From the paper: Dynamic force-induced direct dissociation of protein complexes in a nuclear body in living cells

1. TEMPLATE DESIGN © 2008
www.PosterPresentations.com
Dynamic force-induced direct dissociation of protein complexes in a nuclear
body in living cells
Yeh-Chuin Poh, Sergey P. Shevtsov, Farhan Chowdhury, Douglas C. Wu, Sungsoo Na, Miroslav Dundr & Ning
Wang
Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Champaign, Illinois 61801, USA.
Introduction
Material & Methods
Results Results Results
Summary
Reference
Mechnotransduction has been implied as an
important field relating physical environments with
cellular physiology. It has been shown that
mechanical force regulates a wide variety of cellular
processes such as cell morphology, migration,
differentiation and apoptosis, etc. . Recently, it was
shown that force applied on the cell apical surface
could display mitochondria within the cytoplasmic
matrix of cells, and even the nucleolus within the
nucleus.
• Can force directly alter nuclear function?
• Cajal body is nuclear protein complex consist of
different proteins.
• Cajal body helps biogenesis of RNPs, maturation of
telomerase and maintenance of telomere
HeLa cells were transfected with plasmids of
fluorescent proteins of interest. Fluorescent levels of
CFP- and YFP-proteins were recorded simultaneously
before and after force application on cells. Force
application was done by magnetic twist cytometry.
CFP/YFP emission ratio was measured by a
customized Matlab program. For substrate stiffness
dependent experiments, cells were plated on glass
and different stiffness polyacrylamide gels coated with
type I collagen. For cellular traction experiments,
fluorescent beads were embedded into gels, bead
positions were recorded before and after cells were
removed. The displacement field induced by each
individual cell’s tractional forces was determined by
comparing the fluorescent bead positions before and
after removing cells.
CFP
YFP
FRET
433nm 476nm 433nm
527nm
Figure 1. FRET expreiment.
Cells are co-transfected with
YFP-Coilin and CFP-SMN.
YFP and CFP are FRET pairs.
Movements of SMN and Coilin
were synchronized with the
force application through
magnetic twist cytometry
CFP-SMNYFP-Coilin CFP-SMNYFP-Coilin
Figure 2. Dissociation of Cajal bodies was observed
~0.3s after force application.
-2 -1 0 1 2 3 4 5 12 15 18 21 24
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
NormalizedFRETRatio
Time (s)
Stress on
Stress off
-2 -1 0 1 2 3 4 5 12 15 18 21 24
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
NormalizedFRETRatio
Time (s)
Stress on
Stress off
Dissociation of Cajal bodies in response to force
Dissociation is Magnitude dependent
0.00 0.64 1.28 1.92 2.56 3.20
-600
-400
-200
0
200
400
600 Bead Stress (peak=24.5 Pa)
Coilin SMN
Displacement(nm)
Time (s)
-2 -1 0 1 2 3 4 5
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30 1.8 Pa
14.0 Pa
17.5 Pa
NormalizedFRETRatio
Time (s)
Figure 4. There is a threshold magnitude of force to
trigger the dissociation of Cajal bodies in livinh cells
between 14.0 Pa and 17.5 Pa.
Structural basis of force-induced dissociation
***
-Stress
+Stress
Lat A
Bleb
Colch
Lamin A/C -/-
Plectin -/-
1.00
1.05
1.10
1.15
1.20
NormalizedFRETRatio
*
**
+Stress, t = 0.35s
*
***
-Stress
+Stress
Lat A
Bleb
Colch
Lamin A/C -/-
Plectin -/-
1.00
1.05
1.10
1.15
1.20
NormalizedFRETRatio
*
**
+Stress, t = 0.35s
*
Figure 5. By drug inhibition different force-transmitting
components in cells, it is shown that F-actin, myosin II
and Lamin A/C are necessary for force-induced
dissociation of Cajal bodies.
Substrate stiffness dependent
-2 -1 0 1 2 3 4 5
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30 0.6 kPa Substrate
2.0 kPa Substrate
8.0 kPa Substrate
NormalizedFRETRatio
Time (s)
500 Pa
0
0.6 kPa 2.0 kPa 8.0 kPa
500 Pa
0
500 Pa
0
0.6 kPa 2.0 kPa 8.0 kPa
0.6 2 8
0
50
100
150
200
250
Substrate Stiffness (kPa)
Tractions(Pa)
**
*
0.6 2 8
0
50
100
150
200
250
Substrate Stiffness (kPa)
Tractions(Pa)
**
*
0.6 2 8 Glass
0.0
0.5
1.0
1.5
2.0
Substrate Stiffness (kPa)
CellStiffness(kPa)
*
*
***
0.6 2 8 Glass
0.0
0.5
1.0
1.5
2.0
Substrate Stiffness (kPa)
CellStiffness(kPa)
*
*
***
Figure 6. Force-induced dissociation of Cajal bodies was
substrate stiffness dependent. Only on stiffer substrate,
cells would exhibit dissociation of Cajal bodies in
response to stress.
