Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like...
Transcript of Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like...
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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you for
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attention!