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A Quantitative Method to Track Protein Translocation Between Intracellular Compartments in Real-Time in Live Cells Usin !

ei hted Local "ariance Ima e Anal#sis

Abstract The enetic e$pression o% cloned %luorescent proteins coupled to time-lapse %luorescence microscop# has opened the door to the direct visuali&ation o% a wide ran e o% molecular interactions in livin cells' In particular( the d#namic translocation o% proteins can now )e e$plored in real time at the sin le-cell level' *ere we propose a relia)le( eas#-toimplement( +uantitative ima e processin method to assess protein translocation in livin cells )ased on the computation o% spatial variance maps o% time-lapse ima es' The method is %irst illustrated and validated on simulated ima es o% a %luorescentl#-la)eled protein translocatin %rom mitochondria to c#toplasm( and then applied to e$perimental data o)tained with %luorescentl#-la)eled he$okinase , in di%%erent cell t#pes ima ed )# re ular or con%ocal microscop#' The method was %ound to )e ro)ust with respect to cell morpholo # chan es and mitochondrial d#namics -%usion( %ission( movement. durin the time-lapse ima in ' Its ease o% implementation should %acilitate its application to a )road spectrum o% time-lapse ima in studies'

Introduction Proteins are o%ten motile( shuttlin )etween di%%erent su)cellular destinations to per%orm various %unctions' "isuali&ation and +uanti%ication o% a protein/s su)cellular location and movement over time is there%ore important %or understandin how proteins %unction inside cells' Traditional approaches %or monitorin protein translocation have relied on )iochemical methods to measure protein levels in various cellular compartments over time -e' ' western )lottin su)cellular %ractions at se+uential time points.' !hile these approaches have produced seminal advances in elucidatin molecular mechanisms o% protein %unction( the# also have limitations' 0or e$ample( the# are not suita)le %or trackin protein translocation in sin le cells( limitin kinetic anal#sis to populations o% cells' The# also have limited time resolution due to tissue destruction re+uired %or )iochemical assa#s at each time point' In addition( protein locali&ation ma# )e sensitive to the concentration o% c#tosolic ions and meta)olites that are likel# to )e lost durin the %ractionation procedure ' The development o% techni+ues to la)el proteins with eneticall#-encoded

%luorescent ta s( com)ined with advances in live cell %luorescent microscop#( has circumvented man# o% these limitations' In particular( these new techni+ues permit direct visuali&ation o% )iochemical processes in livin cells in real1time' A current challen e is to couple ima e processin techni+ues with statistical and computational tools to

interpret and e$tract +uantitative in%ormation %rom the vast amounts o% unstructured ima e data enerated )# time-lapse ima in e$periments' *ere we demonstrate a relia)le and eas#-to-implement +uantitative ima e

processin method to assess protein translocation )etween su)cellular compartments in livin cells( )ased on the computation o% the spatial variance o% time-lapse microscope ima es( which minimi&es user-introduced )iases' To demonstrate the use%ulness and advanta es( we %irst validated the method usin simulated ima es and then applied the techni+ue to anal#&e the translocation o% %luorescentl#-la)eled he$okinase -*2.( a ke# l#col#tic en&#me which shuttles )etween the c#toplasm and mitochondria' Currentl#( translocation o% %luorescentl#-la)eled proteins )etween intracellular compartments is most commonl# +uanti%ied as ratio o% %luorescence intensit# )etween two user-de%ined intracellular re ions o% interest -R3I. in microscop# ima es' In the case o% *2( an R3I with a hi h concentration o% mitochondria is compared to an ad4acent R3I with %ew mitochondria ' !hile this method o% measurement is enerall# use%ul( it su%%ers %rom three ma4or draw)acks5 -i. The choice o% the R3I is ar)itrar# and su)4ect to investi ator )ias -ii. Appropriate R3I can onl# )e de%ined i% the discrete or anellar compartments are easil# identi%ia)le and separa)le in the ima es( as( %or e$ample( in Chinese *amster 3var# -C*3. cells or neonatal cardiac m#oc#tes in which mitochondria are concentrated in the perinuclear &one and sparse elsewhere' *owever( the method is pro)lematic %or cell t#pes with a uni%orml# distri)uted or anellar network( such as the mitochondrial network in adult cardiac m#oc#tes' -iii. The R3I is also sensitive to chan es in cell shape and mi ration o% or anelles throu hout the cell durin the time course o% the e$periment( makin read4ustment o% the R3I necessar# to avoid error' The spatial variance method descri)ed herein minimi&es all o% these shortcomin s'

Methods

Ethics statement. This stud# was approved )# the UCLA Chancellor6s Animal Research Committee -ARC ,778-798-,8B. and per%ormed in accordance with the :uide %or the Care and Use o% La)orator# Animals pu)lished )# the United ;tates <ational Institutes o% *ealth -<I* Pu)lication <o' =>-,8( revised ?@@9. and with UCLA Polic# @@7 on the Use o% La)orator# Animal ;u)4ects in Research -revised ,7?7.'

