1. Microscopy Based Biosensors and Functional Assays Receptor Inputs Signaling Proteins Second Messengers Secretion Endocytosis Pinocytosis Phagocytosis Cell Polarity Cell-Cell Contact Adhesion Migration Chemotaxis Cell Cycle Apoptosis Cell Size (DNA Damage, Nutrition State) Functional Outputs

2. Dissecting cellular signaling systems: Perturbations and Biosensors Perturbations Biosensors, Functional assays Receptor Inputs

3. 1. Microscopy Strategies to Explore Signaling Systems 1.Epifluorescence Imaging 2.Confocal Imaging 3.Total Internal Reflection Microscopy

4. Anti FLAG M1 monoclonal Ab YFP Automated assays for receptor endocytosis Epinephrin Internalization (~30%) YFP + EDTA 2mM YFP Fix, perm, 2 ndary Ab A568

5. Automated immunofluorescence analysis (epifluorescence) YFP Texas Red CFP CFP-Arf1 (DN) YFP-  2-adrenergic receptor Internalized receptor

6. Reduced endocytosis rates in the presence of some constitutively active small GTPases Ratio I 594 /I YFP (% CTR) CTR Potentially involved in regulating endocytosis ARL4 ARF1 ARF3

7.  2-adrenergic receptor internalization kinetics (confocal imaging) 45 minute movie, YFP-tagged beta2-adrenergic receptor; epinephrine stimulation after 15 minutes

8. GFP-PKC  Calcium- Crimson Calcium signals versus PKC translocation (confocal imaging) Antigen stimulation of tumor mast cells

9. Membrane translocation measured by total internal reflection fluorescence microscopy +Receptor stimulus PKC-GFP The evanescent wave field in TIRF imaging

10. Monitoring plasma membrane translocation in large numbers of cells Evanescent wave Single Cell Array Technology (E-SCAT) Adherent cells Laser Teflon ring Low magnification projection (9mm x 7mm imaged area) Camera

11. Recording of GFP-C2 domain plasma membrane translocation in many cells PAFionomycin 0 1 Plasma membrane Cytosol 50 s Rel. fluorescence Teruel and Meyer, Science, 2002

12. 2. Perturbation strategies suitable for microscopy 1.RNAi 2.Expression constructs (wt, DN, CA) 3.Small molecule perturbations (for example induced translocation)

13. Generating d-siRNA sets based on in vitro dicing * * Transfection Pool of dsiRNAs r-DicerLarge dsRNA Myers, Jones, Meyer and Ferrell Nature Biotech, 2003 In Vitro Dicing and Purification PCR In Vitro Txn X 24 = 2304 siRNA pools targeted to signaling domain selected proteins In vitro Dicer method developed by Jason Myers in Jim Ferrell’s lab

14. R-RAS2 R-RAS R-RAS3 H-RASK-RASN-RAS RITRIN RAP1ARAP1BRAP2BRAP2ARALARALB RAD GEM REM ARHI AGS1 RRP22 kB-RAS1kB-RAS2 RHEB2RAC3RAC2RAC1 RHOG CDC42h CDC42TC10 TCL RHOE ARHERHO7RHO6 RHOC RHOA RHOBRHOH RHOD ARF1ARF3ARF5ARF4ARF6 ARL1 ARL5 ARL3ARL2ARL7A ARL7BARL4ARF4L ARFRP1 SAR1 SARA RAGARAGB RAB8BRAB8RAB10RAB13SEC4L RAB1A RAB1BRAB35 RAB27ARAB27BRAB26 RAB3BRAB3ARAB30 RAB33ARABRP RAB4 RAB4BRAB14 RAB2 RAB11ARAB11B RAB25RAB18 RAB5A RAB5C RAB5B RAB22A RAB22B RAB21 RAB9 RAB9L RAB7 RAN RABL2B RAB32RAB38RAB7L1RAB23 RAB6RAB6CRAB28 Polar Eyelashes Lamellipodia Filopodia Rounding Stress fibers Multiple Shrunk MultipleLocal spread CA small GTPases and cell morphology Heo and Meyer, Cell, 2003

15. Rac PM translocation induced by a rapamycin analog + FKBP-YFP-Rac1(CA) Rapamycin analog synthesized by Tom Wandless NIH3T3 cells w. PM-FRB

16. 3. Automated Microscopy Based Biosensors and Functional Assays 1.FRET Biosensors 2.Phosphospecific Antibody and Related Fixed Cell Assays 3.Translocation Biosensors 4.Live and Fixed Cell Functional Assays (Outputs) 5.Many critical assays are lacking

17. Activation of c-jun by CA small GTPases (example of rapid survey assay) Constitutively active small GTPases (HS68-cells) Activation of c-jun phosphorylation by small GTPases 0 200 400 600 800 1000 1200 1400 1600 1800 2000 NO TNF-a CDC42 TC10 CDC42h RAC1 ARL7 RAC2 RAB40BRAB7L1 RALB RAB26 RAB2 RAC3 RAB5C RHOG GEM N-RAS RHOB ARHE R-RAS2 RAD K-RAS TCL AGS1 ARL5 RHOD RAB23 RAB8B ARL4 SAR1B RHO7 R-RAS1R-RAS3 RAB21 RHO6 RAGB RHOA RAB5A SAR1ARAP1A RRP22 RHOC ARHI H-RAS RHO8 RAB3A RAB33A RAB1A RAB39L ARL7 RAB22B RAB4B RHEB2 RAB6A RAB9B RAN RAB25 RAGA RAB18 RAB11A RAB10 RAB8 RAB4A RAB27A RAB22B RAB38 RIN RHOH RAB1B RABL2B ARF4 RAB22A ARF3 RIT ARL3 ARF1 RALA RAB27B RAB7 RAB5B RAB35RAB30RAB28 RAP2B RAB6C REM RAB11B RAB9 RAB3B Intensity of phospho-c-jun in nucleus No/low expression High expression 

