Cell Mechanosensitivity is Enabled by the
LINC Nuclear Complex
InspireME Seminar Series
Boise State University
November 6th 2015
Gunes Uzer
LINC Complex
(Linker of Nucleoskeleton and Cytoskeleton)
Cell
Membrane
β
Sun
Focal
Adhesion
CellNucleus
membrane
Cytoskeleton
Nesprin
α
ECM
Cytoskeleton
Nuclear
Envelope
Nucleus
Lamin
Actin
Nesprin
Sun
LINC complex
Lamin
Chromatin
Mechanical signals regulate bone mass

Bone will adapt to the loads under
which it is placed1

Playing arm of tennis players have
higher bone mass2

During microgravity, astronauts can
lose up to 2% hip density per month3
1. Wolff, J. SpringerVerlag,1986 Berlin. 2.Huddleston, A. L. et al. JAMA(1980) 244(10): p.1107., 2.Lang, T.et al. JBMR,2004 19(6): p.1006.
Bone cells are mechanosensitive
MESENCHYMAL
STEM CELLS
Osteocyte
OSTEOBLASTS
Osteoblast
ADIPOCYTES
Mesenchymal stem cell
Osteoclast
Adipocyte
Amplitude
Low Intensity Vibration (LIV) – small but effective
Wavelength
Fritton, S. P+, J.Biomech, 2000
LIV 1
rest
Control
Control
LIV
Luu, Y. K.+, JBMR, 2009
LIV 2
LIV
Adiponectin
aP2
PPARγ
Tubulin
Sen, B.,+, J.Biomech, 2011
How do cells sense these very small vibrations?
Vibration
Mechanical environment during LIV
Cell membrane
Cytoskeleton
Nucleus
ECM strain
Cell deformation
Uzer, G+, Curr Osteoporos Rep, 2013
Fluid Shear
Membrane deformation
Accelerations
Inertia
Stiffer & denser nucleus
Can we determine vibration-induced fluid shear?
Particle Image Velocimetry (PIV)
Speckle Photography
Horizontal vibrations
Finite Element Modeling (FEM)
MSC osteogenesis is not regulated by fluid shear
14
Mineralization (Alizarin Red, mM)
Mean mRNA Activity
12
10
8
Control
6
4
2
LIV
0
0.00
5.00
Uzer, G +, J. Biomech. , 2013
†
3
2
¥
R² = 0.90
†
2.5
*
*
*
1.5
1
0.5
0
10.00
15.00
20.00
Rate of acceleration (km/s3)
25.00
30.00
Vibration
Mechanical environment during LIV
Cell membrane
Cytoskeleton
Nucleus
ECM strain
Cell deformation
Uzer, G+, Curr Osteoporos Rep, 2013
Fluid Shear
Membrane deformation
Accelerations
Inertia
Stiffer & denser nucleus
What is the Primary Deformation Mode of LIV ?
Component
Modulus
Nucleus
6kPa
Membrane
Cell membrane
Cytoplasm
Membrane
Cytoplasm
1.5kPa
Cell
membrane
1kPa
Nucleus Nucleus
Nucleus
Cytoplasm
Contact surface
Cytoplasm
Fluid Shear
Acceleration
Acceleration
magnitude
Relative Nuclear Motion
Uzer, G +, PLoS one. , 2014
0.15g
258nm
1g
1554nm
Fluid shear stress
0.14Pa
0.94Pa
(30Hz-0.15g)
(30Hz-1g)
3.36nm
22.50nm
Vibration
Hypothesis: LIV induced acceleration of the
nucleus generates forces on the cytoskeleton
Zyxin
Zyxin
Src
Increased cytoskeletal
tension?
Src
Plasma Membrane
β α
Integrins
α β
Extracellular Matrix
35μm
LIV induces perinuclear actin remodeling
Accelerations
Stiffer & denser nucleus
Nucleus
Increased cytoskeletal
tension?
pFAK397/T-FAK
Cell
p-FAK
membrane
T-FAK
Cytoskeleton
8
6
4
2
0
Ctrl
2X
1X
***
***
***
Control
Uzer + Stem Cells, 2015
15μm
LIV
15μm
Perinuclear actin
formation (%)
Ctrl LIV 1XLIV 2X
60
40
¥
20
0
Strain (100 cycles)
Ctrl
LMS
LIV
LIV-induced signaling requires an intact actin
cytoskeleton
**
Ctrl
2
1.5
1
*
0.5
0
3
LIV
p-FAK397/T-FAK
p-FAK397/T-FAK
2.5
CytochalasinD
DMSO Cytocalasin
D
Actin
*
Ctrl
LIV
2
1
0
p-FAK397/T-FAK
35μm
3
2
Ctrl
*
LIV
*
1
0
DMSO
Y27632
Y2732
RhoA
DMSO
Colchicine
Microtubules
LINC between actin cytoskeleton and nucleus
LINC complex
Actin
Nesprin
Sun
Nuclear
leaflets
Nucleus
Increased cytoskeletal tension?
Lamin
siCtrl
0
*
*
1
0
siCtrl
G. Uzer +Stem Cells, 2015
Ctrl
500
LIV
400
300
200
100
0
siSUN
siCtrl
siSUN
pFAK397/T-FAK
2
2
***
Nuclear Area (μm2)
4
pFAK397/T-FAK
Nuclear Height (μm)
6
siSUN
600
10
8

