Measure of surface protein mobility with u-paint technique

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2 Measure of surface protein mobility with u-paint technique

3 How dynamic image can solve the situation? Random distribution or cluster?

4 Why live super-resolution microscopy can solve the situation With mobility

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9 25 nm

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13 Electron microscopy STED STORM sptpalm u-paint Photoswitching befor e after Wavelength

14 What do you need to do proteins tracking High efficacy (to avoid photodamages, and allow fast and high precision localization) Relative stability to obtain trajectories Bio-compatible and able to conjugate with a probe/ tag Relatively small size Spt-PALM works well with pro and cons

15 U-PAINT : universal point accumulation for imaging in nanoscale topography, stochastic labelling Sharonov, A., and R. M. Hochstrasser. 26. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA. 13: Giannone, Hosy et al, 21. Biophysical journal Giannone et al, 213. Method Mol Biol 15

16 Microscope objective Target protein

17 Non fluorescent ligand Target protein Microscope objective

18 Non fluorescent ligand ~5 Target protein Microscope objective Off axis excitation beam

19 ~5 Microscope objective Off axis excitation beam

20 T= ~5 Microscope objective Off axis excitation beam Recording

21 T=1 s ~5 Microscope objective Off axis excitation beam

22 T=1 s ~5 Microscope objective Off axis excitation beam

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24 Different strategies to label the target protein Poly-ATTO-antibody TNTA-ATTO SEP or His-tagged protein

25 Nature methods 25

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27 AP tag + biotin ligase 27

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29 Super Resolution using Ultivue s DNA-PAINT Ultivue reagents Nanometer-scale Resolution Images + Widely available microscopes = Fluorophores with DNAmodulated affinity Non-imaged section Imaged optical section Recording of hybridization events Net result: a blinking movie + Objective with optical sectioning i.e. TIRF Fluorophores are separately introduced in excess after antibody staining Targets are observed one hybridization event at a time over multiple snapshots The movie is then processed into a super-resolved image

30 What questions and parameters before starting spt experiments Do I really need live imaging? What technique, surface, density, overexpression? What probe/ fluorophore : size, brightness, wavelength, etc What solution? What type of illumination Then for first steps: Characterization of the noise of the system (environmental noise and cell background) Determination of the pointing accuracy Determination of the trajectory length Optimization of recording properties (laser power, acquisition time, etc ) How to analyse and what parameters do we need to extract? 3

31 Some examples First step of the U-PAINT Giannone, Hosy et al, 21. Biophysical journal 31

32 A Fibroblasts TM-6His B C 2145 trajectories 73 1 µm 1 nm A Fibroblasts GFP B C 294 trajectories 73 1 µm

33 D Cos E F GPI-GFP 344 trajectories µm 1 nm D Cos E F GFP 555 trajectories µm

34 Properties of these trajectories Density of trajectories Lenght of trajectories Trajectory density (µm- 2 ) Fibroblasts TrisNTA-atto Cos cells AntiGFPatto trajectories (µm- 2 ) pdisplay-6his gpi-gfp GFP pdisplay-6his GFP Gpi-GFP Trajectory duration (s)

35 A TM-6His B 3 TM-6His control GFP Occurrence (%) D D (µm²/s) C 4 Occurrence (%) 3 2 Edge Cell body 2 µm 1 Whole cell <.7 µm Step length (µm)

36 Analyses Détection des centroïdes f(x,y) I e (x x )² (y y )² 2 ² Reconstruction des images Image en épifluorescence Image haute résolution Image des trajectoires

37 Diffusion parameters.1 Confinement Inst. Diffusion MSD (µm²) Time (s).4.5 MSD ( t)= < dist ( t)² >

38 Various diffusion types directed Brownian.1 MSD (µm²).5 Confined.1.2 Time (s).3.4.5

39 Analysis Déplacement Quadratique Moyen (MSD) => zone de confinement Confinement t t 3 t 4 MSD (µm²).1.5 t 1 t 2 t n Temps (s) MSD ( t)= < dist ( t)² >.5.3 t Diamètre de confinement: 26nm t 85 MSD (µm²) nm t t 53 1nm Temps (s)

40 Analysis Coefficient de diffusion (D) => vitesse.3 2 pa y( n ) 4Dn 3 MSD (µm²) Temps (s) Cellules individuelles 6 Moyenne Occurrence (%) Occurrence (%) Log D Occurrence (%) Log D Occurrence (%) Occurrence (%) Log D

41 L application de glutamate réduit le confinement des rampa Suivi de rampa endogènes synaptiques contenant la sous unité GluA2 Avant Après application de glutamate Homer 1c

42 How to represent this data Occurrence (%) Immobile Ctrl Glu (1µM) Mobile Log(D) Ratio mobile/immobile Ctrl * Glu (1µM) MSD (X1-2 µm²) Temps (s) *

43 Receptor dynamic inside nanodomains Pattern 1: synaptic Pattern 2: mixte Pattern 3: inside nanodomain 25 nm

44 Receptor dynamic inside nanodomains Pattern 1 Pattern 2 Pattern 3

45 A Closed/resting conformation : GluA2 T686A.5 8 % mobile C 6 * B Instantaneous D µm Time (s) Desensitized conformation : GluA2 S729C.5 19 % mobile D Immobile fraction (%) 4 2 T686A S729C 1 ** 1µm Instantaneous D Time (s) % of time immobile T686A S729C 45