud, elongated cell (WT in FG conditions)

30. Morphology Assignments Assigned morphologies to each clump Looked at 50-150 clumps per mutant Grouped mutants based on % clump assignments

31. Morphology# mutants >70% Axial Round (AR)6 >70% Polar Round (PR)2 >70% Polar Elongated (PE)15 Intermediate AR/PR9 Intermediate PR/PE1 Intermediate AR/PE0 Intermediate AR/PR/PE5 Morphology Results for 38 Mutants

32. Invasive Growth: Another piece of the morphology puzzle WT yeast can invade media when growing filamentously Plate washing can reveal scars left by this growth Pre-washPost-wash Cross-section of invasive growth

33. Invasive Growth: Another piece of the morphology puzzle WT yeast can invade media when growing filamentously Plate washing can reveal scars left by this growth Pre-washPost-wash Cross-section of invasive growth

34. 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 1 small A mutants 2-1 Alpha mutants 10-8 29-1 Mostly show weak or no invasive growth Small sample, but seem to show stronger invasive growth

35. 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

36. Thank yous! George Sprague Claire Romelfanger The Sprague Lab UO SPUR program