Research

programmeYuka Ikeda

Department of Histology and Cell biology

Kagawa University

School of Medicine 4th year

Outline

Rac1

The objective of our experiment

What is Rac1?

GTP and GDP cycling

Methods and procedure

Pictures of microscopes

Conclusion

XL147

What is XL147?

The relationship between XL147 and cancer

Methods and procedure

Conclusion

Conclusion

The objective of our experiment

(Rac1)

How does Rac1 work?

Hypothesis; Constitutively active Rac1

promotes phagocytosis

Dominant negative inhibits phagocytosis

The role of active and inactive Rac1 in the

course of phagocytosis

What is Rac1?

Molecular switch

GTP binding protein

Regulate actin filaments at the plasma

membrane

Lamellipodia

Phagocytosis and macropinocytosis

Cellular motility and adhesion

GTP and GDP cycling

Inactive

GDP

“inhibit”

Active

GTP

“promote”

Macrophage can

complete phagocytosis!

GAP

GEF

Methods and Procedure-1

Cells ; RAW 264.7

Monocytic leukemia –macrophage-like cells

Easy to get

cDNA ; mCitrine-Rac1-Q61

Yellow

Transfection ; electroporation

neon

Methods and Procedure-2

Electron microscopy

4% Glutaraldehyde in 0.1mol phosphate buffer

0.1mol phosphate buffer

1% OsO4

1% tannic acid

CO2 Critical-point dry

Osmium plasma coater

Optical microscopy

4% paraformaldehyde

0.1% glutaraldehyde

0.1mol phosphate buffer

5% sucrose

RAW control 30min and Wild type-1

Control type

Macrophage

phagocytes a lot of

RBC!

RAW control 30min and Wild type

Cup and RBC

macrophages

Higher

magnification

RAW Rac1 CA-1

Active form

It does NOT

phagocyte

RBC.

RAW Rac1 CA-2

Ruffles like cups

Macrophages can make

ruffles. (NOT phagocytic)

→Expression of Rac1 Q61L

inhibited phagocytosis of

IgG-RBC.

RAW264 cells Rac1T17N (DN)

30 min phagocytosis of IgG-RBC

Dominant negativeNon-expressing (control)

Optical microscope-1

Live cell Rac1 CA

Red : RBC

Green : mCitrine-Rac1-Q61L

• Ruffles

• No cups or superficial

• RBC : Just binding

Optical microscope-2

Live cell imaging of Rac1 CA

expressing cell

Red : RBC

Green : mCitrine-Rac1- Q61L

Many ruffles

were formed

around RBCs.

Ruffles moves

toward the

cell’s center.

Optical microscope-3

Live cell image of Rac1 CA

It can make ruffles, however it can

NOT phagocyte RBCs.

Optical microscope-4

Live cell image of Rac1 Dominant Negative

It can NOT make ruffles, however it CAN uptake

RBCs.

The way of uptake RBCs is different from Control.

Optical microscope-5

Green; macrophage

(DN)

Red; RBCs

Optical microscope-6

Live cell images of wild Type.

It can make some cups for uptake RBCs.

Active form

It can NOT uptake RBCs.

Phase F-actin

YFP-Rac1Q61L

Rac1Q61L-expressing cell ( upper left)

It can NOT uptake RBCs.

Non-expressing cell (lower right, arrow)

It can uptake RBCs.

Phase YFP-Rac1Q61L

Conclusion-1

CA Rac1

Ruffles ◎

Cups ☓

Can NOT uptake RBCs

DN Rac1

Ruffles ☓

Cups ☓

CAN uptake RBCs

Conclusion-2

Active only ; can NOT make cups

Negative only ; can NOT make cups

Different way of the uptake RBCs

CR3 complement recognition

Need both!

active and negative

Importance of GTP and GDP cycling

What’s XL147?

Orally available inhibitor of phosphoinositide-3-kinase

(PI3K).

PI3K ; cell proliferation and survival

Phase 2

Methods and Procedure

RAW

Alveolus macrophages

Peritoneal macrophages

XL147

Optical microscope-1

RAW XL147

Optical microscope-2

Peritoneal macrophage

Conclusion

Relationship between cancer cell and lights

PI3k

Skin cancer

Sun light

Bladder cancer

Endoscope

Normal cells and cancer cells

Reference

Current Biology Vol 21 No 14

2013 John Wiley & Sons A/S

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Thank

you for

your kind

attention!