Cell preparation. Animals were anestheti&ed with ,A iso%lurane' Ade+uac# o% anesthesia was assessed )# monitorin the respirator# rate as well as the loss o% response to toe pinch' Animals were then in4ected with sodium pento)ar)ital -?77m Bk ( i'p'. and hearts were rapidl# removed to isolate ventricular m#oc#tes' <eonatal rat ventricular m#oc#tes -<R"M. were en&#maticall# isolated )# standard methods ' Brie%l#( hearts harvested %rom ,- to 8-da#-old neonatal ;pra ue-Cawle# rats were di ested with colla enase -7'7,AD !orthin ton Biochemical Corp( Lakewood( <E. and pancreatin -7'79AD ;i maAldrich( ;t' Louis( M3.' M#oc#tes were isolated with the use o% a Percoll -Pharmacia Biotech AB( Uppsala( ;weden. radient and plated on 8> mm lass )ottom culture dishes' Adult rat ventricular m#oc#tes -AR"M. were en&#maticall# isolated %rom the hearts o% 8-to F-month old male 0isher rats as descri)ed previousl# ' Brie%l#( %ollowin anesthesia( hearts were removed and per%used retro radel# at 8G HC in Lan endor%% %ashion with nominall# Ca,I-%ree T#rode6s )u%%er containin ?', m Bml colla enase t#pe II -catalo num)er F?G9D !orthin ton. and 7'?, m Bml protease t#pe JI" -catalo

num)er P>?FGD ;i ma. %or ,>1,= min' A%ter washin out the en&#me solution( hearts were su)se+uentl# removed %rom the per%usion apparatus and entl# a itated to

dissociate the m#oc#tes' The Ca,I concentration was raduall# increased to ?'= mmolBL

over 87 min' This procedure t#picall# #ielded F7197A o% rod-shaped( Ca ,I-tolerant m#oc#tes that were then plated on 8> mm lass )ottom culture dishes' <R"M and AR"M were cultured in CMKM hi h -,>mM. lucose medium supplemented with 9A -vBv. %etal )ovine serum -0B;.( penicillin -?77 unitsBml.( streptom#cin -?77 unitsBml. and ,mM lutamine.

Gene expression. Rat *2I and *2II were enerousl# provided )# Cr' E' !ilson' 0usion o% rat *2s to L0P was accomplished )# insertin a Bam *? site at the last amino acid o% the codin se+uence and su)clonin into a modi%ied pKL0P<-?vector -Clontech.' The modi%ied pKL0P<-? carried the mutations Q=92 and A,792' All constructs were su)cloned into the mammalian e$pression vectors utili&in the CM" promoter' 3vere$pression o% *2I and *2II in <R"M and AR"M was achieved with en ineered adenoviruses encodin the constructs' K$pression o% the constructs was

su%%icientl# hi h a%ter 89-F=h -<R"M. and G,-@9h -AR"M. to per%orm microscop# ima in '

Standard and confocal microscopy imaging. ;tandard microscop# ima es were ac+uired usin an 3l#mpus IJG7 inverted microscope -3l#mpus( America Inc'. %itted with an 3l#mpus plan apo 97J( ?'F <'A' oil immersion o)4ective and a cooled CCC camera -Model Quanti$( Photometrics( Tucson( AM.' Ima in !ork)ench so%tware was used %or data ac+uisition' L0P -J0?7F-,. %ilter cu)e was purchased %rom 3me a 3ptical Inc' Con%ocal ima es were ac+uired usin on a Meiss A$iovert ?77 L;M inverted microscope %itted with a 97J water immersion o)4ective -Meiss C-Apochromat 98B?', ! Corr.' Meiss

Pascal > ima e so%tware -Carl Meiss( Inc'( Thornwood( <L. was used %or data ac+uisition'