Linker of Nucleoskeleton and Cytoskeleton
Provides mechanical coupling
siCtrl
2 ***
¥
1
0 siSUN
Ctrl
1800
1600
1400 LIV
Ctrl
1200
1000
800
600
400
200
0
siCtrl
Nuclear Volume(μm3)
Cytoplasm

DNKASH
LIV
signals
require
a
LINCed
nucleus
siSUN
Strain does not require LINC
Strain
pFAK397/T-FAK
Cell deformation
2
Ctrl
*
Strain
*
1
0
Ctrl
DNKASH
Inside Inside
Nesprin Sun
Focal
Adhesion
Integrin
β α
Strain
Outside Inside
Lamin
Chromatin
LIV
Strain
Mechanical βcatenin
Akt
GSK-3β
β
β
Actin
MESENCHYMAL
STEM CELLS
Nesprin
S
Nuclear
leaflets
ADIPOCYTES
Sun
WT
Lamin
Nuclear Pore
Complex
Progeria
TCF/LEF
Nuclear-cytoskeletal connections important for MSC fate
LINC complex
OSTEOBLASTS
Loss of LINC promotes MSC adipogenesis
Gene Expression
siCtrl
siSUN
LIV
AP-2
β-tubulin
Ctrl
LIV
LMS
***
**
0.5
0
siCtrl
Uzer et al. Stem Cells, 2015
***
Ctrl
1.5
1
***
***
***
SUN-1 SUN-2 APN AP-2
siSUN
Gene Expression
AP-2/β-tubulin
2
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
siSUN
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
DNKASH
***
***
APN
***
AP-2
PPAR-γ
Does LINC play a role in MSC βcatenin signaling?
Mechanical signals increase nuclear βcatenin
Nuclear compartment
LIV
-
+
Nuclear compartment
Strain
βcatenin
βcatenin
PARP
PARP
2
2.5
2
**
βcatenin/PARP
βcatenin/PARP
-
1.5
1
0.5
+
**
1.5
1
0.5
0
0
Ctrl
LIV
Ctrl
Strain
Disrupting LINC prevents LIV signaling
GSK-3β
β
β
3
Nesprin Sun
β α
Lamin
Integrin
LIV
Strain
p-Akt473/T-Akt
β
2
1
0
siCtrl
*
Ctrl
LIV
p-Akt473/T-Akt
βcatenin/PARP
2.5
2
**
1.5
1
0.5
0
siCtrl
siSUN
siSUN
2
*
1
0
Ctrl
DNKASH
Mechanical signals that do not depend on LINC connectivity ?
LINC mediates nuclear βcatenin entry
Ctrl
Strain
p-Akt473/T-Akt
Strain
*
2
**
**
1
0
β
siSUN
Control
siCtrl
β
siSUN
p-GSK3β/T-GSK3β
Cell deformation
2
Ctrl
**
Strain
**
1
0
siSun1/2
Strain
Act βcat
PARP
siCtrl
-
+
siSUN
+
-
Nuclear
Nucleus
β
β
Act βcat
LDH
Cytoplasmic
How might LINC mediate βcatenin translocation?
+
+
βcatenin associates with LINC
Nesprin pulldown
Nesprin-2G
LIV
Emerin
-
+
Nesprin-2G
Βcatenin
(Active)
Nuclear Envelope
LIV
βcatenin
βcatenin
Nesprin
S
Sun
Lamin
Nuclear Pore
Complex
1
0.8
0.6
***
0.4
0.2
0
Ctrl
Strain
LIV
