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