Quantitative analysis of single molecule tracking in live...

ProfFredericA.MeunierQueenslandBrainInstitute

TheUniversityofQueenslandAustralia

Quantitativeanalysisofsinglemoleculetrackinginlivecells:fromBrownianmotiontofunction

2018WinterSchoolinMathematicalandComputationalBiologyJuly,2018

Frederic A. Meunier | Queensland Brain InstituteClem Jones Centre for Ageing Dementia Research | Single Molecule Neuroscience laboratory

"Progress in science depends on new techniques, new discoveries and new ideas, probably in that order." Sydney Brenner

- Crashcourseonopticalmicroscopy

- Browsethroughthesinglemoleculelocalizationtechniques

- Singlemoleculeanalysisofthemechanismofneurosecretion(exocytosis)

Thislecture

Superresolutionmicroscopy

FluorescencemicroscopyFluorescence microscopy allows visualisation of the distributions of fluorescently-labelledmoleculeswithinasampleinarelativelynon-invasiveandspecificwayincellsortissues.

Oneofthemajorlimitationistheresolutionlimitsetbythediffractionoflight,whichrestrictstheamountofinformationthatcanbecapturedwithstandardobjectives.Thisisoftenreferredtoasthediffractionbarrier,whichrestrictstheabilityofopticalinstrumentstodistinguishbetweentwoobjectsseparatedbyalateraldistancelessthanapproximatelyhalfthewavelengthoflightusedtoimagethespecimen.Basically:twonearbymolecules=1fluorescentblob…)

Super-resolutiontechniques:inthepastfewyears,anumberofnovelapproacheshavebeenemployedtocircumventthediffractionlimit,includingnear-fieldscanningopticalmicroscopy(NSOM),stimulatedemissiondepletionmicroscopy(STED),stochasticopticalreconstructionmicroscopy(STORM)andstructuredilluminationmicroscopy(SIM).Thesetechniqueshaveallachievedimprovedlateral(x-y)resolutiondowntotensofnanometers,morethananorderofmagnitudebeneaththatimposedbythediffractionlimit,buteachmethodhasauniquesetoflimitations.

Single-moleculelocalizationmicroscopy(SMLM)bypassesthelateralresolutionlimitbyseparatingthefluorescenceemittersintimesothattheyAPPEARoneafteranotherstochasticallyinlocationsthataresufficientlyfarapartfortheiraccuratelocalization.Forthispurpose,photoactivatableorphotoswitchablefluorophoresareused,thusenablingthereconstructionofhigh-resolutionimages.

Typically,SMLMexperimentsareeitherperformedinawide-fieldconfiguration,orintotalinternalreflection(TIR).TheadvantageofTIRfluorescencemicroscopyisthattheexcitationoffluorophoresisrestrictedtoabout100nmalongtheaxialplaneandthusreducestheobservedaxialdepth.Twoconceptsthatrelyonphotoswitchingorphotoactivationaredirectstochasticopticalreconstructionmicroscopy(dSTORM)andphotoactivatedlocalizationmicroscopy(PALM).

Singlemoleculelocalisationtechniques

dSTORMThekeyofSMLMistolimitthenumberoffluorophoresdetectedatagiventimetoallowaccuratelocalizationofsingle-moleculesindividually.InthedSTORMapproach,thisisachievedbyoperatingconventionalsyntheticfluorophores(i.e.commerciallyavailable,forexampleasantibodyconjugates)asphotoswitchesinthepresenceofreducingbuffers.

dSTORMexploitsthefactthatmostfluorophoresarepronetoreductionthatpromotetransitintoalong-lived,non-fluorescentradicalorotherreducedstate(egtripletstate).Thetransitionintothis“dark”stateisgovernedbytheintensityoftheexcitationlight(typicallylaserinduced),andthereducingpotentialofthebufferreagents(typicallythiolssuchasmercaptoethylamine).

Theback-transition(orreturnintothe“bright”(orfluorescent)stateisgovernedbyresidualoxygen,thethermalstabilityoftheoff-stateandtheintensityemittedfluorescence(typicallyblue-shiftedtoabout100nmtotheexcitationlight).ThedSTORMconceptallowsreversiblephotoswitchingofmanycommerciallyavailablefluorophoressuchasareAlexaFluor®647,AlexaFluor®532,ATTO647Netc.

PrincipleofdSTORM.Switchingfluorophoresbetweenadarkandabrightstateenablestemporalseparationofnearbyfluorophores.Fittingofpoint-spreadfunctionsofsingleemittersallowsaccuratepositiondetermination.Super-resolutionimagescanthenbereconstructed.

PALMandsptPALM

Lippincott-Schwartz, J. and Manley, S.

Putting super-resolution fluorescence microscopy to work.  Nature Methods 6: 21-23 (2009).  A nice overview of the potential benefits and pitfalls of superresolution imaging with emphasis on PALM and related single-molecule techniques. The authors suggest a set of guidelines for presenting images and point out inconsistencies in the current literature.

Daniel Choquet

Nanoscale dynamic organisation of exocytic molecules

Ravi Kasula

Frederic A. Meunier | Queensland Brain InstituteClem Jones Centre for Ageing Dementia Research | Single Molecule Neuroscience laboratory

Li et al., TiBC in press

Martin et al., 2013, J Cell Sci.