Nuclear Envelope
βcatenin/PARP
Nuclear
leaflets
βcatenin/Nesprin
S
+
PARP
1.2
Actin
-
3
2.5
2
1.5
1
0.5
0
***
Ctrl
LIV
LINC preserves nuclear βcatenin
siSUN
βcatenin
PARP
SUN
-
+
βcatenin/PARP
1.2
1
0.8
**
0.6
0.4
0.2
0
siCtrl
siSUN
Axin-2 Gene Expression
Nuclear compartment
1.2
1
**
0.8
0.6
0.4
0.2
0
siCtrl
How does LINC enable nuclear βcatenin entry?
siSUN
Perinuclear βcatenin accumulation in LINC deficient cells
siSUN
**
Nesprin-2
Peri-nuclear
Colocalization
siControl
100%
Nuclear
βcatenin
Peri-nuclear
DAPI
Colocalization
Peri-nuclear
Nuclear
DAPI
Colocalization
0%
Peri-nuclear
Nuclear envelope as a signaling platform
regulating cell function and fate
Lamin
β
α
Extracellular Matrix
Focal Adhesion
Plasma Membrane
Chromatin
Cytoskeleton
Nuclear Envelope
How LINC is regulated?
Nucleus
Mechanical regulation of LINC & nucleoskeleton
β
α
Actin
Nesprin
Force
Focal adhesions
Ctrl LIV
Vinculin
Paxilin
T-Akt
Aging
Nucleus
Cytoskeleton
Lamin
Sun
Nucleoskeleton
LINC
Progeria
DNKASH
Lamin A/C
βtubulin
LaminAC/βtubulin
Nuclear
Envelope
Integrin
-
+
1.5
1
**
0.5
0
Ctrl DNKASH
Microgravity ?
Role of LINC in maintenance of MSC βcatenin signaling under microgravity
15 rpm
1-D Clinostat
Uddin et al. PLoS One, 2013
S

Healthy aging

Progeria (Lamin & LINC
mutations)

Cancer
Acknowledgements
Janet Rubin , MD
Maya Styner, MD
Stefan Judex , PhD
Aditi Nivedita Senthilnathan
Buer Sen, MD
Clinton Rubin , PhD
Kaushik Puranam
Zhihui Xie, MD
Yi-Xian Qin, PhD
Sophia Kim
Guniz Bas Uzer
Junaid Qureshi
Speckle photography – macro fluid motion
60Hz, 1g @250fps
20Hz
10Hz
Vertical Surface Elevation (mm)
2
1.5
B
1
0.5
0A
10
Uzer, G +, Cell. Mol. Bioeng. , 2012
20
30
40
50
Frequency (Hz)
60
10
Fluid shear near the cell surface
30µm
30µm
Fluorospheres,1µm
37x106/cm2
Overnight
drying
Shear Rate per g (sec-1.g-1)
Slide Surface
Fluorospheres,1µm
40,000/ml
In αMEM
Particle Image Velocimetry
(PIV)
75 µm from
Slide Surface
600
500
400
R² = 0.998
300
200
100
0
-50
50
150
250
Distance from Slide Surface (µm)
Uzer, G +, Cell. Mol. Bioeng. , 2012
11
FEM of fluid sloshing
 Out of phase with well velocity
 Non‐homogenous  Viscosity effects
 Acceleration and frequency
Velocity (mm/s)