Solutions and experimental techniques. The )ath solution %or cell ima in consisted o% -in mM. ?F7 <aCl( > 2Cl( ?'? M Cl,( ,'> CaCl ,( ?7 *KPK;( :lucose( ?7( with the p* ad4usted to G', with 23*' 0or the ano$iaBreo$# enation e$periments( the cells were per%used with the same solution containin > mM o% sodium dithionite -an o$# endepletin a ent( . %or ?> min %ollowed )# ?7 min super%usion with the ori inal solution' ;olutions were per%used directl# over the cells usin a ravit# %ed ei ht wa#s per%usion device -!arner Instruments( *amden( CT. with electricall# controlled solenoids -The Lee Compan#( !est)rook( CT.' Input and output o% solution volumes to the recordin cham)er -8> mm lass )ottomed culture dish. were e+uili)rated to maintain constant %low rates and pressures within the recordin cham)er' K$periments were carried out at room temperature -,>oC.'

Numeric treatment and algorithm generation. Ima e anal#sis( al orithm eneration( statistical anal#sis and simulations were implemented in the P#thon pro rammin lan ua e ( usin the <ump# and ;cip# packa es that provide support %or arra#

manipulation and eneral scienti%ic computation respectivel# '

Statistical analysis. QQ-plot and ;hapiro-!ilk tests were per%ormed to assess the normalit# o% the samples under anal#sis' The conventional percentile )ootstrapresamplin approach with ?7777 replications was used %or estimatin @>A CI as well as

e$aminin the si ni%icant di%%erence )etween roups -e%%ect si&e statistics. ' All anal#ses were per%ormed )# su)routines %or )ootstrappin developed in the P#thon pro rammin lan ua e( usin the <ump# and ;cip# packa es( )ased on the code we previousl#

pu)lished ' K%%ects were also anal#&ed with Mann-!hitne# U tests' A P value N7'7> was considered statisticall# si ni%icant'

Supplementary formulas - K+uation o% a two-dimensional :aussian %unction

f ( x, y ) = Ae

( x x0 )2 ( yy0 )2 ) 2 2 2 x 2 y

with A( the amplitude o% the :aussianD $7 and #7( the center o% the :uassian in the plane -$( #.D and O$ and O#( the spreads o% the :aussian in the $ and # directions'

- "olume under a ,C :aussian5 v = A 2 x y

Method overview In cells( mitochondria %orm a network o% discrete or anelles( o)serva)le in microscop# ima es in the %orm o% lo)ules andBor %ilaments when la)eled usin

mitochondria-speci%ic d#es such as TMRM( or e$pressin %luorescentl#-ta ed proteins which locali&e to mitochondria -%i ure ?B.' In the latter case( i% the protein molecules translocate %rom mitochondria to c#toplasm( a concomitant decrease o% the mitochondrial %luorescence and increase o% %luorescence inside the c#toplasm will )e o)served( resultin

in the dilution o% the mitochondrial %luorescence and makin the interior o% the cell more homo eneousl# %luorescent in the ima es' There%ore( in addition to the commonl# used ratio-)ased measurement trackin the %luorescence intensit# chan es over time )etween an R3I containin a hi h concentration o% mitochondria( and an R3I representin the ad4acent c#tosolic area with spare mitochondria ( assessment o% %luorescent protein translocation can )e also handled as a %eatures detection pro)lem( i'e' assessin the disappearance o% the mitochondrial o)4ects' Ima e derivative )ased approaches such as the ;o)el and Cann# operators are amon the most popular ima e processin al orithms usin ed e detection %or o)4ect detection ' *owever( spatial variance mappin -)ased approaches have )een shown to )e superior %or o)4ect detection under conditions o% )lurred or low contrast ima es ' B# comparin the variation in pi$el intensit# %rom re ion to re ion throu hout the entire cell( spatial variance also provides a +uantitative measure o% the overall spatial comple$it# inside a cell' ;ince mitochondria are discrete or anelles( the spatial variance o% re# scale values in a iven window is lar e when %luorescence %rom a protein arises predominantl# %rom mitochondria( and decreases as it is released and di%%uses evenl# throu hout the c#toplasm' In the spatial variance al orithm( the variance operator is a local nei h)orhood operation that calculates the sum o% s+uares o% the )ri htness di%%erences %rom the mean %or the nei h)orhood surroundin each pi$el in the ori inal ima e ' The variance value is small in re ions o% the ima e with uni%orm )ri htness and )ecomes lar e whenever sharp dark-)ri ht transitions occur( which allow eas# detection o% or anelles such as mitochondria -%i ure ?B and C.' In our al orithm( %or each pi$el P$(# in the input ima e

