1. Imaging signal transduction in single dendritic spines during synaptic plasticity Ryohei Yasuda (HHMI, Duke)

2. Spine Spine: Biochemical compartment Small ~0.1 fL. Narrow neck (~100nmΦ) : Diffusional barrier Ca 2+ signaling in spines  Synaptic plasticity  Memory

3. Signaling networks R. Iyenger

4. Imaging signaling in single spines Measure FRET with 2-photon fluorescence lifetime imaging (2-photon FLIM) Develop and use FRET sensors optimized for 2-photon FLIM Image signal transduction, while inducing plasticity in single spine with 2-photon glutamate uncaging

5. FRET and fluorescence lifetime FRET decreases fluorescence lifetime. Use donor fluorescence only. –Independent of fluorophore concentrations. –Independent of wavelength-dependent light scattering Multiple populations can be deconvolved. Easy to combine with 2-photon microscopy 2 4 6 8 10 12 Log (fluorescence) Time (ns) Laser pulse FRET Mixture Donor

6. 2-photon fluorescence lifetime imaging microscopy High resolution and sensitivity in deep tissue. Quantitative measurements of FRET

7. Stimulate single spines using 2-photon glutamate uncaging Synapse-specific Ca 2+ elevation Matsuzaki, Ellis-Davies, Kasai

8. 2p-uncaging to produce long lasting synaptic potentiation and spine growth 30-60 times, 0.5-1 Hz in Zero extracellular Mg 2+ Matsuzaki 2001, 2004

9. Imaging activity of CaMKII Ca 2+ /Calmodulin-dependent kinase II

10. Ca 2+ /Calmodulin-dependent kinase II: biochemical memory? Kinase domain Low Ca 2+ Ca 2+ Activation Memory? Ca 2+ /CaM CaM P Ca 2+ P Inactive GFP FRET d-YFP Takao et al, 2004 Lisman 2002 Autoinhibitory domain Kinase domain

11. Fluorescence lifetime change in lysates Time (min) 0 0.1 0.2 0.3 0.4 0.5 -5051015 CaM+, ATP+ CaM+, ATP+(T286A) CaM+, ATP– CaM–, ATP+ CaM–,ATP– Fluorescence lifetime change (ns) Ca 2+ EGTA Lee et al., Nature 2009

12. CaMKII activation during structural plasticity of spines Lee et al., Nature 2009

13. Transient and spine-specific activation of CaMKII Lee et al., Nature 2009

14. Transient and spine-specific activation of CaMKII -505101520253035 ?0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 CaMKII Activity Change Stimulated Spine Adjacent Spine Dendrite Uncaging 35 2 min CaMKII Uncaging Volume Lee et al., Nature 2009

15. Ca 2+ /Calmodulin-dependent kinase II: biochemical memory? Kinase domain Low Ca 2+ Ca 2+ Activation Memory? Ca 2+ /CaM CaM P Ca 2+ P Inactive Not for 1 hour What is the role of phosphorylation?

16. T286A Wild-type Fast fluorescence lifetime imaging Lee et al., Unpublished -202468101214 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.7 s 5.8 s Time (s) Fluorescence lifetime (ns) 1 μM Δ[Ca 2+ ] ~0.1 s Glutamate uncaging (4 ms)

17. Ca 2+ /Calmodulin-dependent kinase II: biochemical memory? Kinase domain Low Ca 2+ Ca 2+ Activation Memory? Ca 2+ /CaM CaM P Ca 2+ J. Lisman P Inactive Yes, but only for 6 s ~ 6 s

18. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour1 min Previous view of LTP

19. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour1 min Now ….

20. Imaging the activity of Ras superfamily proteins

21. Small GTPase signaling Several major subgroups: Ras, Rho, Rab, Rap, Arf, Ran etc… Acts as signaling switch. Regulate organization of actin cytoskeleton, membrane trafficking etc. Important for morphogenesis of dendritic spines and plasticity Mutations in the pathway are associated with mental retardation

22. Imaging binding between Ras and Ras binding domain (RBD) of Raf1 Yasuda et al., Nat.Neurosci. 2006 Harvey et al., Science 2008 CaMKII Ras

23. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour1 min Ras  ERK AMPAR exocytosis

24. General approach to make sensors for Ras superfamily Cdc42 / RhoA: Important for regulation of actin cytoskeleton and dendritic spine morphology. 65-118 a.a. S79A, F89A mGFPCdc42 Pak3 mRFP P 8-89 a.a.. WT mGFP RhoA RTKN mRFP P Cdc42 sensor RhoA sensor

25. Making small GTPase sensors 1. Screen RXX Binding Domain in cuvette K d ~ 1 – 5 uM for GTP form (RBD inhibits Rho inactivation) K d > 50 uM for GDP form for low background 2. Test sensitivity & specificity in cell line 3. Test sensitivity & kinetics in neurons

26. Step1: Screen RBD and mutants FRET between GFP-CDC42 and PAK2-mCherry

27. GFP-RasCdc42 Rac1 RhoA mCherry-RBDGDPGMPPNPGDPGMPPNPGDPGMPPNP PAK16.90.1170.4 PAK1 (F89A)740.6784.6 PAK1 (F89A, F96A)913.4> 30041 PAK2100.2220.5 PAK3570.3691.8 PAK3 (F89A)1391.014312.5 PAK3 (S79A, F89A)1501.87722 WASP432.9 RTKN473.9

