Cell Mechanosensitivity is Enabled by the LINC...

Gunes Uzer

InspireME Seminar SeriesBoise State University

November 6th 2015

Cell Mechanosensitivity is Enabled by the LINC Nuclear Complex

β

α

ECM

CellMembrane

Focal Adhesion

Cytoskeleton

Lamin Chromatin

NucleusNuclear Envelope

Nesprin

Sun

Cell membrane

Cytoskeleton

Nucleus

LINC complex

LaminSunNesprinActin

LINC Complex(Linker of Nucleoskeleton and Cytoskeleton)

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.

OSTEOBLASTS

ADIPOCYTES

Bone cells are mechanosensitive

MESENCHYMAL STEM CELLS

Osteocyte

Mesenchymal stem cell

Adipocyte

Osteoblast

Osteoclast

Low Intensity Vibration (LIV) – small but effective

Control LIV

How do cells sense these very small vibrations?

LIV 1 LIV 2rest

Luu, Y. K.+, JBMR, 2009Sen, B.,+, J.Biomech, 2011

Fritton, S. P+, J.Biomech, 2000

AdiponectinLIVControl

aP2PPARγTubulin

Am

plitu

de

Wavelength

Mechanical environment during LIV

Cell membrane

CytoskeletonNucleus

Vibr

atio

n

Fluid Shear

Membrane deformation

Accelerations

InertiaStiffer & denser nucleus

ECM strain

Cell deformation

Uzer, G+, Curr Osteoporos Rep, 2013

Can we determine vibration-induced fluid shear?

Horizontal vibrations

Speckle Photography

Particle Image Velocimetry (PIV)

Finite Element Modeling (FEM)

MSC osteogenesis is not regulated by fluid shear

0

0.5

1

1.5

2

2.5

3

Min

eral

izat

ion

(Aliz

arin

Red

, mM

)

**

†¥

*†

Control

LIV

R² = 0.90

0

2

4

6

8

10

12

14

0.00 5.00 10.00 15.00 20.00 25.00 30.00

Mean mRN

A Ac

tivity

Rate of acceleration (km/s3)

Uzer, G +, J. Biomech. , 2013

Mechanical environment during LIV

Cell membrane

CytoskeletonNucleus

Vibr

atio

n

Fluid Shear

Membrane deformation

Accelerations

InertiaStiffer & denser nucleus

ECM strain

Cell deformation

Uzer, G+, Curr Osteoporos Rep, 2013

What is the Primary Deformation Mode of LIV ?

Nucleus

Cytoplasm

Membrane

Fluid Shear Acceleration

Nucleus

Cytoplasm

MembraneCell membrane

Cytoplasm

Nucleus

Contact surface

Component ModulusNucleus 6kPa

Cytoplasm 1.5kPaCell

membrane1kPa

Relative Nuclear Motion

Acceleration magnitude

Fluid shear stress

0.15g 1g 0.14Pa(30Hz-0.15g)

0.94Pa(30Hz-1g)

258nm 1554nm 3.36nm 22.50nmUzer, G +, PLoS one. , 2014

Increased cytoskeletal tension?

Hypothesis: LIV induced acceleration of the nucleus generates forces on the cytoskeleton

Vibr

atio

n

Integrins

Extracellular Matrix

αβ

Src

Zyxin

α β

Src

Zyxin

Plasma Membrane

35μm LIV induces perinuclear actin remodeling

15μmLIV0

20

40

60

Ctrl LMS

¥

LIVPer

inuc

lear

actin

form

atio

n (%

)

02468

Ctrl LIV 1XLIV 2X

***

***

***

pFA

K39

7/T-

FAK

1X 2XCtrlp-FAK

T-FAK

15μmControl

Uzer + Stem Cells, 2015


AccelerationsStiffer & denser nucleus Cell

membraneCytoskeleton

Nucleus

Strain (100 cycles)