-%i ure ?A.( the variance was calculated %or a iven window si&e usin the %ormula )elow which allows local computations to )e per%ormed simultaneousl# and e%%icientl# within windows o% man# si&es 5

2 window

n i= 1

xi2

(in=1 xi ) n2

where $ and n are the intensit# o% each pi$el and the num)er o% pi$els within the window( respectivel#' In practice( most windows are s+uared( providin uni%orm wei ht to all points within the window area( and &ero wei ht to points outside this area' Computations per%ormed within a s+uared window( however( ma# )e overl# sensitive to ima e points near the ed e o% the window( so that estimates o% ima e properties chan e a)ruptl# as the window is moved across pi$els that represent ima e noise' This e%%ect ma# )e avoided )# measurin properties within :aussian-like windows in which the reatest wei ht is iven to the pi$els near the sample position and pro ressivel# less wei ht is iven to more distant pi$els ' The :aussian outputs a Pwei hted avera e6 o% each pi$el6s nei h)orhood( with the avera e wei hted more towards the value o% the central pi$els' This is in contrast to the mean %ilter6s uni%orml# wei hted avera e' Because o% this( a :aussian %ilter provides entler smoothin and preserves ed es )etter than a similarl# si&ed mean %ilter -this is illustrated in the supplemental %i ure ;?.' There%ore( in our al orithm( the :aussian-wei hted variance -O ,. o% all the pi$els in a chosen window is computed( and the value is attri)uted to the central pi$el -P $(#. o% this window -%i ure ?A.' The window is then moved )# one pi$el( and the operation is repeated( and so %orth %or all the pi$els in the input ima e to o)tain the variance map o% the ori inal ima e -%i ure ?B.'

Results Method validation Simulated images of protein translocation. To test our method( we %irst enerated arti%icial ima es simulatin translocation o% a protein %rom mitochondria to c#toplasm over time -%i ure ,.' Mitochondrial protein-)ound areas were modeled as isotropic or anisotropic ,C :aussians with added random noise( and translocation was simulated throu h redistri)ution o% the volume under the :aussians over the ad4acent area in the ima e -decrease in amplitude and increase in width( so that the volume is kept constant. -%i ure ,.' In our e$perimental ima es( the mitochondria are appro$imatel# > pi$els wideD there%ore( in our computational ima es -87$87 pi$els.( we modeled mitochondria with 8 to G pi$el-wide ,C :aussians -%i ure ,.' Computation o% the spatial variance %or the se+uence o% ima es in %i ure ,C simulatin protein dissociation %rom mitochondria is presented in %i ure 8A' In our

al orithm( the variance map command computes a map o% the input ima e( where the intensit# o% a pi$el in the output map represents the variance within the pi$els window centered at the correspondin pi$el o% the input ima e' This operation is applied to all the pi$els o% the ori inal ima e throu h a local nei h)orhood operation' ;ince mitochondria are discrete or anelles( the spatial variance is lar e when %luorescence arises predominantl# %rom mitochondria -%i ure 8A( %irst ima e.' As the protein is released and di%%uses evenl# throu h the c#toplasm( intensit# o% the variance ima e decreases and o)4ects )ecome less and less detecta)le in the ima es -%i ure 8A( su)se+uent ima es.' Calculation o% the mean o% the variance values %or each ima e allows to +uanti%# the

decrease in variance si nal as protein translocates to the c#toplasm over time -%i ure 8B.' Cissociation time constant can )e then e$tracted %rom the data -%i ure 8B.' To compensate %or arti%acts arisin %rom )leachin o% the preparation( the variance o)tained %or each ima e is normali&ed to the total amount o% %luorescence in the ima e -supplemental %i ure ;,.' This is 4usti%ied )ecause we are interested in measurin the relative redistri)ution o% a %luorophore )etween di%%erent cellular compartments( rather than the a)solute chan es in %luorescence variance which include )leachin as well as redistri)ution' <ote that ourmethod is more sensitive than the re ion-o%-interest -R3I. ratiometricmethod -ratio o% %luorescence intensit#)etween two intracellular R3I( usuall# an R3I on cellular area with hi h concentration o% mitochondria( and an R3I representin the ad4acent c#tosolic area -Carrin ton et al'( ?@@>.. -see supplemental