Munc18-1Δ317-333 unable to rescue neuroexocytosis in DKD-PC12 cells

Activation Imaging Tracking

20Hzimaging+trackingwithPALM-traceronMetamorph

DKD cells + Munc18-1-mEos

sptPALMRaviKasula

Kasula et al., 2016, Journal of Cell Biology

Munc18-1WTmEos

sptPALM

Kasula et al., 2016, Journal of Cell Biology

Munc18-1WT Munc18-1LM

ControlStimulated

Munc18-1Δ317-333

PALM autocorrelation analysisYeJinChai

Munc18-1WT

Munc18-1Δ317-333

Munc18-1Δ317-333

Munc18-1WT Munc18-1Δ317-333

EffectofMunc18-1onSyntaxin-1nanoscaleorganisation

Kasula et al., 2016, Journal of Cell Biology

Giannoneetal.,BiophysJ2010

Kasula et al., 2016, Journal of Cell Biology

�33Kasula et al., 2016, Journal of Cell Biology

Munc18-1WT + Sx1 Munc18-1Δ317-333 + Sx1

BoNT/ETeTx

Munc18-1WT + Sx1 Munc18-1WT + Sx1

Kasula et al., 2016, Journal of Cell Biology

Munc18-1 domain 3a hinge loop controls the opening of Sx1 and it engagement into the SNARE complex.

Kasula et al., 2016, J Cell Biol.; Bademosi et al., Nature Communications, 2017; Bademosi et al., Jove, 2018, Bademosi et al., Cell Reports, 2018.

- Syntaxin-1 nanoclusters are controlled by NSF and a-SNAP and the PIP2/PIP3 binding domain KARRA of Sx1A (Bademosi et al., Nature Communications, 2017) and… general anaesthetics (Bademosi et al., Cell Reports, 2018).

Opened bunch hypothesis French kiss hypothesis

Morphing potential VAMP2 binding sites of Munc18-1

Residues Identified:

Munc18-1 A297 Munc18-1 T304

VAMP2

We identified two residues that may m e d i a t e M u n c 1 8 - 1 a n d VA M P 2 interaction, closely resembling Vps33 and Vam3 interaction in yeast. (Baker et al. 2015)

Using this we cloned Munc18-1 A297 and T304 residues with Histidine to create steric hindrance and affect its binding with VAMP2

Munc18-1

Increase of Munc18-1 mobility due to its release from nanodomain confinement in DKO-PC12 cells

In agreement with our previous findings in Kasula et al., 2016.

Activity-dependent release of Munc18-1 from nanodomains

Munc18-1 binding to VAMP2 underpins an activity-dependent release of Munc18-1

Mobility of VAMP2 binding deficient Munc18-1 mutants does not increase after stimulation.

Unpublished data

Syn

taxi

n-1A

+Mun

c18-

1WT

Syntaxin-1A-GFP (uPAINT) + Munc18-1WT-mcherry

-5 -4 -3 -2 -1 0 10.00

0.05

0.10

0.15

0.20

0.00 0.05 0.10 0.15 0.200.00

0.05

0.10

0.15

0.5

0.6

0.7

0.8

0.9

1.0

Syn

taxi

n-1A

+Mun

c18-

1A29

7H

MS

D (µ

m2 )

Freq

uenc

y di

strib

utio

n(fr

actio

ns)

Mob

ile fr

actio

n

Time(s) Log(D)

Syntaxin-1A + Munc18-1A297H

-5 -4 -3 -2 -1 0 10.00

0.05

0.10

0.15

0.20

Syn

taxi

n-1A

+Mun

c18-

1T30

4H

0.00 0.05 0.10 0.15 0.200.00

0.05

0.10

0.15

0.6

0.7

0.8

0.9

1.0

MS

D (µ

m2 )

Freq

uenc

y di

strib

utio

n(fr

actio

ns)

Mob

ile fr

actio

n

Time(s) Log(D)

Syntaxin-1A + Munc18-1T304H

Munc18-1WT undergoes an activity-dependent conformational change leading to Sx1 opening and engagement in the SNARE complex.

Conclusions

Munc18-1 domain 3A extended conformation is probably favoured by VAMP2 binding leading to Syntaxin1A opening. Syntaxin-1A opening therefore occurs within the context of vesicular docking (French kiss hypothesis) which may favour proper templating of the SNARE complex during assembly.

Ravi Kasula Adekunle Bademosi Ye Jin Chai Rachel Gormal Sally Martin Andreas Papadopulos Mahdie Mollazade Callista Harper Pranesh Padmanabhan Anmin (Jeff) Jiang Merce Salla-Martret Merja Joensuu Vanessa Lanoue Tristan Wallis Ailisa Blum Chris Small Isabel Morrow Tong Wang Ramon Martinez Marmol

IMB Brett Collins Rob Parton

IINS Bordeaux: Daniel Choquet, Eric Hozy and JB Sibarita UNSW: Yann Gambin, Kat Gaus VIB-KU Louven: Patrik Verstreken Elsa Lawers

Acknowledgements

PhD and post-doc positions available