Download Xinnan Wang Stanford University School of Medicine

XinnanWang
StanfordUniversitySchoolofMedicine
Cellular Organelles are Highly Dynamic
COS7Cell,MitoTracker
• Fission-fusion,changingmorphologyandlocaBon
• Tomeetever-changingenergydemand
ElectricalSignals
Proteins,
Organelles,
mRNAs
WheelsandEngine:
Kinesin,
Dynein,
Myosin
Tracks:MicrotubulesorAcBn
HowtoStudyAxonalTransport?
LiveImaging!
Axonal Mitochondria
Retrograde
RFP-mito
Anterograde
Heatedstage:
37degree
CO2independent
Medium
hUp://www.labessenBals.com/
hUps://www.microscopyu.com/
GlutamateReceptorAcBvaBon
Ca2+
Glutamate
?
Ca2+
Ca2+
Ca2+
Elevation of Cytosolic Ca++ Inhibits
Mitochondrial Motility
• Calcimycin: a calcium ionophore, used to increase intracellular calcium
Retrograde
Anterograde
RFP-mito
10um
WangandSchwarz,Cell,2009
Parkinson’sDisease
• Secondcommonmovementdisorder
• SelecBvelossofdopaminergic(DA)neuronsinthemidbrain
• GeneBcandenvironmental
AutosomalRecessiveEarly-OnsetParkinson’sDisease
PINK1,locusPARK6,onHumanChromosome1p35-p36,Valentaetal.,2006
Parkin,locusPARK2,onHumanChromosome6q25.2-q27,Kitadaetal.,1998
PINK1andParkinInhibitMitochondrialMoBlityinRatAxons
Retrograde
Anterograde
crtl
Mito-dsRed
PINK1Overexpression
Mito-dsRed
ParkinOverexpression
Mito-dsRed
Rathippocampalaxons
Scalebars=10microns
PINK1-dependentMitochondrialArrestRequiresParkin
OverexpressionofPINK1nolongerstopsmitochondriainParkin-/-axons
Retrograde
Parkin+/+
Anterograde
Micehippocampalaxons
Scalebars=10um
Drs.J.SchleheandM.LaVoie
Mito-dsRed
Parkin+/+; PINK1Overexpression
Mito-dsRed
Parkin-/-
Mito-dsRed
Mito-dsRed
Parkin-/-; PINK1Overexpression
PINK1andParkinInhibitMitochondrialMoBlityinFlyAxons
AllaxonshaveCCAP-G4,UAS-mitoGFP
Retrograde
Control
Anterograde
10um
UAS-PINK1
UAS-PINK1RNAi
UAS-Parkin
UAS-ParkinRNAi
Flythirdinstarlarvae
Livecellimagingiscrucialto
understandingthefundamental
regulaBonsoforganellemovement!
Kymograph
*agraphicalrepresentaBonofspaBalposiBonoverBmeinwhichaspaBalaxis
representsBme.
Kymograph
10um
Step1:HowtomakeKymograph
UsingImageJ?
ImageJ/FiJiisfreelydownloadable.
hUp://imagej.nih.gov/ij/
hUp://fiji.sc/Downloads
MulBStackRegPluginifinterestedinDrimCorrecBon
hUp://bigwww.epfl.ch/thevenaz/turboreg/
•  Loadlive-imaging
fileinImageJ
–  Plug-in:Bio-Formats
Importer
•  Select
neuronof
interestand
straighten
curvedaxon
–  Plug-in:
Straighten
–  Reslicethe
imagefrom
topto
boUom
(Bme)
Image!Stacks
!Reslice
•  Image!Stacks!Zproject
AutomaBcallyMakingKymograph
Manyprocessescanbeautomatedbyrecordingandsavingthestepsinaprocess.
Tostart,openthe“Macros”Pluginand“Record”thestepsinaProcess
Torecord,gothrough
eachstepintheprocess
Onceallstepsarerecorded,hit“Create”
Tomakekymograph,adda
linetoyourvideoandhit“Run”
Onceallstepsarerecorded,hit“Create.”
Oncecomplete,
DrimCorrecBon
Mitochondria;mitoDsRed
Neuron;GFP
•  MulBStackReg
–  AllowsfortheautomaBcalignmentofasourceimageora
stack(greenchannel)toatargetimage(redchannel)
Openmovieand
splitchannels
RunMulstStackReg
forsourceimage
(greenchannel)
Check“SaveTransformaBonFile”,
hit“OK,”andsavethefiletoDesktop.
Thetransformedfilewillturnwhite.
ClickonthetargetimageandapplythetransformaBonfiletoit.
Then,loadthetransformaBon
filetoapplytothetarget
channel.Hit“OK.”
Result:Corrected
SaveanduBlizedthiscorrectedfileforanalysis.
SavingLinesusingROIManager
OpenROIManager
Changeto“SegmentedLine”
“Add”linetoROIManager
“Add”addiBonallines
“Rename”lines
“Rename”lines
Highlightlinesandsaveby
goingto“More”and“Save”
Opennewimageandapply
linebyclickingonthelinename
Clickonwhitedottoadjustlineifnecessary.
“Update”tosavechanges.
Step2:Howtoanalyzemovement
parametersusingkymographs?
=>withBmeanddistancewecancalculatevelocity,pausing,etc.
Principlesforourself-wriUenmacro
0.1um
2s
Drawalineinthemiddle
ofamito,allocate11
pointsequallyalongthe
line,calculate
instantaneousvelocity
foreachpoint.
Resizekymograph:x:1pixel=0.1um;y:1pixel=2seconds,total50pixel
Matlabscripts
-direcBons(+anterograde;-retrograde)
-instantaneousvelocityforeachpoint
-averagevelocityineachdirecBon
-percentageBmeinmoBonineachdirecBon=
numberofmovingpoints/11
-stopfrequency=numberof{nmoving,(n+1)
zero}/11
-turnbackfrequency=numberof{nand(n+1)
changedirecBon}/11
•  Drawalineinthecenterofthewhiteline(onemitochondrion)
•  Click“Finishthisline”
•  Finishallmitochondria
–  Remembertoclick“Finishthisline”onebyone
•  Click“Finishthisfigure”
•  AMicrosomExcelfilewillpopup
–  6parametersofmitochondriamoBlitywillbeshowed
• 
• 
• 
• 
averagevelocity(um/s):anterograde/retrograde
%ofBmeinmoBon:anterograde/retrograde
stopfrequency
turnbackfrequency
PINK1
OE
OE
OE
OE
Parkin
Mitochondrial
Arrest
OE
OE
OE
OE
Acknowledgement