%i ure ;8A and ;8B.' As shown in %i ure 8C( plottin the values o)tained )# the anal#sis a ainst the %unction used to model the protein translocation reveals that our anal#sis tends to )e more sensitive to small chan es in variance when the variance is hi h' This propert# is an advanta e to detect earl# translocation o% the protein %rom mitochondria' Moreover( the window si&e used to compute the spatial variance o% the ima es does not stron l# a%%ect the output o% the anal#sis( as shown )# the similar results o)tained usin windows %rom si&e 8-)#-8 to ,G-)#-,G' Influence of mitochondrial motility. !e then tested the ro)ustness o% our anal#sis to mitochondrial motilit#( phenomenon commonl# o)served in time-lapse microscope ima in ' In this aim( we enerated simulated ima es in which the location o% the ,C

:aussians has )een randoml# assi ned( without simulatin protein translocation -si&e and width o% the ,C :aussians unchan ed over time.' An e$ample o% se+uence o% ima es recapitulatin the movement o% mitochondria over time that could )e o)served in

e$perimental ima es is presented in %i ure FA' Unlike the ratiometric method( which per%orms poorl# when the o)4ects in the ima es are motile -see supplemental %i ure ;8C.( our al orithm is insensitive to mitochondrial movement and a similar variance is calculated over time %or this se+uence o% ima es -%i ure FB.' 0i ure FC( in which the similar anal#sis has )een e$tended to ?777 simulations with di%%erent de rees o% mitochondrial motilit#( shows that our method is ro)ust toward mitochondrial motilit# as similar spatial variance is o)served %or all the ima es' To compare a :aussian kernel over a unit# kernel to compute the variance in our method( we did a statistical pair-wise comparison usin the Bland-Altman %ramework o% the results o)tained with the two kernels %or di%%erent se+uences o% ima es simulatin protein dissociation %rom mitochondria( without or with mitochondrial motilit#' As seen in the supplemental %i ure ;FA( when the data o)tained %rom the anal#sis o% ?777 se+uences o% ima es simulatin protein dissociation %rom nonmotile mitochondria usin the :aussian kernel are plotted a ainst the data o)tained with a unit# kernel( all the points lie on the e+ualit# line' Moreover( the raphical depiction o% di%%erences )etween paired o)servations %rom the two methods versus their avera e -supplemental %i ure ;FB. shows that( even i% the unit# kernel tends to ive sli htl# hi her values( the calculated mean di%%erence )etween measurements -oran e s+uare in the %i ure. is not si ni%icantl# di%%erent %rom &ero( and the slope o% the linear %it o% the di%%erences is hori&ontal' These results su est that there is a ver# hi h de ree o% a reement )etween the two methods

when onl# protein dissociation is modeled in the ima es' *owever( when the same anal#sis is done on se+uences o% ima es simulatin dissociation o% protein %rom motile mitochondria( the plot o% the pair o% measurements o)tained %rom the two methods deviate %rom the e+ualit# line -supplemental %i ure ;>A.' Moreover( )oth the avera e in di%%erences( indicator o% the constant )ias( and the slope o% the re ression o% di%%erences on means( which is a satis%actor# method %or detectin proportional )ias ( are statisticall# di%%erent %rom &ero -supplemental %i ure ;>.' This over-estimation o% the variance values when usin simulatin the unit# kernel compared to the :aussian kernel to anal#&e ima es protein translocation coupled to mitochondrial motilit# mi ht )e the

conse+uence o% the e$cessive sensitivit# o% the unit# kernel to ima e points near the ed e o% the unit# window ivin ver# hi h values when the simulated movin mitochondria suddenl# enter the window'

