Post on 12-Apr-2017
transcript
Kinase effects on kinetochore attachment strength
Jonathan Driver
courtesy of Justin Decarreau
State of the field: attachment error correction
How are incorrect attachments broken? How are incorrect attachments sensed?
Ipl1 kinase Lack of tension
Partial explanation: Question:
Mps1 kinase ???
kinetochore
Outline
1. Tools for a direct test of Mps1 kinase activity
2. Assay design and attachment strength measurement
3. Optimization of conditions
4. Results
5. Next steps
in vitro tools
Kinetochores can be purified from yeast.
Akiyoshi et al., Nature 2010
Active Mps1 kinase copurifies.
London et al., Curr. Biol. 2012
Spc105
Dsn1 Ndc80
Laser trapping
Akiyoshi et al., Nature 2010
Laser trapping
Akiyoshi et al., Nature 2010
Outline
1. Tools for a direct test of Mps1 kinase activity
2. Assay design and attachment strength measurement
3. Optimization of conditions
4. Results
5. Next steps
Assay Design
Goal: Create KT-MT attachments prior to the introduction of ATP. Rationale: (1) Mps1 activity is specifically tested on attached KTs; (2) precursor to tension-sensing experiment.
microtubule
biotin 1.
2.
microtubule
biotin 1.
2.
3.
3.
time (s)
Forc
e (p
N)
time (s)
Forc
e (p
N)
Outline
1. Tools for a direct test of Mps1 kinase activity
2. Assay design and attachment strength measurement
3. Optimization of conditions
4. Results
5. Next steps
10 µL/min
25 µL/min
Rupture force distributions at two different flow rates are similar
Must strike a balance between drag force from flow and total exchange time.
10 µL/min
25 µL/min
Rupture force distributions at two different flow rates are similar
Must strike a balance between drag force from flow and total exchange time.
ATP-triggered weakening/loss of attachments
A lower ATP concentration might allow for more measurements.
Varying nucleotide concentration
2 mM ATP seems to give the right response.
Outline
1. Tools for a direct test of Mps1 kinase activity
2. Assay design and attachment strength measurement
3. Optimization of conditions
4. Results
5. Next steps
Results from three different preps
Outline
1. Tools for a direct test of Mps1 kinase activity
2. Assay design and attachment strength measurement
3. Optimization of conditions
4. Results
5. Next steps
1. Inhibit Mps1 using an analog-sensitive mutant
2. Block phosphorylation at putative Mps1 sites
3. Apply tension prior to and during ATP introduction to test whether tension is sensed by Mps1
Next steps
1. Inhibit Mps1 using an analog-sensitive mutant
2. Block phosphorylation at putative Mps1 sites
Spc105
Dsn1 Ndc80
Acknowledgments
Asbury Lab Biggins Lab Wordeman Lab Chip Asbury Sue Biggins Justin Decarreau Andy Powers Nitobe London Krishna Sarangapani Nicole Duggan Andrew Frank
The Raymond and Beverly Sackler Scholars Program