GFP Assays

Transcription

1 GFP Assays

2 GFP Assays Akt1 SMAD2 STAT3 NFAT-C1 MAPKAP-k2 Rac-1 PLCδ PH domain FYVE G2M CCPM Cell survival, proliferation, apoptosis, insulin response pathways TGF-beta signalling, growth, differentiation, cell survival Acute-phase response pathways, cytokine signalling, leptin signalling Immune responses (IL2, IL4, TNFa gene expression), prostaglandin signalling Stress response pathways Cytokinesis, transformation, adhesion, migration PI(4,5)P2 signalling pathways, adrenoreceptor function Growth factor (RTK) and cytokine signalling pathways, PI(3)P levels (class III PI3Kinase sensor) Cell cycle analysis FYVE DNA PLC PH dom

3 Analysis modules Assay Algorithm Compatibility Cell line Analysis Module ( software ) Translocation MAPKAP-k2 BHK cell Nuclear Trafficking Nucleus to cytoplasm RAC1 CHO cell Plasma Membrane Trafficking Cytoplasm to cell surface ruffles PLCδ-PH domain CHO cell Plasma Membrane Trafficking Cell surface to cytoplasm FYVE U-2OS cell Granularity Endosomes to cytoplasm AKT1 CHO Plasma Membrane Trafficking Cytoplasm to cell surface ruffles STAT3 BHK Nuclear Trafficking Cytoplasm to nucleus NFAT c1 U-2OS Nuclear Trafficking Cytoplasm to nucleus SMAD2 CHO Nuclear Trafficking Cytoplasm to nucleus G2M CCPM U-2OS Cell Cycle Trafficking Cell Cycle

4 The MAPKAP-k2 GFP assay Agonist Anisomycin Antagonist SB Unstimulated cell: majority of MAPKAP-k2 GFP is in the nucleus Stimulated cell: translocation of MAPKAP-k2 GFP to the cytoplasm Inhibited cell: concentration of MAPKAP-k2 GFP in the nucleus Cells: Translocation: BHK stably expressing MAPKAPK2-GFP fusion Nucleus to Cytoplasm (agonist-stimulated) 1.2 Translocation Index (C:N) Log [Anisomycin] (M) No Treatment LOGEC50 HILLSLOPE EC e µ M Anisomycin Image acquisition & analysis: IN Cell Analyzer 3000

5 Rac1 GFP Assay Agonist (Insulin) Unstimulated cell Stimulated cell Insulin is used as a reference agonist GFP-Rac1 redistributes from cytoplasm to form membrane ruffles Wortmannin can be used as an inhibitor of GFP-Rac1 fusion protein

6 Rac1 GFP Assay Agonist-induced Rac-1 translocation No Treatment 50 nm Insulin

7 GFP-PLC PLCδ PH domain membrane to cytoplasm redistribution assay Agonist, 5-10 sec sec Un-stimulated cell: majority of GFP-PLCδ localized on inner surface of plasma membrane. Stimulated cell: GFP-PLCδ redistributes transiently to cytoplasmic space, Post-stimulation cell: GFP-PLCδ re-accumilates at plasma membrane

8 FYVE Domain Assay Inhibition by wortmannin, a broad-spectrum PI3-Kinase inhibitor No treatment + Wortmannin IN Cell Analyzer 3000 IN Cell Analyzer 1000 U2OS stable cell line

9 AKT1 Assay Agonist (IGF-1) 4 mins Un-stimulated cell: Majority of AKT1-EGFP localized in cytoplasmic space Stimulated cell: AKT1-EGFP redistributes to plasma membrane A B

10 SMAD2 Assay TGFβ signalling: Cell growth, proliferation, apoptosis No treatment + TGFβ 10 ng/ml CHO-derived stable cell line Z = 0.6 Image acquisition & analysis: IN Cell Analyzer 3000

11 STAT3 Assay Agonist (IL-6) 25 min Un-stimulated cell: Majority of EGFP-STAT3 localized in cytoplasm. Stimulated cell: EGFP-STAT3 redistributes to nucleus. A T he B H K de rive d E G F P -S T A T 3 e x p re s s in g ce lls 2 5 m in a fte r s tim u la tio n w ith (A ) c o n tro l b u ffe r on ly a n d ( B ) 3 0 ng/m l I L -6. B

12 NFAT-c1 Assay 10 mins Agoinst Agonist (Ionomycin) (Ionomycin) Top image: U-2 OS derived EGFP-NFATc1 expressing cells 10 minutes after the addition of 0.1 % DMSO (control cells) Bottom image: U-2 OS derived EGFP-NFATc1 expressing cells 10 minutes after the addition of 1 µm ionomycin (stimulated cells).

13 G2M Cell cycle phase marker Enabling dynamic, non-destructive reporting of live cell cycle status G1/S G2 Prophase Mitosis

14 G2M Cell cycle phase marker Cells imaged every two minutes for 2 hours 3 cells to through mitosis 4 other cells remain in G2

15 Cell cycle distribution analysis Control + Colchicine G2 26% P 2% M 2% G1/S 14% G2 6% P 7% G1/S 70% M 73% The analysis module determines the % of cells in each phase

16 G2M Cell cycle phase marker Applications: Screening; desired cell cycle blockers, e.g. as anti-proliferatives Toxicology; undesirable cell cycle blocks Multiplexed marker for cell cycle dependent assays, e.g. cell cycle variation in receptor:g-protein coupling

17 GFP Translocation Assays Assay Response pathways Example Therapeutic Areas MAPKAP-k2 Rac-1 PLCδ PH domain 2XFYVE domain (domain from Hrs) Stress response pathways Cytokinesis, phagocytosis, pinocytosis, axon outgrowth, morphogenesis, cellcell contacts, cell polarity, transformation, adhesion, migration PI(4,5)P2 signalling pathways, adrenoreceptor function, cytoskeletal organization, responses to purinergic agonists, mechanical stress pathways Growth factor (RTK) and cytokine signalling pathways, PI(3)P levels (class III PI3Kinase sensor) Inflammation, neuronal growth and differentiation, pain, CNS disorders, ischemia, seizures, skeletal muscle regulation Colon and breast cancer, cardiac hypertrophy, myofibrillogenesis, proinflammatory signalling, leukemia, rheumatoid arthritis, progression to AIDS Cardiac diseases/ hypertrophy, cardiac injury, Alzheimer s disease, bipolar disorders Identification of class-specific PI3K inhibitors for development of cancer treatments, immuno-suppressants and anti-inflammatory drugs (Slide 1 of 2)

18 GFP Translocation Assays Assay Response pathways Example Therapeutic Areas Akt1 SMAD2 Cell survival, proliferation, apoptosis, insulin response pathways TGF-beta signalling, growth, differentiation, cell survival, regulation of cell proliferation and migration, elaboration of extracellular matrix Cancer (breast, prostate, ovarian, gastric), tumor progression, drug and radiation resistance in cancer therapy, invasion, angiogenesis, diabetes Oncogenesis, tumor progression, cardiac hypertension, embryonic development, tissue repair, immune function STAT3 NFAT-c1 Acute-phase response pathways, cytokine signalling, leptin signalling Immune responses (IL2, IL4, TNFα gene expression), prostaglandin signalling Acute-phase responses, oncogenesis (hematologic, breast, head, neck, prostate cancers), obesity Immunosuppression, muscle growth (Slide 2 of 2)