Xinnan Wang Stanford University School of Medicine

Transcription

1 Xinnan Wang Stanford University School of Medicine

2 Cellular Organelles are Highly Dynamic COS7 Cell, MitoTracker Fission-fusion, changing morphology and locabon To meet ever-changing energy demand

3 Electrical Signals Proteins, Organelles, mrnas

4 Wheels and Engine: Kinesin, Dynein, Myosin Tracks: Microtubules or AcBn

5 How to Study Axonal Transport? Live Imaging!

6 Axonal Mitochondria Retrograde Anterograde RFP-mito

7 Heated stage: 37 degree CO 2 independent Medium hup://

8 hups://

9 Glutamate Receptor AcBvaBon Glutamate Ca 2+? Ca 2+ Ca 2+ Ca 2+

10 Elevation of Cytosolic Ca ++ Inhibits Mitochondrial Motility Calcimycin: a calcium ionophore, used to increase intracellular calcium Retrograde RFP-mito Anterograde 10 um Wang and Schwarz, Cell, 2009

11 Parkinson s Disease Second common movement disorder SelecBve loss of dopaminergic (DA) neurons in the midbrain GeneBc and environmental Autosomal Recessive Early-Onset Parkinson s Disease PINK1, locus PARK6, on Human Chromosome 1p35-p36, Valenta et al., 2006 Parkin, locus PARK2, on Human Chromosome 6q25.2-q27, Kitada et al., 1998

12 PINK1 and Parkin Inhibit Mitochondrial MoBlity in Rat Axons Retrograde crtl Anterograde Mito-dsRed PINK1 Overexpression Mito-dsRed Parkin Overexpression Mito-dsRed Rat hippocampal axons Scale bars=10 microns

13 PINK1-dependent Mitochondrial Arrest Requires Parkin Overexpression of PINK1 no longer stops mitochondria in Parkin-/- axons Retrograde Mito-dsRed Parkin+/+ Parkin+/+; Anterograde Mice hippocampal axons Scale bars=10 um Drs. J. Schlehe and M. LaVoie PINK1 Overexpression Mito-dsRed Mito-dsRed Mito-dsRed Parkin-/- Parkin-/-; PINK1 Overexpression

14 PINK1 and Parkin Inhibit Mitochondrial MoBlity in Fly Axons Retrograde All axons have CCAP-G4, UAS-mitoGFP Control Anterograde 10 um UAS-PINK1 UAS-PINK1RNAi UAS-Parkin UAS-ParkinRNAi Fly third instar larvae

15 Live cell imaging is crucial to understanding the fundamental regulabons of organelle movement!

16 Kymograph * a graphical representabon of spabal posibon over Bme in which a spabal axis represents Bme.

17 Kymograph 10um

18 Step 1: How to make Kymograph Using Image J?

19 ImageJ/FiJi is freely downloadable. hup://imagej.nih.gov/ij/ hup://fiji.sc/downloads MulBStackReg Plugin if interested in Drim CorrecBon hup://bigwww.epfl.ch/thevenaz/turboreg/

20 Load live-imaging file in ImageJ Plug-in: Bio-Formats Importer

21 Select neuron of interest and straighten curved axon Plug-in: Straighten

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24 Reslice the image from top to bouom (Bme) Image!Stacks!Reslice

25 Image!Stacks!Z project

26 AutomaBcally Making Kymograph Many processes can be automated by recording and saving the steps in a process. To start, open the Macros Plugin and Record the steps in a Process

27 To record, go through each step in the process

28 Once all steps are recorded, hit Create

29 To make kymograph, add a line to your video and hit Run Once all steps Once are complete, recorded, hit Create.

30 Drim CorrecBon Mitochondria; mitodsred Neuron; GFP

31 MulBStackReg Allows for the automabc alignment of a source image or a stack (green channel) to a target image (red channel)

32 Open movie and split channels

33 Run MulstStackReg for source image (green channel)

34 Check Save TransformaBon File, hit OK, and save the file to Desktop. The transformed file will turn white.

35 Click on the target image and apply the transformabon file to it.

36 Then, load the transformabon file to apply to the target channel. Hit OK.

37 Result: Corrected Save and ublized this corrected file for analysis.

38 Saving Lines using ROI Manager Open ROI Manager

39 Change to Segmented Line

40 Add line to ROI Manager

41 Add addibonal lines

42 Rename lines

43 Rename lines

44 Highlight lines and save by going to More and Save

45 Open new image and apply line by clicking on the line name

46 Click on white dot to adjust line if necessary. Update to save changes.

47 Step 2: How to analyze movement parameters using kymographs? => with Bme and distance we can calculate velocity, pausing, etc.

48 Principles for our self-wriuen macro 2s 0.1um Draw a line in the middle of a mito, allocate 11 points equally along the line, calculate instantaneous velocity for each point. Resize kymograph: x: 1 pixel=0.1um; y: 1 pixel=2 seconds, total 50 pixel

49 Matlab scripts -direcbons (+ anterograde; - retrograde) -instantaneous velocity for each point -average velocity in each direcbon -percentage Bme in mobon in each direcbon= number of moving points/11 -stop frequency=number of {n moving, (n+1) zero}/11 -turn back frequency=number of {n and (n+1) change direcbon}/11

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51 Draw a line in the center of the white line (one mitochondrion) Click Finish this line

52 Finish all mitochondria Remember to click Finish this line one by one Click Finish this figure

53 A Microsom Excel file will pop up 6 parameters of mitochondria moblity will be showed average velocity (um/s): anterograde/retrograde % of Bme in mobon: anterograde/retrograde stop frequency turn back frequency

54 PINK1 OE OE OE OE Parkin Mitochondrial Arrest OE OE OE OE

55 Acknowledgement