18. Apoptosis induced by d-siRNA against human signaling proteins (important control) Plate #2 siRNA-1 (MCF-7) 0 10 20 30 40 50 60 70 A01A03A05A07A09A11A13A15A17A19A21A23 B01B03B05B07B09B11B13B15B17B19B21B23 C01C03C05C07C09C11C13C15C17C19C21C23D01D03D05D07D09D11D13D15D17D19D21D23 E01E03E05E07E09E11E13E15E17E19E21E23 F01F03F05F07F09F11F13F15F17F19F21F23 G01G03G05G07G09G11G13G15G17G19G21G23 H01H03H05H07H09H11H13H15H17H19H21H23 well Apoptotic cells/total cells (%) Anti-Lamin B antibody apoptosis assay

19. Fluorescent Translocation Biosensors: Non-perturbing versus endpoint indicators SH2-domains to monitor local tyrosine phosphorylation (Stauffer et al., JCB 1997) C1-domains to monitor localized diacylglycerol signals (Oancea et al., JCB 1998) C2-domains to monitor local Ca 2+ /PS-signals (Oancea et al., Cell 1998) PH-domains to monitor local changes of phosphoinositides (Stauffer et al., Current Biology 1998, PLC-delta; Kontos et al., Mol. Pharm. 1998, Akt) Potentially many other useful domains (FYVE, PTB, …)

20. PH Domain Binding selectivity CTH3(PH1A)Akt3(PH1A)PLCd1(PH1A)RalGPS2(PH1A)Hapip1(PH1A) PI(3,4,5)P 3 PI(3,4,)P 2 PI(3,4,5)P 3 PI(4,5)P 2 PI(3,4)P 2 PI(4,5)P 2 PI(3,4,5)P 3 PI(3,4)P 2 127 PH Domain constructs tested: PI(3,4,5)P 3 CTH3(PH1A), Myo10(PH1A), ITK(PH1A), H056(PH2A), EtOHD4(PH1A), APS(PH1A), Afap(PH1A), TEC(PH1A) PI(3,4)P 2 and PI(3,4,5)P 3 Gab1(PH1A), Gab2(PH1A), Bam32(PH1A), CTH2(PH1A), IRS-1(PH1A), Osbp13(PH1A), Plek(PH1A), TNFidp(PH1A), Akt2(PH1A), Akt3(PH1A), Akt1(PH1A), LL5(PH1A), Arl61(PH1A), BCRa(PH1a) PI(4,5)P 2 and PI(3,4,5)P 3 PLCd1(PH1A), Spnb2(PH1A), RalGPS2*(PH1A), Centb5(PH1A), Cnk2(PH1A) PI(3,4)P 2 Plek2(PH2A), Hapip1(PH1A) Biosensors & Perturbations PH-domain selectivity Wei Sun Park, James Whalen, Takako Mukai & Nancy O’Rourke

21. PIP3 production by constitutively active small GTPases ECFP H-RAS K-RAS RALA RAP2B RAP2A RAC1 RHOG CDC42RHOH TC10 RAB1A RAB2 RAB23 RAB30 ARF1 Ras subfamily Rho subfamily Rab & Arf subfamily Morphology changes make automated analysis more difficult

22. Functional assays (Outputs) YFP NLS PM targeting Mitotic biosensor Jones, Myers, Ferrell & Meyer, Nat. Biotech., 2004

23. Watching 3 hours in the life of cycling cells Mitosis biosensor

24. Measuring cell cycle timing Mitosis in RBL’s

25. Mitosis is accelerated by a loss of the spindle checkpoint 25 nM 15 min 5 nM 25 min 3 nM 35 min Untreated 45 min 0 10 20 30 40 50 60 0GL3351015255075 [d-siRNA] nM Time (min) NEB-Anaphase Prometaphase Metaphase Mad2 targeted by d-siRNA

26. Examples of mitosis defects observed with the mitosis biosensor Cytokinesis Defects (Rab 21) Abnormal Spindles (Rab 3B) Normal

27. Automated measurements of dynamic parameters in cell migration Dendritic cell migration in presence of C5a

28. TIRF measurements of secretion, endocytosis and signaling processes CFP-PH(Akt) GLUT4-YFP TIRF Assay for PIP3 and the PM insertion and endocytosis of GLUT4 transporter

29. Acknowledgments Josh Jones Angie Hahn Onn Brandman Annette Salmeen Cecile Arrieumerlou Takanari Inoue Marc Fivaz Madeleine Craske Thierry Galvez Michael Bradshaw Chuck Fink Mary Teruel Man Lyiang Kim Won Do Heo Jen Liou Calif. Ave. AfCS Microscopy lab: Grischa Chandy Nancy O’Rourke Wei Sun Park Jim Whalen Takako Mukai Mary Verghese Liz Gehrig Sarah Lim James Ferrell, Jason Myers, Michal Ronen Tom Wandless

30. Automated microscopy based signaling and functional assays 1.Phosphospecific antibodies and other fixed cell assays (analysis procedures can readily be developed; more suitable phosphospecific antibodies needed) 2.FRET biosensors (implementation of automated assays of existing biosensors is first needed) 3.Translocation biosensors (PM, nucleus, Golgi and vesicular structures could be automatically analyzed; development of new assays and implementation of automated assays needed) 4.Functional output assays (apoptosis, cell cycle, secretion endocytosis, phagocytosis, pinocytosis, cell migration, cell adhesion; Automated assays still need development) 5.Still fairly low biosensors coverage. More assays needed (how many?). Microscopy can provide suitable assays for many of them.