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

研究例 ２
未分化細胞の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

分化制御○Natsumi Hagiwara , Yohei Hirai
Dep. Bioscience, Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.
Background and our question＜OUR QUESTION＞
a)

Extracellularly presented syntaxin4
affects anaplastic cell behaviors?

a) Subpopulations of certain syntaxins including syantaxin4
translocate across the cell membrane in response toexternal stimuli.
b) The extracellularly presented syntaxins regulateb)morphology and differentiation of several epithelial cell types. ex) epithelial cells

stx4d) Mouse teratocarcinomae) The method for the unidirectional differentiation to theendodermal linage has been well established withusing all-trans retinoic acid (RA).

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

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

morphology
RAcont. +RA- 34 kDa stx4 -b-actin -cont.

recombinantstx4- 34 kDacont.

F9b-actincont.

+ RAb-actin stx4

F9 cells
Extracellular subpopulationof stx4 localized at the lateral
membrane.

Colonies → Flattened morphology

sig-stx4stx4
150μm

2nd Ab only

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

Differentiation induction perturbed stx4’s polar localization.
③ Effect of stx4 on cell-substrate adhesion⑤ Effect of stx4 on
migration and vinculin
expression④ Effect of stx4 on cell-cell adhesion

Long term adhesion (starved)
sig-stx4

Functional core peptide

Localization of E-cadherin

15**

50cont.

r-stx4 cont. sig-stx4 RAn=6***300.40.3
0.2
0.1
0EPn0

sig-stx4

empty

ST4n1r-stx4* : P<0.05** : P<0.01*** : P<0.001

10E-cadherin-Catenin complex***

3530252015
1050input**②

Extracellularly suppulied Stx4
enhance cell-substrate adhesion

spread

①

empty

②

sig-stx4

・preceding study

early differentiation
marker－＋

gata4
gapdh

gata4 expression

Differentiation markers
RT

n=3

1.5
1
0.50⋆

RT

stx3

intracellular stx3⇒pseudopodia ↑ex)melanoma cells
epithelial cells

+RA＋

Stx3
AFPZO-1α+Villin0.20.100.50
empty sig-stx4

IgG
empty sig-stx4 empty- E-cadherin- a-catenin- b-catenin

vinculin F-actin

Stx4
migration ↑＜down-regulation of Vinculin＞

F9 cell

anaplastic cellafp

P19 cell

stx3

stx3 F-actin DAPI

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

1***IP ECCD2⇒ over expression of syntaxin3 in F9 cells

gapdh

1.5

cell shapeadhesioncell-substrate↑cell-cell↓

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

0.3

sig-stx4

W.B.
stx3

zo-1α+

F9

0.4

Conclusion

・extracellular stx4⇒stx3↑ (protein・mRNA）－actinempty- a-catenin- b-catenin

Stx4cell-cell adhesion↓＜Cadherin-Catenin complex was not affected＞⑦involvement of stx3

⑥ Effect of stx4 on
differentiation・migrateon
・cell-substratecell-celladhesion- E-cadherin-①

Vinculin
focal
adhesion

empty sig-stx4 empty** : P<0.01*** : P<0.001

spherical**

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

E-cadherinn=6empty sig-stx4 empty sig-stx4

Sig-stx4

20μm

sig-stx4n=7*200Empty

0.5*

cont***

Day1

sig-stx4

Suspension culture

Number of cell clusters

10empty

Functionalcore peptide

empty

Adhesion / Spreading
onto petri dish
Adherent cells (relative)①＞0.3㎜②≦0.3㎜**
Adherent cells (relative)

Adherent cells (relative)**

Wound healing assay
Day0stx4
G AI
AIEPQK
CE(Functional core ) CK QP
ST4n1-34kDa

T7cell shapechange
empty vector

20μｍempty

parent sig-stx4

r-stx4Amount of stx4 was not affected bydifferentiation induction.

non-permeabilized+ RAF9cont.
(r-GFP)b-actin -well-packed colonies→ flattened morphology (substrate –adhesion↑）

permeabilized

Signal peptide-stx4:
For forced expressionof cell surface stx4

Addition tomedium

stx2(epm) stx3

150μm

DAPI ZO-1 F-actin② Effect of stx4 on morphology

endogenouscont.

differentiation

Expression level(relative)

Anaplastic cellse)

Model: F9 cells

cornification

RACell migration(relative)

syntaxins (stxs)

extracellular stx4

intracellular stx3 ↑stx3

pseudopodia↑, spread＜stx3 partly mediates stx4’s extracellular function＞

differentiation
markers
endoderm
gata4 afp
stx3 etc.↑

stx2
ectoderm

motility
migration↑
Vinculin
stx3

Check
supplementary results

mesoderm

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