researchanatomy2/kadai2013/kadai2013ikeda.pdfcells ; raw 264.7 monocytic leukemia –macrophage-like...
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![Page 1: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/1.jpg)
Research
programmeYuka Ikeda
Department of Histology and Cell biology
Kagawa University
School of Medicine 4th year
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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
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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
![Page 4: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/4.jpg)
What is Rac1?
Molecular switch
GTP binding protein
Regulate actin filaments at the plasma
membrane
Lamellipodia
Phagocytosis and macropinocytosis
Cellular motility and adhesion
![Page 5: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/5.jpg)
GTP and GDP cycling
Inactive
GDP
“inhibit”
Active
GTP
“promote”
Macrophage can
complete phagocytosis!
GAP
GEF
![Page 6: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/6.jpg)
Methods and Procedure-1
Cells ; RAW 264.7
Monocytic leukemia –macrophage-like cells
Easy to get
cDNA ; mCitrine-Rac1-Q61
Yellow
Transfection ; electroporation
neon
![Page 7: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/7.jpg)
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
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RAW control 30min and Wild type-1
Control type
Macrophage
phagocytes a lot of
RBC!
![Page 9: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/9.jpg)
RAW control 30min and Wild type
Cup and RBC
macrophages
Higher
magnification
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RAW Rac1 CA-1
Active form
It does NOT
phagocyte
RBC.
![Page 11: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/11.jpg)
RAW Rac1 CA-2
Ruffles like cups
Macrophages can make
ruffles. (NOT phagocytic)
→Expression of Rac1 Q61L
inhibited phagocytosis of
IgG-RBC.
![Page 12: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/12.jpg)
RAW264 cells Rac1T17N (DN)
30 min phagocytosis of IgG-RBC
Dominant negativeNon-expressing (control)
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Optical microscope-1
Live cell Rac1 CA
Red : RBC
Green : mCitrine-Rac1-Q61L
• Ruffles
• No cups or superficial
• RBC : Just binding
![Page 14: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/14.jpg)
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.
![Page 15: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/15.jpg)
Optical microscope-3
Live cell image of Rac1 CA
It can make ruffles, however it can
NOT phagocyte RBCs.
![Page 16: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/16.jpg)
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.
![Page 17: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/17.jpg)
Optical microscope-5
Green; macrophage
(DN)
Red; RBCs
![Page 18: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/18.jpg)
Optical microscope-6
Live cell images of wild Type.
It can make some cups for uptake RBCs.
![Page 19: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/19.jpg)
Active form
It can NOT uptake RBCs.
Phase F-actin
YFP-Rac1Q61L
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Rac1Q61L-expressing cell ( upper left)
It can NOT uptake RBCs.
Non-expressing cell (lower right, arrow)
It can uptake RBCs.
Phase YFP-Rac1Q61L
![Page 21: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/21.jpg)
Conclusion-1
CA Rac1
Ruffles ◎
Cups ☓
Can NOT uptake RBCs
DN Rac1
Ruffles ☓
Cups ☓
CAN uptake RBCs
![Page 22: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/22.jpg)
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
![Page 23: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/23.jpg)
What’s XL147?
Orally available inhibitor of phosphoinositide-3-kinase
(PI3K).
PI3K ; cell proliferation and survival
Phase 2
![Page 24: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/24.jpg)
Methods and Procedure
RAW
Alveolus macrophages
Peritoneal macrophages
XL147
![Page 25: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/25.jpg)
Optical microscope-1
RAW XL147
![Page 26: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/26.jpg)
Optical microscope-2
Peritoneal macrophage
![Page 27: Researchanatomy2/kadai2013/kadai2013ikeda.pdfCells ; RAW 264.7 Monocytic leukemia –macrophage-like cells Easy to get cDNA ; mCitrine-Rac1-Q61 Yellow Transfection ; electroporation](https://reader033.vdocument.in/reader033/viewer/2022050314/5f76efeb97ecd7638e578ec5/html5/thumbnails/27.jpg)
Conclusion
Relationship between cancer cell and lights
PI3k
Skin cancer
Sun light
Bladder cancer
Endoscope
Normal cells and cancer cells
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Reference
Current Biology Vol 21 No 14
2013 John Wiley & Sons A/S
荒木先生のパワーポイント
加藤先生のノート
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Thank
you for
your kind
attention!