28. Step 2: Test sensitivity & specificity in HeLa cells

29. Step 3: Test sensitivity & reversibility in neurons

30. Cdc42/RhoA activation during LTP

31. Cdc42 activation is compartmentalized and sustained Hideji Murakoshi Stimulated spine Adjacent spine

32. RhoA activation spreads and sustained Hideji Murakoshi Stimulated spine Adjacent spine AP5 RhoA activation (%) 0102030 Time (min) 0 5 10 Uncaging

33. Cdc42 0 5 10 Binding fraction change (%) Cdc42 8-56 s1-5 min5-10 min10-20 min 0510 Distance (µm) 051005 05 Before 24 s 2.15 ns 2.65 5 µm Spine Dendrite Spatial profile of Cdc42

34. Spatial spreading of RhoA RhoA 24 s Before 2.0 ns 2.6 0510 Distance (µm) RhoA 0 5 10 15 051005 5 µm 1005 8-56 s1-5 min5-10 min10-20 min Binding fraction change (%) Spine Dendrite

35. Effects of overexpression on length constant Length constant (µm) 050100 0 2 4 6 8 [mCherry-RBD-mCherry] (µM) 02468 0 2 4 6 8 [mEGFP-RhoA] (µM) RhoA A n = 20/18 r = 0.05 n = 20/18 r = 0.16

36. Diffusion coupling at the spine neck Lee, Harvey, Murakoshi Ras proteins: ~5 s CaMKII: ~60 s PA-GFP tagged Ras *Constitutively active mutants * * * *

37. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour Cdc42 1 min RhoA

38. RhoA activation is CaMKII dependent (NMDA receptor inhibitor) (CaMKII inhibitor) Late phase is CaMKII dependent Partial inhibition at early phase 0102030 Time (min) 0 5 10 RhoA activation (%) Ctrl (stim) KN62 AP5 Uncaging Hideji Murakoshi

39. Control Hideji Murakoshi AP5 (NMDA receptor inhibitor) KN62 (CaMKII inhibitor) 02040 0 5 10 Cdc42 activation (%) Time (min) Uncaging Cdc42 activation is CaMKII dependent

40. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour Cdc42 1 min RhoA

41. Cdc42 is required for long-term structural maintenance Hideji Murakoshi Cdc42Sustained growth

42. Cdc42 is required for long-term structural maintenance Hideji Murakoshi Cdc42Sustained growth Volume change (%) Time (min) Cdc42 binding domain (24 hours)

43. RhoA is required for transient phase Hideji Murakoshi RhoATransient growth

44. Stronger inhibition of RhoA inhibits both transient and sustained phases Volume change (%) Time (min) RhoATransient/Sustained growth

45. RhoA/Cdc42 does not alter Ca 2+ -CaMKII Cdc42 RhoA CaMKII Ca 2+ CaMKII activation Structural plasticity Control C3: Rho inhibitor WASP: Cdc42 inhibitor -50510 0 0.05 0.1 0.15 Time (min) Lifetime change (ns) 012 0 0.05 0.1 0.15 -50510 0 100 200 300 400 Time (min) Volume change (%) 012 0 100 200 300 400 Time (min) Spine growth

46. Regulation of spine volume by Rho GTPases Cdc42/RhoA Spine growth CaMKII Hideji Murakoshi Cdc42 Volume CaMKII 2 min Cdc42 Volume CaMKII 10 min Uncaging RhoA

47. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour Cdc42 Actin 1 min RhoA Actin Transient plasticity

48. Downstream of RhoA/Cdc42

49. Ca 2+ CaMKII Long-term plasticity 0.1 s10 s10 min1 hour Cdc42  PAK Actin 1 min RhoA  ROCK Actin Transient plasticity Ras  ERK AMPAR exocytosis

50. There are 100 more small GTPase proteins…

51. Rac1 sensor mGFPRac1 PAK1 mRFP P Rac1 activity Before Stimulation 30 s High Low

52. RhoA/Cdc42/Rac1 activation time courses RhoA Cdc42 Rac1 RhoA Cdc42 Rac1

53. Rap1 sensor 0 60 Binding fraction (%) -2 min 6 min Control Forskolin IBMX 0 20 40 WTG12VS17N Control Forskolin IBMX 10 um Binding fraction (%) mGFPRap1A RalGDS mRFP P Neuroblastoma cell

54. Signaling networks R. Iyenger

55. Ca 2+ Hong Wang CaMKII Seok-Jin Lee Ras/ERK/Rap Ana Oliveira Erzsebet Szatmari Shenyu Zhai Rho Hideji Murakoshi Nathan Hedrick AMPAR Michael Patterson Technical assistance Airong Wan David Kloetzer Funding NIH/NIMH NIH/NINDS NIH/NIDA NSF HHMI Alzheimer’s Association Brandeis Univ. J. Lisman HHMI/Janelia Farm Christopher Harvey Karel Svoboda Yasuda lab Duke Sridhar Raghavachari Michael Ehlers Scott Soderling