Nature - USA (2020-02-13)

292 | Nature | Vol 578 | 13 February 2020

Article

was a global cell-wide phenomenon. Fourth, we examined the phos-
phorylation of vinculin at Y822, a site known to be phosphorylated
when force is applied on E-cadherin^16 , and observed a significant
increase in its activation after force application on PLXND1 (Fig. 2d).
These mechanoresponses were specific to PLXND1, as ECs incubated
with beads coated with another transmembrane receptor (CD44)
or poly-l-lysine did not respond to force. These data demonstrate
that PLXND1 is a direct force sensor that can elicit robust and global
mechanical signalling in ECs.

PLXND1Control

025 02 5 025 025

PLXND1CD44 PLXND1CD44

025 025 025025

p-VEGFR2

VEGFR2

p-Akt

Akt

p-ERK1/2

ERK1/2

Bright eld forceNo Force

PL

XND

1

Contro

l

a

Force–+–+

b

No

force Force

cd

PLXND1 CD44

PLXND1CD44 PLXND1CD44 PLXND1CD44

p-Vinculin

Vinculin

PLXND1CD44

forceNoForce

****

Forctimee

(min)^025025

Forctimee

(min)

0

1

2

3

4

Forctimee

(min)

*

*

##

0

2

4

6

*

*

##

Force

(min)time

0

2

4

6

025 025

0

0. 5


1. 0


2. 5


3. 0

0.8

1.0

1.2

1.4

1.6

Time(s)

0102030405060

PLXND1

Control

***

0

2

4

6 Force time(min)

Force time(min)

0250 25

PLXND1 CD44

0

1

2

3

4

*

*

##

*

*

##

Fold change Fold change Fold change

Fold change

Normalized FA

Mean amplitude (AU)

Ca

2+(

F/F

) 0

025025

Fig. 2 | PLXND1 is a mechanosensor that mediates the EC response to force.
a, Mouse ECs were incubated with anti-PLXND1 or C44 (negative control)
antibody-coated beads and subjected to force (10 pN) for the indicated time
periods. Phosphorylation of VEGFR2, Akt and ERK1/2 was determined by
western blotting and quantified using Image Studio Lite v.5.2. n = 3 biological
repeats. Phosporylated proteins are indicated by ‘p-’. *P < 0.05 relative to the no
force condition, #P < 0.05 relative to the respective force application time point
with PLXND1. b, Bovine aortic ECs were loaded with Fluo-8AM dye and then
incubated with beads coated with an antibody against the extracellular domain
of PLXND1 or poly-l-lysine (negative control). The beads were then subjected to
force (1 nN). Calcium responses were measured by calculating the f luorescent
intensity of individual cells before (10 s), during (20 s) and after (30 s)
stimulation. Representative images are shown along with quantification.
n = 18 cells for PLXND1 and n = 19 cells for control across 3 independent
biological replicates. *P < 0.001 relative to unstimulated controls. Scale bar,
10 μm. A representative trace of the calcium inf lux response over time is also
shown. The arrow marks the start of the stimulation. AU, arbitrary units.
c, Bovine aortic ECs were incubated with anti-PLXND1-coated beads and
subjected to force (10 pN) for 30 min. ECs were fixed and stained with an anti-
vinculin antibody to mark focal adhesions. Focal adhesion (FA) numbers were
quantified using ImageJ software. Values were normalized to the no force
condition. Locations of the beads are highlighted in yellow circles. n = 50 cells
for each condition from 3 independent biological replicates. **P < 0.0001.
Scale bar, 10 μm. d, Mouse ECs were incubated with anti-PLXND1 or C44
antibody-coated beads and subjected to 10 pN force for the indicated time
periods. Phosphorylation of vinculin was determined by western blotting and
quantified using Image Studio Lite v.5.2. n = 3 biological repeats. *P < 0.05
relative to the no force condition, #P < 0.05 relative to the respective force
application time point with PLXND1. Data are mean ± s.e.m. P values were
obtained using two-tailed Student’s t-tests using GraphPad Prism.