Application to experimental data !e validated our method on ima es o)tained %rom C*3 cells( <"RM and AR"M e$pressin he$okinase , -*2,. linked to L0P ' C*3 cells were su)4ected to ano$iaBreo$# enation to induce *2, dissociationBreassociation %rom mitochondria -%i ure >.' The ano$iaBreo$# enation episode was mimicked )# e$posin the cells %or ?> min to a solution containin the o$# en scaven er dithionite -? mM.( %ollowed )# ?7 min super%usion with the control solution -%i ure >.' At the )e innin o% thee$periment( a

lar e %raction o%*2, was )ound tomitochondria( and upone$posure to ano$ia( *2, rapidl# translocated to thec#tosol -%i ure >A.' This e%%ect wasreversi)le as *2, reassociatedwith mitochondriaa%ter reo$# enation -%i ure >A.' Application o% our spatial

variance anal#sis al orithm to the ima es -%i ure >B. allowed us to +uanti%# *2, dissociation -%i ure >C. and re-association -%i ure >C. with mitochondria durin the ano$iaBreo$# enation episode( data %rom which use%ul in%ormation can )e o)tained( such as the hal%-time constant o% dissociationBre-association' ;imilar %indin s were o)tained in <R"M -0i ' 9C( control trace.' In this case( the R3I method also per%ormed reasona)l# well -0i ' 9C( control trace. even i% the sensitivit# o% dissociation detection was lower -7'F7?( @>A CIs Q7'8G( 7'F8R( %or the variance method versus 7',@( @>A CIs Q7',>( 7'8FR( %or the ratiometric method( pN7'7>. -R3I S? and S, noted in the ima es were used respectivel# as area o% hi h mitochondrial densit# and c#toplasmic area to per%orm the ratiometric method.' To show that the measured dissociation was not an arti%act o% the method( we repeated the e$periments o% *2, dissociation in <R"M a%ter e$posin the cells to ano$ic preconditionin ( which prevents *2, dissociation %rom mitochondria ' In this case( no *2, dissociation was o)served in the ima es -0i ' 9B. or detected )# our ima e anal#sis method -0i ' 9C( APC trace. and the R3I ratiometric method -0i ' 9C( APC trace.' A"RM( on the other hand( represent a more challen in settin ( since the dense uni%orml# dispersed mitochondrial network makes de%inin appropriate R3I complicated( re+uirin a small spatial scale which )ecomes sensitive to chan es in cell position( cell morpholo # and mitochondrial mi ration' *2,-L0P was overe$pressed in AR"M and the cells were su)mitted to ano$ia -0i ' GA( lower series o% ima es.' In those e$periments( the spatial variance method -0i ' GB. clearl# identi%ied translocation o% *2, %rom mitochondria to c#toplasm durin ano$ia -0i ' GC.( and allows accurate

+uanti%ication o% the hal%-time %or dissociationBreassociation' In contrast to *2,( *2? is

known to remain )ound to mitochondria durin

meta)olic stresses

and does not

translocate to the c#toplasm upon ano$iaBreo$# enation' !hen *2?-L0P was overe$pressed in A"RM and ano$iaBreo$# enation was applied -0i ' GA( upper series o% ima es.( the spatial variance measurement did not chan e -0i ' GB and GC.' 0inall#( we also demonstrate that the spatial variance method works e+uall# well %or %luorescent pro)es which are loaded into mitochondria )# other means than enetic encodin ' 0i ' = shows an e$ample o% an AR"M in which mitochondria were selectivel# loaded with the %luorescent d#es TMRM -87nM. and calcein-AM' !hen e$posed to Phen#larsine 3$ide -PA3. to induce the mitochondria permea)ilit# transition -MTP.( mitochondria depolari&e causin loss o% TMRM %luorescence %rom the mitochondrial matri$( with little chan e in c#toplasmic %luorescence -0i ' =A( lower panel. -Cue to its ne ative char e( TRMR is concentrated more than a thousand-%old in the matri$ o% polari&ed mitochondria( )ut when released is rapidl# diluted.' !hen calcein leaves the matri$( however( c#toplasmic %luorescence increases as mitochondrial %luorescence decreases -0i ' =A( upper panel.' Cespite these di%%erences in the pattern o% %luorescence chan es( )oth spatial variance measurements #ielded compara)le hal%-times o% dissociation ->'G9min vs >'=8min %or the variance calculated %rom the calcein and TMRM data respectivel#.( indicatin the ro)ustness o% the techni+ue -0i ' =B.'

