Lattice Structure of the Yeast Centrosome as Explored by FRET in Living Cells Trisha N. Davis Yeast...

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

Mitosis & Chromosome Segregation

(Harold Fisk, U. Colorado, Boulder)

centrosomesmicrotubulesDNAkinetochores

The Yeast Centrosome (Spindle Pole Body) is Layered

Microtubules

Inner Plaque

Central PlaqueOuter Plaque

Intermediate Layer 2

Giddings, McIntosh & Winey

Yeast Spindle Pole Body

• Challenges– 1 - 2 per cell

– 0.5 gigadaltons

• Advantages– ~500 copies of 5 different proteins

– Crystalline array

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.

€

FRETR =FRETChannel

Spillover

C-terminusOuter PlaqueIntermed. Layer 1Intermed. Layer 2Central PlaqueInner Plaque

CytoplasmicMicrotubules

NuclearMicrotubules

γ -T ubulin Complex

γ -T ubulin Complex

Core Components SPB OrganizationS 72pc p1Nud p67Cnm pS 42 , 67pc p Cnm pS 42 , pc p S29pc pS 1pc10 , 1p Cmd pS 1pc10p

S 97 , pc p S98 , pc pT 4ub p

S 97 , pc p S98 , pc pT 4ub p-N terminus-C terminus-N terminus-C terminus

1Spc10p29Spc p42Spc p67Cnm p1Cmd p

-N terminus

500 Å

YC

C-terminusOuter PlaqueIntermed. Layer 1Intermed. Layer 2Central PlaqueInner Plaque

CytoplasmicMicrotubules

NuclearMicrotubules

γ -T ubulin Complex

γ -T ubulin Complex

Core Components SPB OrganizationS 72pc p1Nud p67Cnm pS 42 , 67pc p Cnm pS 42 , pc p S29pc pS 1pc10 , 1p Cmd pS 1pc10p

S 97 , pc p S98 , pc pT 4ub p

S 97 , pc p S98 , pc pT 4ub p-N terminus-C terminus-N terminus-C terminus

1Spc10p29Spc p42Spc p67Cnm p1Cmd p

-N terminus

500 ÅY

CYY

Y

YYYC

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.

€

FRETR =FRETChannel

Spillover

SpilloverSpilloverCFP CFP = FRET Channel/CFP = .446= FRET Channel/CFP = .446

CFP alone: Spc110p-CFP CFP alone: Spc110p-CFP

The Contribution from CFP to the Intensity in the FRET Channel

EmissionEmissionwavelength:wavelength:

ExcitationExcitationwavelength:wavelength:

DICDIC

Channel:Channel:

500 nm500 nm

545 nm545 nm

YFPYFP

440 nm440 nm

545 nm545 nm

FRETFRET

440 nm440 nm

480 nm480 nm

CFPCFP

SpilloverSpilloverYFP YFP = FRET Channel/YFP = .232= FRET Channel/YFP = .232

YFP alone: Spc110p-YFP YFP alone: Spc110p-YFP

The Contribution from YFP to the Intensity in the FRET Channel

EmissionEmissionwavelength:wavelength:

ExcitationExcitationwavelength:wavelength:

Channel:Channel:

500 nm500 nm

545 nm545 nm

YFPYFP

440 nm440 nm

545 nm545 nm

FRETFRET

440 nm440 nm

480 nm480 nm

CFPCFP

DICDIC

Spc110p-YFP-CFP Spc110p-YFP-CFP

The Positive FRET Control

EmissionEmissionwavelength:wavelength:

ExcitationExcitationwavelength:wavelength:

Channel:Channel:

DICDIC

Intensity:Intensity:

500 nm500 nm

545 nm545 nm

YFPYFP

50945094

440 nm440 nm

545 nm545 nm

FRETFRET

1058710587

440 nm440 nm

480 nm480 nm

CFPCFP

71947194

Spillover = (.446 x 7194) + (.232 x 5094) = 4103Spillover = (.446 x 7194) + (.232 x 5094) = 4103

Dealing with Spillover

SpilloverFRETFRET channelS −=

channelchannel

channelN YFPCFP

SpilloverFRETFRET

×−

=

Spillover

FRETFRET Channel

R =UW

Youvan et al.

Gordon et al.

Muller et al.

Spillover

Positive Control

YFP-Spc110-CFP4200 4113 1.02±0.07

Spc110-YFP-CFP

10590 4390 2.42±0.22

Negative Control700 Å

FRETRFRET channel

FRETR Signals with Different Pairs of CFP & YFP Tagged SPB Proteins

BE

SY

10

2-2

AB

ES

Y2

5E

MY

17

3E

MY

17

8E

MY

17

9E

MY

18

0B

ES

Y3

8D

HY

71

EM

Y1

67

-1D

EM

Y1

75

EM

Y1

81

-1D

EM

Y1

90

BE

SY

22

BE

SY

91

-2C

BE

SY

95

-6D

BE

SY

97

-3D

BE

SY

98

-2D

DH

Y4

1D

HY

47

-6B

EM

Y1

76

EM

Y1

92

EM

Y1

94

-2C

BE

SY

31

BE

SY

34

DH

Y2

12

DH

Y3

8B

ES

Y1

00

-3B

BE

SY

10

1-4

CB

ES

Y1

09

BE

SY

18

BE

SY

89

-1C

DH

Y4

3D

HY

87

EM

Y1

85

BE

SY

23

BE

SY

40

BE

SY

45

BE

SY

86

-12

DB

ES

Y8

8-8

AB

ES

Y9

6-1

DB

ES

Y9

9-6

C

DH

Y2

08

DH

Y2

09

EM

Y1

64

-1D

EM

Y1

95

-9A

FR

ET

R

0.5

1.0

1.5

2.0

2.5

3.0

DH

Y1

50

DH

Y1

51

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:None None Lowest Low Moderate HighC:High