Force^0 –+ –+

1

2

3

0

1

2

3

Force –+ –+

Shear

time (min)0 2 5

ac

d

VEGFR2

VE-cadherin

PECAM-1

αβ

b

h

p-VEGFR2

Shear

time (min) 0 2 5

p-VEGFR2

PI3K/p85

VE-cadherin

VEGFR2

ScramblePlxnd1

Shc

αvβ 3

ScramblePlxnd1

Junctional complex

Integrins

IP: VEGFR2

Fold change Fold change Fold change

IP:

αβv

3

Shear stress Shear stress

COS-7 PLXND1, VEGFR2, NRP1COS-7 +

Shear stress –+–+–+

DMSO SU1498

Force –+ –+

p-Vinculin

Vinculin

*

DMSOSU1498

ScrambleNRP1

Non-

blocking

NRP1

blocking

* *

ScrambleNRP1

Force –+ –+ Force –+ –+

Non-

blocking

NRP1

blocking

efIP: NRP1

i

g

VEGFR2

PLXND1

Shear

time (min)0 2 5 0 2 5

Scramble NRP1

P

VEGFR2

NRP1

PLXND1

P

Src Src

IP: VEGFR2

IP: VEGFR2

VEGFR2

NRP1

PLXND1

VEGFR2

p-VEGFR2

NRP1

PLXND1

Src Src

IP: VEGFR2

g

PI3K

Shc

0

1

2

3

0

1

2

3

0

1

2

3

Force –+ –+

StaticShearStaticShear StaticShearStaticShear

UntransfectedNRP1 +VEGFR2PLXND1(WT) +NRP1 +VEGFR2

siRNA

siRNA

siRNA

siRNA

siRNA

Fig. 3 | The PLXND1, NRP1 and VEGFR2 mechanocomplex functions

upstream of known mechanosensory hotspots and is suff icient for the

response to shear stress. a, Schematic showing signalling at the junctional

complex and integrins. b, Mouse ECs were transfected with scrambled or

Plxnd1 siRNA, exposed to shear stress for 2 min before immunoprecipitating

VEGFR2 and analysis of the phosphorylation and association of VEGFR2 with

the p85 subunit of PI3K and VE-cadherin. n = 3. IP, immunoprecipitation.

c, Mouse ECs were transfected with scrambled or PLXND1 siRNA, exposed to

shear stress for 30 min before immunoprecipitating integrin αVβ 3 and analysis

of the association of integrin αVβ 3 with Shc. n = 3. d, Mouse ECs were treated

with the VEGFR2 kinase inhibitor SU1498, transfected with siRNA against Nrp1

or treated with a NRP1-blocking antibody, incubated with anti-PLXND1-coated

beads and subjected to force for 5 min before analysis of the phosphorylation

of vinculin. (n = 3; *P < 0.05) Data are mean ± s.e.m. P values were obtained using

two-tailed Student’s t-test using GraphPad Prism. e, Mouse ECs were exposed

to shear stress before immunoprecipitating VEGFR2 and analysis of the

phosphorylation and association of VEGFR2 with PLXND1, NRP1 and Src. n = 3.

f, Mouse ECs were exposed to shear stress before immunoprecipitating NRP1

and analysis of the association of NRP1 with PLXND1 and VEGFR2. n = 3.

g, Mouse ECs transfected with either scrambled or Nrp1 siRNA were exposed to

shear stress before immunoprecipitating VEGFR2 and analysis of the

association of VEGFR2 with PLXND1. n = 3. h, Schematic showing that

reconstitution of PLXND1, VEGFR2 and NRP1 in COS-7 cells confers shear-stress

sensitivity to these cells. i, COS-7 cells were left untransfected or transfected

with NRP1 and VEGFR2, with or without PLXND1 before being subjected to

shear stress for 2 min and VEGFR2 was immunoprecipitated. Shear-stress

sensitivity was assessed by analysing the levels of phosphorylated VEGFR2, the

complex formation between VEGFR2 and Src and the complex formation of

PLXND1, VEGFR2 and NRP1. n = 3. All shear-stress experiments were at

1 2 dy nes cm−2 using a parallel plate system.