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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