5mm
Vibration induced fluid shear– in silico
A
14mm
Well Velocity
Fluid Velocity
120
Fluid Shear
0.02
0.10
0.01
0.00
0.000
‐0.01
0.05
0.005
0.010
0.015
0.020
0.025
0.00
0.030
‐0.05
‐0.02
‐0.10
‐0.03
‐0.15
Shear Rate per g (sec‐1.g‐1)
0.15
Fluid Shear (Pa)
Horizontal Velocity (m/s)
0.03
PIV
100
FEM
80
60
40
20
0
100
200
300
400
500
Distance from Slide Surface (µm)
600
700
Time (s)
Uzer, G +, Cell. Mol. Bioeng. , 2012
12
Fluid shear is modulated by vibration frequency, acceleration and fluid viscosity
3
Fluid Shear (Pa)
2.5
0.01g
2
0.1g
0.5g
1g
1.5
1
0.5
0
0
10
20
30
40
50
60
70
80
90
100
Frequency (Hz)
Uzer, G +, Cell. Mol. Bioeng. , 2012
13
Novel cell mechano-characterization
Cell receptors
 Focal adhesions
 Plasma membrane
 Microtubules

Control
3D reconstruction
Simulation #2
Move nucleus
X- direction 300nm
z
x
14000
15
10500
12
7000
9
Simulation #1
Move nucleus
Z- direction 300nm
Static simulations
Digitized FE mesh
Cell membrane
Cytoplasm (not shown)
Nucleus
3500
3
Nucleus
Strain
Max Principal (x10-5)
Cytoplasm
0
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3.0
3.3
3.6
3.9
4.2
4.5
4.8
Membrane
Number of Elements (x103)
Multi-plane Scanning
Low Intensity Vibration
Maximum Principal Strain (x10
Dynamic
simulations
-3)
33
Multiscale experimental biomechanics
CCD
20X
Actuator
Cells
Micro-etched slide
Vibration Direction
Microscope
60Hz- 0.3g
32
LINC and cell structure
IMARIS
siCtrl
600
***
8
6
4
2
0
Nuclear Area (μm2)
Nuclear Height (μm)
10
500
***
400
300
200
100
0
siCtrl
siSUN
siCtrl
siSUN
Nuclear Volume(μm3)
siSUN
1800
1600
1400
1200
1000
800
600
400
200
0
siCtrl
siSUN
Does fluid shear modulate LIV response ?
No Dextran
6% Dextran
(Viscosity Increase)
Cells
24hr.
LIV
One time LIV, 30 minutes
1g acceleration @ 10, 30, 60, 100 Hz
In-vitro response
0.5-1Pa
Fluid Shear (Pa)
Low Shear
(0% dextran)
High Shear
(6% dextran)
100Hz
0.28 Pa
0.8 Pa
60Hz
0.47 Pa
1.3 Pa
30Hz
0.94 Pa
2.6 Pa
3-fold increase
in fluid shear
14
COX-2 mRNA levels are higher in low fluid shear
in MC3T3 osteoblasts
0% Dextran
Uzer, G +, Cell. Mol. Bioeng. , 2012
6% Dextran
15
Gap junctional intracellular communication (GJIC) in MLO‐Y4 osteocytes is unresponsive to fluid shear
Parachute Assay
Control
GJIC+ cells
% of Total of total cell #
140
***
***
LIV
***
***
90
40
‐10
Uzer, G +, PLoS one. , 2014
Control
30Hz‐0.15g
30Hz‐1g
100Hz‐0.15g 100Hz‐1g
16
Engineering mechanically functionalized cartilage
Cartilage
Bone
Loading direction
Functionalized scaffold
b-a = 100 μm ±1%
Petri Dish
Dynamic loading
Multiscale testing
34
Engineering mechanically functionalized cartilage
MSC
Gap junctional intracellular communication (GJIC)
in MLO-Y4 osteocytes
10,000/cm2
72 hr.
MLO-Y4
Osteocyte
cells
Vibrations 30 min 25oC
30Hz-0.15g
100Hz-0.15g
30Hz-1g
1 hr.
MC3T3
cells
(Calcein stained)
Stained/Unstained
~1:500
Flow Cytometry
(n=18, minimum)
Negative Control
Uzer, G., et al. (2013). PLoS One (in-press).
100Hz-1g
Positive Control
GJIC + cells
Manufacturing Composite Scaffolds
1mm
1.5mm
Upside down
Theriform®
UV
PLGA
(85:15)
Transition Zone
PLGA(85:15)/TCP
55% Porosity
~200μm pore size
~400μm channels
E= 54 MPa
Mixin
g
Photo-initiator
2wt% MeHA
Cells
Pre-wetting
1.5 hr.
3mm
0.5mm
PLGA(85:15)
90-95% Porosity
~120μm pore size
E= 2 MPa
10 min.
3mm
Cell-MeHA
Solution
Cell-MeHAPLGA
Scaffold
Dynamic Compression Loading (DCL)
Loading regime.





