Transcript Presentation Title

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