1. Kinase effects on kinetochore
attachment strength
Jonathan Driver

2. courtesy of Justin Decarreau

4. 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

5. 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

6. 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

7. Laser trapping
Akiyoshi et al., Nature 2010

8. Laser trapping
Akiyoshi et al., Nature 2010

9. 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. 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.

11. microtubule
biotin
1.
2.

12. microtubule
biotin
1.
2.

13. 3.

14. 3.

15. time (s)
Force(pN)

16. time (s)
Force(pN)

17. 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

18. 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.

19. 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.

20. ATP-triggered weakening/loss of attachments
A lower ATP concentration might allow for more measurements.

21. Varying nucleotide concentration
2 mM ATP seems to give the
right response.

22. 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

23. Results from three different preps

24. 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

25. 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

26. 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