Figure 7. Actin bunles formation, cellular traction force and
cell stiffness are proportional to substrate stiffness,
implying prestress on cytoskeletons play a role in force
propagation into nucleus
ARTICLENATURE COMMUNICATIONS | DOI: 10.1038/ncomms1873
CFP-SMNYFP-Coilin
1.2
1.4
1.6
0.9
1.0
1.1
1.2
1.4
1.6
CFP-SMN/YFP-Coilin CFP-SMN
–200
0
200
400
600 Bead Stress (peak=24.5 Pa)
Coilin SMN
1.10
1.15
1.20
1.25
1.30 1.8 Pa
14.0 Pa
17.5 Pa
1.05
1.10
1.15
1.20
1.25
1.30
Stress off
1.0
Normalized
CFPintensity
0.8
Normalized
YFPintensity
1.0
Normalized
FRETratio
Time (s)
–600
–400
Displacement(nm)
Time (s)
0.90
0.95
1.00
1.05
NormalizedFRETratio
Time (s)
0.90
0.95
1.00
NormalizedFRETratio
Time (s)
Stress on
0
0.15 µm
SMNCoilin
8 Coilin
× 104
Before stress
0
2.0
+ Stress– Stress
0
2
4
6
SMN
MSD(nm2)
t (s)
0.00 0.64 1.28 1.92 2.56 3.20
–2 0 2 4 6 8
Time (s)
–2 0 2 4 6 8
Time (s)
–2 0 2 4 6 8
YFP-Coilin
–2 –1 0 1 2 3 4 5 –2 –1 0 1 2 3 4 5 12 15 18 21 24
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 71 2 3 4 5 6
t (s) t (s)
During stress After stress
igure 1 | A local surface force directly dissociates coilin from SMN in the CB in the nucleus. (a) Fluorescence images of a HeLa cell transfected with
CFP-SMN and YFP-coilin (inset is the bright-field image of the cell; black dot shows the bead). Nucleus is outlined with dashed line. Scale bar, 10 m.
b) Displacements of the magnetic bead and of the CB proteins SMN and coilin as a function of cyclic forces (0.3Hz). Displacements of the bead, coilin
nd SMN were all synchronized with the applied stress (peak magnitude=24.5Pa). (c) Displacement maps of coilin and SMN within the nucleus of the
ame cell. White arrows indicate direction of displacement, and the colour bar indicates the displacement magnitude. Pink arrow represents the direction
f bead centre displacement (not drawn to scale). Scale bar, 10 m (d) FRET ratio map of force-induced dissociation of coilin and SMN. Inset shows an
nlarged CB with FRET changes when stress is applied. (e) A representative time course plot of CFP-SMN and YFP-coilin anti-correlation in response
Figure 3. Cajal bodies did not reassembly after force
application was turned off. Stress-induced structural
change to CB protein pairs is ‘plastic’
• Mechanical Force is possible to directly altered
nuclear functions
• Mechanotransduction in living cell nucleus depends
on force magnitude, intact F-actin, cytoskeletal
tension (prestress), Lamin A/C, or substrate rigidity
• Justified the tensegrity model of cells
• It is possible to manipulate cell growth, cell
development by force-induced change in gene
expression profile
Other protein pairs exhibit different changes
Figure 8. Other protein pairs in Cajal body has different
sensitivity to force, implying that they have different
interactions within the Cajal body.
Poh, Y.-., Na, S., Chowdhury, F., Ouyang, M., Wang, Y., and Wang, N.
(2009) Rapid activation of Rac GTPase in living cells by force is
independent of Src. PLoS ONE. 4(11)
Wang, N., Tytell, J.D., and Ingber, D.E. (2009) Mechanotransduction
at a distance: Mechanically coupling the extracellular matrix with the
nucleus. Nature Reviews Molecular Cell Biology. 10, 75-82
Wang, N., Butler, J., and Ingber, D. (1993) Mechanotransduction
across the cell surface and through the cytoskeleton. Science. 260,
1124-1127