Sterile technique.
Loading inside an incubator.


Displacement control.
1Hz, 4hr a day.
10% Strain.
b-a = 100 μm ±1%
Loading direction

5% CO2, 37oC
Up to 70 scaffolds.
Petri Dish
1. Stem Cells. 2004;22(3):313-23.
Migration Assay

Investigation of the effects of initial cell density and subsequent ECM
accumulation on the migration ability of MSCs.
Scaffolds
PKH labeled MSCs (1M cell/ml)
PolyHEMA
coating
Transfer to
standard
culture
MSC
attachment
2 hr.
Cell
counting
Spinning
5 Section
per sample
Time points
3 Days
migration density 
Sectioning
Hoechst 33258
Blue Staining
Migrated cell
1. Tissue Eng. 2007;13(7):1525-37.
migration capability 
Resident cell
# of migrated cells
# of attached cells
migration density (sample)
x100
migration density (control)
Surgical Procedure
 Approved by IACUC.
 5 months old rabbits.
 Full thickness defects by
drilling.
 Second knee as control
 Free cage activity
Patellar surface
Medial condyle
Lateral condyle
Image source: ref. 3
Cartilage
3mmx3mm
defect
Bone
Medial parapatellar approach
1. J Bone Joint Surg-Am Vol. 1994;76A(4):579-92., 2. Tissue Eng. 1998;4(4):429-44.,
3. http://www.aofoundation.org/srg/33/04-Approaches/A20-med_parapat_appr.jsp
Bone remodeling
-OB-OC balance is lost = OP
1. Kapur, S.et al., Bone, 2003. 32(3): p. 241, 2.Bancroft, G.N.et al., PNAS, 2002 99(20): p. 12600, 3.Rubin, J., JBMR, 2002 17(8): p. 1452, 4.Mullender, M. G.et al. JOR, 2006 24(6): p.1170,
5.Zhou, Y.et al., Eur Cell Mater, 2011 22: p.12., 5.Bonewald, L. F., JBMR,2011 26(2): p. 229., 7.Luu, Y. K.et al., JBMR,2009 24(1): p.50.
Mechanical control of MSC fate
Akt
GSK-3β
β
β
OSTEOBLASTS
MESENCHYMAL
STEM CELLS
Nucleus
ADIPOCYTES
TCF/LEF
S
Nuclear-cytoskeletal connections important for MSC fate
Plasma
Membrane
Extracellular Matrix
β α
Integrins
β
β
GSK-3β
Akt
β
β
RhoA
Actin
LIV
induced
force
OSTEOBLASTS
Nuclear
leaflets
Nesprin
S
Sun
Lamin
NPC
S
TCF/LEF
ADIPOCYTES
Mechanical regulation of LINC & nucleoskeleton
Actin
15 rpm
Force
1-D Clinostat
Uddin et al. PLoS One, 2013