lattice structure of the yeast centrosome as explored by fret in living cells trisha n. davis yeast...
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![Page 1: Lattice Structure of the Yeast Centrosome as Explored by FRET in Living Cells Trisha N. Davis Yeast Resource Center Department of Biochemistry University](https://reader036.vdocument.in/reader036/viewer/2022062407/56649cdc5503460f949a6b44/html5/thumbnails/1.jpg)
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)
centrosomesmicrotubulesDNAkinetochores
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The Yeast Centrosome (Spindle Pole Body) is Layered
Microtubules
Inner Plaque
Central PlaqueOuter 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.
€
FRETR =FRETChannel
Spillover
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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
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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
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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.
€
FRETR =FRETChannel
Spillover
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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
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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
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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
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Dealing with Spillover
SpilloverFRETFRET channelS −=
channelchannel
channelN YFPCFP
SpilloverFRETFRET
×−
=
Spillover
FRETFRET Channel
R =UW
Youvan et al.
Gordon et al.
Muller et al.
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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
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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
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Only FRETR is Independent of the Level of Spillover
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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
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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.€
FRETR =FRETChannel
Spillover
![Page 18: Lattice Structure of the Yeast Centrosome as Explored by FRET in Living Cells Trisha N. Davis Yeast Resource Center Department of Biochemistry University](https://reader036.vdocument.in/reader036/viewer/2022062407/56649cdc5503460f949a6b44/html5/thumbnails/18.jpg)
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)
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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
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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
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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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Spc42 forms a 2-D crystal
Bullitt, Rout, Kilmartin & Akey
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Model for the C-terminus of Spc42
36
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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-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)
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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