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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

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

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

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

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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.

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Y C

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YCYYYYYY C

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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.

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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

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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

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

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

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

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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 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

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Only FRET R is Independent of the Level of Spillover

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

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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.

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C-TERM Spc42 C-TERM Cnm67 N-TERM Spc42 C-TERM Spc29 C-TERM Cmd1 C-TERM Spc110 C-term Spc C-term Cnm N-term Spc C-term Spc C-term Cmd C-term Spc N-term Spc lethalND1.75lethal N-term Spc FRET Donor (CFP tag) FRET Acceptor (YFP tag)

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C-term Spc42 C-term Cnm67 N-term Spc42 C-term Spc29 C-term Cmd1 C-term Spc110 C-term Spc C-term Cnm N-term Spc C-term Spc C-term Cmd C-term Spc N-term Spc lethalND1.75lethal N-term Spc FRET between IL2 layer and Central Plaque Components

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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

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58 Å

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

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Model for the C-terminus of Spc42

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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

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

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C-TERM Spc42 C-TERM Cnm67 N-TERM Spc42 C-TERM Spc29 C-TERM Cmd1 C-TERM Spc110 C-term Spc C-term Cnm N-term Spc C-term Spc C-term Cmd C-term Spc N-term Spc lethalND1.75lethal N-term Spc FRET Donor (CFP tag) FRET Acceptor (YFP tag)

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

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

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

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Model of the Central Plaque Spc29 Spc42 Spc110 CaM

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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.

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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

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