Background and our question

RA cont.

a) Subpopulations of certain syntaxins including syantaxin4 translocate across the cell membrane in response to external stimuli. b) The extracellularly presented syntaxins regulate morphology and differentiation of several epithelial cell types.

c)

b)

a) syntaxins (stxs)

Anaplastic cells

ex) epithelial cells

Extracellularly presented syntaxin4 affects anaplastic cell behaviors?

Model: F9 cells

d) Mouse teratocarcinoma e) The method for the unidirectional differentiation to the endodermal linage has been well established with using all-trans retinoic acid (RA).

F9

Signal peptide-stx4: For forced expression of cell surface stx4

Differentiation induction perturbed stx4’s polar localization.

① Morphology in RA treated F9 cells and the surface expression of syntaxin4

Extracellularly stx4 led to flattened and spreading morphology (≒RA).

c) The differentiation is accompanied by the cell shape change.

RA

DAPI ZO-1 F-actin

e)

differentiation

＜OUR QUESTION＞

② Effect of stx4 on morphology

F9 recombinantstx4

Addition to medium

stx4

permeabilized

b-actin stx4 2nd Ab only

+ RA cont. cont. cont. + RA

20μｍ

non-permeabilized

endogenous

stx3 - 34 kDa

stx2(epm)

b-actin -

cont. +RA

stx4 -

b-actin -

- 34 kDa

cont.

(r-GFP)

empty vector

sig-stx4

r-stx4 150μm T7

b-actin

-34kDa

parent sig-stx4

well-packed colonies→ flattened morphology (substrate –adhesion↑）

Colonies → Flattened morphology

morphology

Extracellular subpopulation of stx4 localized at the lateral membrane.

Amount of stx4 was not affected by differentiation induction.

change cell shape

150μm

F9 cells

stx4

cornification

n=6

Extracellularly suppulied Stx4 enhance cell-substrate adhesion

n=7

cont. r-stx4 cont. sig-stx4 RA 0

5

10

15

**

**

**

sig-stx4 empty

*** *** empty sig-stx4

empty sig-stx4 empty sig-stx4

spherical spread

0

10

20

30

0

0.1

0.2

0.3

0.4

cont EPn0 ST4n1 r-stx4

ST4n1

A I E P Q K

G C C

AIEPQK

(Functional core )

③ Effect of stx4 on cell-substrate adhesion

stx4

*

*

Ad

he

ren

t ce

lls (

rela

tive

) A

dh

ere

nt ce

lls (

rela

tive

)

Long term adhesion (starved)

Adhesion / Spreading onto petri dish

Functional core peptide

Ad

he

ren

t ce

lls (

rela

tive

)

Functional core peptide

n=6

* : P<0.05 ** : P<0.01 *** : P<0.001

Conclusion

F9 cell cell-substrate↑ cell-cell↓

adhesion

Vinculin

motility

migration↑

stx3

cell shape anaplastic cell

Stx4 impacts morphology and cell-cell / cell-substrate adhesion to trigger the differentiation.

endoderm differentiation

markers gata4 afp stx3 etc.↑

mesoderm ectoderm

P19 cell

stx2 Check

supplementary results

Extracellular syntaxin4 impacts on cell adhesion and differentiation of embryonal carcinoma F9 cells.

○Natsumi Hagiwara , Yohei Hirai Dep. Bioscience, Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.

細胞外シンタキシンによるEC細胞の接着と分化への影響

④ Effect of stx4 on cell-cell adhesion

empty sig-stx4

0

5

10

15

20

25

30

35

① ② ① ②

empty sig-stx4

①＞0.3㎜ ②≦0.3㎜

IP ECCD2 IgG

input

empty sig-stx4 empty

- E-cadherin

- a-catenin - b-catenin

-

- E-cadherin

- a-catenin - b-catenin

Nu

mb

er

of ce

ll clu

ste

rs

**

***

empty sig-stx4 empty

Stx4 cell-cell adhesion↓ ＜Cadherin-Catenin complex was not affected＞

Suspension culture

empty sig-stx4

E-cadherin-Catenin complex

E-cadherin

Localization of E-cadherin

20μm

n=3

** : P<0.01 *** : P<0.001

Wound healing assay

0

0.1

0.2

0.3

0.4

0.5

N=4 ** P<0.01 *** P<0.001

Cel

l mig

rati

on

(r

elat

ive)

**

Day1 Day0

Empty Sig-stx4

Stx4 migration ↑ ＜down-regulation of Vinculin＞

0

0.5

1

1.5

empty sig-stx4

⑤ Effect of stx4 on migration and vinculin

expression

vinculin F-actin

empty sig-stx4

***

Exp

ressio

n le

ve

l

(r

ela

tive

)

actin

focal adhesion

・migrateon ・cell-substrate cell-cell adhesion

Vinculin

⑥ Effect of stx4 on differentiation

gapdh

zo-1α+

afp

stx3

villin

RT ＋ －

AFP : endoderm ZO-1α+：tight junction Villin ：polarization Stx3：polarization

Villin

AFP

ZO-1α+

Stx3

F9

stx4 differentiation ↑ ＜weaker than RA-treatment, though＞

+RA

Differentiation markers early differentiation

marker ⋆

gapdh

gata4

RT ＋ －

ga

ta4

expre

ssio

n

0

0.5

1

1.5

⑦involvement of stx3

・preceding study

intracellular stx3⇒pseudopodia ↑

・extracellular stx4⇒stx3↑ (protein・mRNA）

⇒ over expression of syntaxin3 in F9 cells

stx3 F-actin DAPI

stx3 ex)melanoma cells epithelial cells

stx3 pseudopodia↑, spread

＜stx3 partly mediates stx4’s extracellular function＞

intracellular stx3 ↑ extracellular stx4

stx3

β-actin

W.B.

研究例 ２

未分化細胞の

分化制御