0

1

2

3

DMSO Y2732

p-FA

K39

7/T-

FAK

Ctrl LIV*

Y27632

RhoAActin

LIV-induced signaling requires an intact actin cytoskeleton

35μm

0

1

2

3

DMSO Colchicine

p-FA

K39

7/T-

FAK Ctrl LIV

**

Microtubules

0

0.5

1

1.5

2

2.5

DMSO Cytocalasin D

p-FA

K39

7/T-

FAK Ctrl LIV**

*

CytochalasinD

Nuclearleaflets Lamin

Nucleus

Cytoplasm

Nesprin

Sun

Actin

LINC between actin cytoskeleton and nucleus

Linker of Nucleoskeleton and Cytoskeleton Provides mechanical coupling

0

1

2

siCtrl siSUN

Ctrl LIV**

pFA

K39

7/T-

FAK

LIV signals require

a LINCednucleus

LIN

C c

ompl

ex

G. Uzer +Stem Cells, 2015


0

1

2

Ctrl DNKASH

Ctrl LIV¥pF

AK

397/

T-FA

K

siCtrl siSUN

0

2

4

6

8

10

siCtrl siSUN

Nuc

lear

Hei

ght (μm

)

0

100

200

300

400

500

600

siCtrl siSUN

Nuc

lear

Are

a (μ

m2 )

0200400600800

10001200140016001800

siCtrl siSUNN

ucle

ar V

olum

e(μm

3 )

******

Strain does not require LINC

β α

0

1

2

Ctrl DNKASH

pFA

K39

7/T-

FAK Ctrl Strain

* *

Strain

Outside Inside

Inside Inside

ChromatinIntegrin

Focal Adhesion

Lamin

Nesprin Sun

StrainLIV

Cell deformation

Strain

Nuclearleaflets

TCF/LEF

Nuclear Pore Complex

S

GSK-3βAktβ

WT

Progeria

Mechanical βcatenin


OSTEOBLASTS

ADIPOCYTES

β

Actin

Nuclear-cytoskeletal connections important for MSC fate

LIN

C c

ompl

ex

Lamin

Sun

Nesprin

Loss of LINC promotes MSC adipogenesis

0

0.5

1

1.5

2

siCtrl siSUN

AP

-2/β

-tubu

lin

Ctrl LMS***

**

LIV

siSUNLIV

AP-2

β-tubulin

0.00.51.01.52.02.53.03.5

APN AP-2 PPAR-γ

Gen

e E

xpre

ssio

n

Ctrl DNKASH

******

***

0.00.51.01.52.02.53.03.5

SUN-1SUN-2 APN AP-2

Gen

e E

xpre

ssio

n

siCtrl siSUN******

******

Does LINC play a role in MSC βcatenin signaling?

Uzer et al. Stem Cells, 2015

Mechanical signals increase nuclear βcatenin

0

0.5

1

1.5

2

2.5

Ctrl LIV

βcat

enin

/PA

RP

βcatenin

LIV - +

PARP

**

0

0.5

1

1.5

2

Ctrl Strain

βcat

enin

/PA

RP

βcatenin

Strain - +

PARP

**

Nuclear compartment Nuclear compartment

β α Integrin Lamin

Nesprin Sun

StrainLIV

0

1

2

Ctrl DNKASHp-

Akt

473/

T-A

kt *

0

1

2

3

siCtrl siSUN

p-A

kt47

3/T-

Akt

**

Disrupting LINC prevents LIV signaling

GSK-3β β ββ

0

0.5

1

1.5

2

2.5

siCtrl siSUN

βcat

enin

/PA

RP Ctrl LIV*

Mechanical signals that do not depend on LINC connectivity ?

Cell deformation

LDHAct βcat

siSun1/2 - - + +Strain - + - +

Act βcatPARP

0

1

2

siCtrl siSUN

Ctrl Strain

p-G

SK

3β/T

-GS

K3β ** **

LINC mediates nuclear βcatenin entry

Strain

Nuclear

Cytoplasmic

How might LINC mediate βcatenin translocation?

β

β

β

Nucleusβ

Controlβ

β

β

Nucleusβ

siSUN0

1

2

siCtrl siSUN

p-A

kt47

3/T-

Akt

Ctrl Strain

** **

*

Nuclearleaflets

Nuclear Pore Complex

S

Actin

Lamin

Sun

Nesprin

S

Nesprin-2G

Emerin

Βcatenin(Active)

00.20.40.60.8

11.2

Ctrl Strain

βcat

enin

/Nes

prin

***

LIV

Nesprin-2G

βcatenin

LIV - +

βcateninLIV - +

00.5

11.5

22.5

3

Ctrl LIV

Nuc

lear

Env

elop

eβc

aten

in/P

AR

P

PARP

***

βcatenin associates with LINC

Nesprin pulldown Nuclear Envelope

00.20.40.60.8

11.2

Axi

n-2

Gen

e E

xpre

ssio

n

siCtrl siSUN

**

How does LINC enable nuclear βcatenin entry?