Only FRETR is Independent of the Level of Spillover

FRETR is Linear at all FRET Strengths

Spillover

FRETFRET Channel

R =ChannelChannel

SN YFPCFP

FRETFRET

×=

Our Method Gordon MethodHighest FRET Category

CFP x YFP

Lowest FRET Category

CFP x YFP

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.€

FRETR =FRETChannel

Spillover

C-TERM Spc42

C-TERM Cnm67

N-TERM Spc42

C-TERM Spc29

C-TERM Cmd1

C-TERM Spc110

C-termSpc42 2.02 2.55 1.23 1.13 1.06 1.05

C-termCnm67 2.06 1.67 1.11 1.11 1.09 1.05

N-termSpc42 1.26 1.15 1.70 2.25 2.02 2.18

C-termSpc29 1.19 1.10 1.86 1.54 1.69 1.32

C-termCmd1 1.19 1.10 2.09 1.70 1.60 2.37

C-termSpc110 1.07 1.02 1.86 1.41 1.96 1.78

N-termSpc29 1.00 0.99 lethal ND 1.75 lethal

N-termSpc110 1.02 0.97 1.04 1.03 1.01 1.02

FRET Donor (CFP tag)F

RE

T A

ccep

tor

(YF

P ta

g)

C-term Spc42

C-term Cnm67

N-term Spc42

C-term Spc29

C-term Cmd1

C-term Spc110

C-termSpc42 2.02 2.55 1.23 1.13 1.06 1.05

C-termCnm67 2.06 1.67 1.11 1.11 1.09 1.05

N-termSpc42 1.26 1.15 1.70 2.25 2.02 2.18

C-termSpc29 1.19 1.10 1.86 1.54 1.69 1.32

C-termCmd1 1.13 1.10 2.09 1.70 1.60 2.37

C-termSpc110 1.05 1.02 1.86 1.41 1.96 1.78

N-termSpc29 0.98 0.99 lethal ND 1.75 lethal

N-termSpc110 1.02 0.97 1.04 1.03 1.01 1.02

FRET between IL2 layer and Central Plaque Components

C-terminusOuter PlaqueIntermed. Layer 1Intermed. Layer 2Central PlaqueInner Plaque

CytoplasmicMicrotubules

NuclearMicrotubules

γ -T ubulin Complex

γ -T ubulin Complex

Core Components SPB OrganizationS 72pc p1Nud p67Cnm pS 42 , 67pc p Cnm pS 42 , pc p S29pc pS 1pc10 , 1p Cmd pS 1pc10p

S 97 , pc p S98 , pc pT 4ub p

S 97 , pc p S98 , pc pT 4ub p-N terminus-C terminus-N terminus-C terminus

1Spc10p29Spc p42Spc p67Cnm p1Cmd p

-N terminus

500 Å

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

Spc42 forms a 2-D crystal

Bullitt, Rout, Kilmartin & Akey

Model for the C-terminus of Spc42

36

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

Central plaque of the SPB

CaM

Proj of Cnm67

N-Spc42 & Projection C-Spc42

C-Spc29

Spc42 coils

C-TERM Spc42

C-TERM Cnm67

N-TERM Spc42

C-TERM Spc29

C-TERM Cmd1

C-TERM Spc110

C-termSpc42 2.02 2.55 1.23 1.13 1.06 1.05

C-termCnm67 2.06 1.67 1.11 1.11 1.09 1.05

N-termSpc42 1.26 1.15 1.70 2.25 2.02 2.18

C-termSpc29 1.19 1.10 1.86 1.54 1.69 1.32

C-termCmd1 1.19 1.10 2.09 1.70 1.60 2.37

C-termSpc110 1.07 1.02 1.86 1.41 1.96 1.78

N-termSpc29 1.00 0.99 lethal ND 1.75 lethal

N-termSpc110 1.02 0.97 1.04 1.03 1.01 1.02

FRET Donor (CFP tag)F

RE

T A

ccep

tor

(YF

P ta

g)

N-Spc42 & Projection C-Spc42

Location of C-Spc110

Spc42 coils

CaM

C-Spc29

Proj of Cnm67

C-Spc110

CaM

N-Spc42 & Projection C-Spc42

C-Spc29

Proj of Cnm67

C-Spc110

N-Spc29

Spc42 coils

Location of N-Spc29

The Central Plaque as viewed by FRET

CaM

N-Spc42

C-Spc29

C-Spc110

N-Spc29

Spc42 coils

Model of the Central Plaque

Spc29

Spc42

Spc110

CaM

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.

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