Stable spatial gradients of cytoskeleton assembly regulators
description
Transcript of Stable spatial gradients of cytoskeleton assembly regulators
![Page 1: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/1.jpg)
Stable spatial gradients of cytoskeleton assembly regulators
David Odde
University of Minnesota
![Page 2: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/2.jpg)
Microtubule Structure
![Page 3: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/3.jpg)
Leng
th (
µm
)
Time (minutes)
“Catastrophe”
“Rescue”
Microtubule “Dynamic Instability” (DI)
Vg
Vs
kc
kr
see VanBuren et al., PNAS USA (2002)
![Page 4: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/4.jpg)
Microtubules in Mitosis
![Page 5: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/5.jpg)
Mitotic Spindle
spindle pole body
chromosome
kinetochore
kinetochoremicrotubule
spindle pole body
1.5 µmIn yeast:
~40 MTs10-20 µm
In animal cells:
~1000 MTs
Interpolarmicrotubule
![Page 6: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/6.jpg)
Hypothesis
Dynamic instability alone is sufficient to explain the observed MT length distribution in the yeast mitotic spindle
![Page 7: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/7.jpg)
Results: Cse4p-GFP Distribution
Experimentally Observed
Theoretically Predicted
?
2 µm
![Page 8: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/8.jpg)
Leng
th (
µm
)
Time (minutes)
“Catastrophe”
“Rescue”
Microtubule “Dynamic Instability” (DI)
Vg
Vs
kc
kr
![Page 9: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/9.jpg)
Point Spread Function (PSF)
• A point source of light is spread via diffraction through a circular aperture
• Modeling needs to account for PSF
-0.4-0.20+0.2+0.4 μm
![Page 10: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/10.jpg)
Simulated Image Obtainedby Convolution of PSF and GWN
with Original Distribution
Original FluorophoreDistribution
Model-Convolution
![Page 11: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/11.jpg)
Spindle Geometry
![Page 12: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/12.jpg)
Results: Distribution of Cse4-GFP fluorescence
Experimentally Observed
Theoretically Predicted
![Page 13: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/13.jpg)
Results: Distribution of Cse4-GFP fluorescence
x=0 x=L
QS QSSE
![Page 14: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/14.jpg)
Results: DI Only Model
1000 nm
![Page 15: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/15.jpg)
Results: DI Only Model
![Page 16: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/16.jpg)
Alternative Models
![Page 17: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/17.jpg)
Microtubule Chemotaxis
ImmobileKinase
MobilePhosphatase
Microtubule
A: Phosphorylated Protein Stabilizes MTsB: Unphosphorylated Protein Destabilizes MTs
Concentration
Position
MT Attractant
MT Repellant
X=0 X=L
k*Surface reaction B-->A
kHomogeneous reaction A-->B
![Page 18: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/18.jpg)
Microtubule Chemotaxis:Op18
ImmobilePlx1
MobilePP2A
Microtubule
A: Op18-hi-PB: Op18-low-P Destabilizes MTs
Concentration
Position
Op18-hi-P
Op18-low-P
Chromatin
![Page 19: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/19.jpg)
Microtubule Chemotaxis: RanGTP
ImmobileRCC1
MobileRanGAP
Microtubule
A: RanGTP Stabilizes MTsB: RanGDP
Concentration
Position
RanGTP
RanGDP
Chromatin
![Page 20: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/20.jpg)
Model for Chemotactic Gradients of Phosphoprotein State
cAt
D 2cAx2
kcA Fick’s Second Law with First-Order HomogeneousReaction (A->B)
DcAx x0
k *cB 0 B.C. 1: Surface reaction at x=0 (B->A)
DcAx xL
0 B.C. 2: No net flux at x=L
cA cB cT Conservation of phosphoprotein
![Page 21: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/21.jpg)
Predicted Concentration Profile
where
Y cA cT
X x L
kL2
D
A*e2
e2 1 * 1 e2 B*
e2 1 * 1 e2 * k
*LD
Y Ae X BeX
If k= 1 s-1, D=10-11 m2/s, and L=10 µm, then =3
![Page 22: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/22.jpg)
Model Predictions: Effect of Homogeneous Reaction Rate
1.0
0.8
0.6
0.4
0.2
0.0
Con
cen
trati
on
, Y
1.00.80.60.40.20.0
Position, X
![Page 23: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/23.jpg)
Model Predictions: Effect of Surface Reaction Rate
1.0
0.8
0.6
0.4
0.2
0.0
Con
cen
trati
on
, Y
1.00.80.60.40.20.0
Position, X
![Page 24: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/24.jpg)
Microtubule Chemotaxis: RanGTP
ImmobileRCC1
MobileRanGAP
Microtubule
A: RanGTP Stabilizes MTsB: RanGDP
Concentration
Position
RanGTP
RanGDP
Chromatin
![Page 25: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/25.jpg)
![Page 26: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/26.jpg)
![Page 27: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/27.jpg)
Results: Chemical Gradient and Polar Ejection Force Models
1000 nm
![Page 28: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/28.jpg)
Cse4 Bleach @ end of simulation, mutant “Tension” model
LeftHalfSpindle
RightHalfSpindle
Figure 2
![Page 29: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/29.jpg)
Cse4 Bleach @ End of Simulation, wild-type, “Gradient-Only” Model
RightHalfSpindle
LeftHalfSpindle
Figure 4
![Page 30: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/30.jpg)
Mitotic Spindle
Conclusion: Spatial gradients in MT DI parameter(s)may play a role in mediating budding yeast mitotis
F FF F
![Page 31: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/31.jpg)
X
X
X
Y
Z
Y
Simulated Actin FilamentDendritic Branching
Simulated Image of Actin FilamentDendritic Branching
Model-Convolution: Application to Dendritic Actin Filament Branching
![Page 32: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/32.jpg)
Simulated Image Obtainedby Model-Convolution of
Original Distribution
Original FluorophoreDistribution
Image Obtained by Deconvolution
of Simulated Image
Potential Pitfalls of Deconvolution
![Page 33: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/33.jpg)
Acknowledgements
• Whitaker Foundation
• National Science Foundation
![Page 34: Stable spatial gradients of cytoskeleton assembly regulators](https://reader036.fdocuments.in/reader036/viewer/2022070411/5681480b550346895db53c1c/html5/thumbnails/34.jpg)
Comparing Models to Microscopy
Molecular Theory Molecular Reality
Microscopic Observations
Model Predictions ???
Fluorescence Microscope
Computer Simulation