LINC preserves nuclear βcatenin

00.20.40.60.8

11.2

siCtrl siSUN

βcat

enin

/PA

RP

**

PARP

SUN

siSUN - +βcatenin

Nuclear compartment

Peri-nuclear Peri-nuclear Nuclear

DAPI Colocalization

Peri-nuclear Peri-nuclear Nuclear

siC

ontro

lsi

SU

N

Nesprin-2 βcatenin

**

DAPI Colocalization

100%

0%

Colocalization

Perinuclear βcatenin accumulation in LINC deficient cells

Lamin

Chromatin

Plasma Membrane

β

α

Focal Adhesion

Extracellular Matrix

CytoskeletonNuclear Envelope Nucleus

Nuclear envelope as a signaling platformregulating cell function and fate

How LINC is regulated?

βα

Integrin

Lamin

NucleusNuclear Envelope

Nesprin

Sun

Ctrl LIVVinculinPaxilinT-Akt

Focal adhesions Cytoskeleton Nucleoskeleton

Actin

LINC

Force

βtubulin

DNKASHLamin A/C

- +

0

0.5

1

1.5

Ctrl DNKASH

**

Lam

inA

C/β

tubu

lin

Mechanical regulation of LINC & nucleoskeleton

Aging ProgeriaMicrogravity ?

Role of LINC in maintenance of MSC βcatenin signaling under microgravity

15 rpm

1-D ClinostatUddin et al. PLoS One, 2013

S

Healthy aging

Progeria (Lamin & LINC mutations)

Cancer

Acknowledgements

Janet Rubin , MD

Maya Styner, MD

Buer Sen, MD

Zhihui Xie, MD

Guniz Bas Uzer

Stefan Judex , PhD

Clinton Rubin , PhD

Yi-Xian Qin, PhD

Aditi Nivedita Senthilnathan

Kaushik Puranam

Sophia Kim

Junaid Qureshi

Speckle photography – macro fluid motion

10

A

B

0

0.5

1

1.5

2

10 20 30 40 50 60

Vertical Surface

Elevation (m

m)

Frequency (Hz)

60Hz, 1g @250fps

10Hz20Hz

Uzer, G +, Cell. Mol. Bioeng. , 2012

Fluid shear near the cell surface

Particle Image Velocimetry(PIV)

11

30µm30µm

OvernightdryingFluorospheres,1µm

37x106/cm2

Fluorospheres,1µm40,000/mlIn αMEM

Slide Surface 75 µm fromSlide Surface

R² = 0.998

0

100

200

300

400

500

600

-50 50 150 250

She

ar R

ate

per g

(sec

-1.g

-1)

Distance from Slide Surface (µm)Uzer, G +, Cell. Mol. Bioeng. , 2012

12

FEM of fluid sloshing Out of phase with well velocity Non‐homogenous Viscosity effects Acceleration and frequency

‐0.15

‐0.10

‐0.05

0.00

0.05

0.10

0.15

‐0.03

‐0.02

‐0.01

0.00

0.01

0.02

0.03

0.000 0.005 0.010 0.015 0.020 0.025 0.030

Fluid Shear (Pa)

Horizon

tal Velocity

(m/s)

Time (s)

Well VelocityFluid VelocityFluid Shear

Velocity (m

m/s)

5mm

14mm

A

0

20

40

60

80

100

120

100 200 300 400 500 600 700

Shear R

ate pe

r g (sec‐1.g‐1)

Distance from Slide Surface (µm)

PIV

FEM

Vibration induced fluid shear– in silico


13

Fluid shear is modulated by vibration frequency, acceleration and fluid viscosity

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50 60 70 80 90 100

Fluid Shear (Pa)

Frequency (Hz)

0.01g 0.1g 0.5g 1g


Novel cell mechano-characterization

Multi-plane Scanning 3D reconstruction Digitized FE mesh

Cell receptors Focal adhesions Plasma membrane Microtubules

Nucleus

Cytoplasm

MembraneCell membrane

Cytoplasm (not shown)

Nucleus

33

0

3500

7000

10500

14000

Maximum Principal Strain (x10-3)

0

3

9

12

15

Num

ber o

f Ele

men

ts (x

103 )

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

Simulation #2Move nucleus X- direction 300nm

Simulation #1Move nucleus Z- direction 300nm

x

z

Static simulations

StrainMax Principal (x10-5)

Dynamic simulations

Control Low Intensity Vibration

Multiscale experimental biomechanicsV

ibra

tion

Dire

ctio

n

32

20X

CCD

ActuatorMicro-etched slide

Cells

Microscope

60Hz- 0.3g

LINC and cell structure

0

2

4

6

8

10

siCtrl siSUN

Nuc

lear

Hei

ght (μm

)

0

100

200

300

400

500

600

siCtrl siSUN

Nuc

lear

Are

a (μ

m2)

0200400600800

10001200140016001800

siCtrl siSUNN

ucle

ar V

olum

e(μm

3)

******

siCtrl siSUN

IMARIS

Does fluid shear modulate LIV response ?

