1. Lattice Structure of the Yeast Centrosome as Explored by FRET in Living Cells Trisha N. Davis Yeast Resource Center Department of Biochemistry University of Washington Seattle, Washington, USA

2. Mitosis & Chromosome Segregation (Harold Fisk, U. Colorado, Boulder) centrosomes microtubules DNA kinetochores

3. The Yeast Centrosome (Spindle Pole Body) is Layered Microtubules Inner Plaque Central Plaque Outer Plaque Intermediate Layer 2 Giddings, McIntosh & Winey

4. Yeast Spindle Pole Body Challenges –1 - 2 per cell –0.5 gigadaltons Advantages –~500 copies of 5 different proteins –Crystalline array

5. Structure of the yeast centrosome (spindle pole body) Improved FRET methodology in living cells Construct a model of the core layers of the SPB by combining FRET data with prior cryo-EM analysis.

6. Y C

7. YCYYYYYY C

8. Structure of the core of the yeast centrosome (spindle pole body) Improved FRET methodology in living cells Construct a model of the core layers of the SPB by combining FRET data with prior cryo-EM analysis.

9. Spillover CFP = FRET Channel/CFP =.446 CFP alone: Spc110p-CFP The Contribution from CFP to the Intensity in the FRET Channel Emissionwavelength: Excitationwavelength: DIC Channel: 500 nm 545 nm YFP 440 nm 545 nm FRET 440 nm 480 nm CFP

10. Spillover YFP = FRET Channel/YFP =.232 YFP alone: Spc110p-YFP The Contribution from YFP to the Intensity in the FRET Channel Emissionwavelength: Excitationwavelength: Channel: 500 nm 545 nm YFP 440 nm 545 nm FRET 440 nm 480 nm CFPDIC

11. Spc110p-YFP-CFP The Positive FRET ControlEmissionwavelength: Excitationwavelength: Channel:DICIntensity: 500 nm 545 nm YFP 5094 440 nm 545 nm FRET 10587 440 nm 480 nm CFP 7194 Spillover = (.446 x 7194) + (.232 x 5094) = 4103

12. Dealing with Spillover UW Youvan et al. Gordon et al. Muller et al.

13. Spillover Positive Control YFP-Spc110-CFP 420041131.02±0.07 Spc110-YFP-CFP 1059043902.42±0.22 Negative Control 700 Å FRET R FRET channel

14. FRET R Signals with Different Pairs of CFP & YFP Tagged SPB Proteins BESY102-2A BESY25 EMY173EMY178EMY179EMY180 BESY38 DHY71 EMY167-1D EMY175 EMY181-1D EMY190 BESY22 BESY91-2CBESY95-6D BESY97-3D BESY98-2D DHY41 DHY47-6B EMY176 EMY192 EMY194-2C BESY31BESY34 DHY212 DHY38 BESY100-3B BESY101-4C BESY109 BESY18 BESY89-1C DHY43DHY87 EMY185 BESY23 BESY40BESY45 BESY86-12D BESY88-8A BESY96-1D BESY99-6C DHY208 DHY209 EMY164-1D EMY195-9A FRET R 0.5 1.0 1.5 2.0 2.5 3.0 DHY150DHY151 Strains There are 4,386 SPB’s from 47 strains represented in the dataset. Normal distributions with standard deviations about 10% of the mean. C:NoneNoneLowestLowModerateHigh C:High

15. Only FRET R is Independent of the Level of Spillover

16. FRET R is Linear at all FRET Strengths Our Method Gordon Method Highest FRET Category CFP x YFP Lowest FRET Category CFP x YFP

17. Structure of the core of the yeast centrosome (spindle pole body) Improved FRET methodology in living cells Construct a model of the core layers of the SPB by combining FRET data with prior cryo-EM analysis.

18. C-TERM Spc42 C-TERM Cnm67 N-TERM Spc42 C-TERM Spc29 C-TERM Cmd1 C-TERM Spc110 C-term Spc42 2.022.551.231.131.061.05 C-term Cnm67 2.061.671.11 1.091.05 N-term Spc42 1.261.151.702.252.022.18 C-term Spc29 1.191.101.861.541.691.32 C-term Cmd1 1.191.102.091.701.602.37 C-term Spc110 1.071.021.861.411.961.78 N-term Spc29 1.000.99lethalND1.75lethal N-term Spc110 1.020.971.041.031.011.02 FRET Donor (CFP tag) FRET Acceptor (YFP tag)

19. C-term Spc42 C-term Cnm67 N-term Spc42 C-term Spc29 C-term Cmd1 C-term Spc110 C-term Spc42 2.022.551.231.131.061.05 C-term Cnm67 2.061.671.11 1.091.05 N-term Spc42 1.261.151.702.252.022.18 C-term Spc29 1.191.101.861.541.691.32 C-term Cmd1 1.131.102.091.701.602.37 C-term Spc110 1.051.021.861.411.961.78 N-term Spc29 0.980.99lethalND1.75lethal N-term Spc110 1.020.971.041.031.011.02 FRET between IL2 layer and Central Plaque Components

21. Assumptions Assumption 1: –Simplify to consider only four distances: a red distance, an orange distance, a green distance and a blue distance. Assumption 2: –Red distance < Orange distance < Green distance < Blue distance

28. 58 Å

29. Spc42 forms a 2-D crystal Bullitt, Rout, Kilmartin & Akey

30. Model for the C-terminus of Spc42

31. Assumptions Assumption 1: –Simplify to consider only four distances: a red distance, an orange distance, a green distance and a blue distance. Assumption 2: –Red distance < Orange distance < Green distance < Blue distance Assumption 3: –Spc42 is arranged in an hexagonal array in IL2

33. Central plaque of the SPB CaM Proj of Cnm67 N-Spc42 & Projection C-Spc42 C-Spc29 Spc42 coils

34. C-TERM Spc42 C-TERM Cnm67 N-TERM Spc42 C-TERM Spc29 C-TERM Cmd1 C-TERM Spc110 C-term Spc42 2.022.551.231.131.061.05 C-term Cnm67 2.061.671.11 1.091.05 N-term Spc42 1.261.151.702.252.022.18 C-term Spc29 1.191.101.861.541.691.32 C-term Cmd1 1.191.102.091.701.602.37 C-term Spc110 1.071.021.861.411.961.78 N-term Spc29 1.000.99lethalND1.75lethal N-term Spc110 1.020.971.041.031.011.02 FRET Donor (CFP tag) FRET Acceptor (YFP tag)

35. N-Spc42 & Projection C-Spc42 Location of C-Spc110 Spc42 coils CaM C-Spc29 Proj of Cnm67 C-Spc110

36. CaM N-Spc42 & Projection C-Spc42 C-Spc29 Proj of Cnm67 C-Spc110 N-Spc29 Spc42 coils Location of N-Spc29

37. The Central Plaque as viewed by FRET CaM N-Spc42 C-Spc29 C-Spc110 N-Spc29 Spc42 coils

38. Model of the Central Plaque Spc29 Spc42 Spc110 CaM

40. Conclusions Combining the the relative distance constraints provided by FRET analysis of living cells with the cryo EM allowed construction of a detailed model of the lattice structure of the core of the SPB.

41. Acknowledgements Yeast Resource Center, UW Eric Muller Brian Snydsman Bryan Sundin Dale Hailey Dept. of Mathematics, UW Isabella Novik Funded by NCRR and NIGMS at the NIH