14

24hr.Cells

No Dextran6% Dextran

(Viscosity Increase)

One time LIV, 30 minutes1g acceleration @ 10, 30, 60, 100 Hz

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

LIV

In-vitro response0.5-1Pa

3-fold increase in fluid shear

COX-2 mRNA levels are higher in low fluid shear in MC3T3 osteoblasts

15Uzer, G +, Cell. Mol. Bioeng. , 2012

6% Dextran0% Dextran

Gap junctional intracellular communication (GJIC) in MLO‐Y4 osteocytes is unresponsive to fluid shear

16

Control LIV

Uzer, G +, PLoS one. , 2014

‐10

40

90

140

Control 30Hz‐0.15g 30Hz‐1g 100Hz‐0.15g 100Hz‐1g

****** *** ***

GJIC

+ cells

% of Total of total cell #

Parachute Assay

Engineering mechanically functionalized cartilage

Cartilage

Bone

Functionalized scaffold

Load

ing

dire

ctio

n 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

MC3T3cells

(Calcein stained)Stained/Unstained

~1:500

MLO-Y4Osteocyte

cells

72 hr.

Vibrations 30 min 25oC

Flow Cytometry(n=18, minimum)

Negative Control Positive Control GJIC + cells

10,000/cm2

30Hz-0.15g 100Hz-0.15g30Hz-1g 100Hz-1g

Uzer, G., et al. (2013). PLoS One (in-press).

1 hr.

Manufacturing Composite Scaffolds

PLGA (85:15)

Cell-MeHASolution

Theriform®

Pre-wetting

Photo-initiator2wt% MeHA

Cells

UV

3mm

3mm

1.5 hr. 10 min.

PLGA(85:15)90-95% Porosity

~120μm pore sizeE= 2 MPa

1mm

PLGA(85:15)/TCP55% Porosity

~200μm pore size~400μm channels

E= 54 MPa

Transition Zone

1.5mm

0.5mm

Mixing

Cell-MeHA-PLGA

Scaffold

Upside down

Dynamic Compression Loading (DCL) Loading regime. Displacement control. 1Hz, 4hr a day. 10% Strain.

Sterile technique. Loading inside an incubator. 5% CO2, 37oC

Up to 70 scaffolds.

Load

ing

dire

ctio

n

b-a = 100 μm ±1%

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.

PolyHEMAcoating

ScaffoldsPKH labeled MSCs (1M cell/ml)

MSC attachment

2 hr.

Spinning

Transfer to standardculture

Hoechst 33258Blue Staining

SectioningTime points

3 Days

Cell counting

cells attached of #cells migrated of #

density migration

100x(control)density migration(sample)density migration

capability migration

Migrated cell Resident cell

5 Sectionper sample

1. Tissue Eng. 2007;13(7):1525-37.

Surgical Procedure Approved by IACUC. 5 months old rabbits. Full thickness defects by

drilling. Second knee as control Free cage activity

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

3mmx3mmdefect

Patellar surface

Medial condyleLateral condyle

Cartilage

Bone

Medial parapatellar approach

Image source: ref. 3

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.

S

GSK-3βAktβ

Mechanical control of MSC fate


OSTEOBLASTS

ADIPOCYTES

β

Nuclear-cytoskeletal connections important for MSC fate

TCF/LEFNucleus

S

NPC

Lamin

Nuclearleaflets

Actin

Nesprin

Sun

Plasma Membrane

Integrins

Extracellular Matrixβ α

GSK-3β

STCF/LEF

β

Akt

β

ββ

LIV induced force

RhoA

ADIPOCYTES

OSTEOBLASTS

Actin

Force

Mechanical regulation of LINC & nucleoskeleton

15 rpm

1-D ClinostatUddin et al. PLoS One, 2013

Cell Mechanosensitivity is Enabled by the LINC...

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