Discussion

3)tainin

accurate +uantitative data %rom live cell ima es is ke# %or testin

mechanistic h#potheses a)out molecular and cellular processes' 0or e$ample( cellT)ased protein translocation assa#s can )e used to pro)e cellular si nalin pathwa#s that are otherwise di%%icult to stud# usin traditional )iochemical assa#s' !hen ta ed with

%luorescent molecules( translocation o% proteins o% interest can )e appreciated )# visual inspection o% ima es %rom %luorescence microscop#' *owever( these su)4ective impressions can )e challen in to +uanti%#' *ere we developed a relia)le and eas#-toimplement ima e processin method to assess protein translocation )etween or anelles and c#toplasm in livin cells( usin mitochondria as a test or anellar s#stem( )ased on the computation o% the spatial variance o% time-lapse ima es' !e have demonstrated that the method is ro)ust with respect to mitochondrial d#namics -%usion( %ission( movement. and chan es in cell morpholo # durin the time-lapse ima in ' !e validated the method )# +uanti%#in *2, dissociation and re-association %rom mitochondria durin

ano$iaBreo$# enation as well as mitochondria transition pore openin in livin cells( demonstratin pro)es' The spatial variance method has the %ollowin advanta es' -i. The method its use%ulness %or )oth eneticall#-encoded and standard %luorescent

minimi&es user )ias )# eliminatin the need to choose and ad4ust R3I inside the cells as re+uired )# the ratiometric method' Indeed( spatial variance provides a lo)al measure o% spatial %luorescence hetero eneit# which is relativel# insensitive to chan es in or anellar and cellular morpholo #' -ii. The method can )e applied to individual sin le cells' ;in le cell ima in techni+ues overcome the avera in e%%ects inherent in ensem)le

measurements and ena)le characteri&ation o% the )iolo ical varia)ilit# )etween individual cells' -iii. The method is capa)le o% ivin hi h detection responses in the settin o% low

contrast ed es( as illustrated in detectin

the mitochondrial network in low contrast

re ions o% ima es' This is )ecause ed e detection is implemented on the )asis o% local variances rather than on the local radients' As a result( ramp ed es with low variation can )e detected e%%icientl#D QivR The method is readil# applied to the whole cell( providin a lo)al measure o% shi%ts in %luorescence compared to the R3I method or calculation o% the variance alon a sin le line drawn throu h the cell -see %i > o% .' In their recent report( "ena)le and al' -,7?8. showed the pit%alls o% usin spatial avera ed mean %luorescence ima in %or detectin su)cellular )ehavior o% %luorescent pro)es( in particular )ecause avera ed spatial %luorescence cannot readil# assess the de ree o% d#e redistri)ution i% the ima es are not resolved enou h to detail all the compartments ' !hile our method is also )ased on the measure o% a chan e in an avera e( usin the variance instead o% the raw %luorescence still permits detection o% a chan e in compartment even i% the total amount o% %luorophoreBd#e remains unchan ed -0i ure ,B.( as the variance ima es hi hli ht the chan es in distri)ution o% the content in %luorescence )etween di%%erent compartments' In addition( the advanta e o% usin the variance is that it can detect either a redistri)ution o% the %luorophore %rom one compartment to another( or a loss o% the %luorophore' This is demonstrated in the 0i ure =( where the variance method per%orms e+uall# well %or )oth scenarios simultaneousl#( detectin the

redistri)ution o% Calcein and the loss o% TMRM durin the induction o% PTP openin in adult cardiac m#oc#tes' In summar#( the ease o% implementation o% this method should ena)le its application to a )road spectrum o% time-lapse ima in studies' Althou h to date we have onl# validated the method %or the mitochondrial network( we e$pect that it will prove

valua)le %or measurin translocation )etween other or anellar compartments as well'

Acknowledgements !e thank Bernard Ri)alet and ;cott Eohn %or their help%ul discussion'

References

Figures

Fig 1: :aussian wei hted local variance computation process' A. Curin the process( the source pi$el is replaced with a :aussian wei hted variance o% all the pi$els inside the chosen window si&e' Kach pi$el inte er value in the source ima e is multiplied )# the correspondin value in the overl#in :aussian kernel( and the variance o% all the resultin products is computed' The ra# value o% the source pi$el -P $(#. is then replaced )# this :aussian wei hted local variance' This operation is repeated %or each pi$el in the ori inal ima e' B. ;ource ima e and C. correspondin output ima e a%ter application o% the al orithm'

Fig : ;imulation o% he$okinase -*2. translocation %rom mitochondria to c#toplasm' A. Area o% hi h %luorescence arisin %rom mitochondrial *2-)ound was simulated )# a series o% ,C :aussians( whose redistri)ution o% the %luorescence simulates the translocation %rom mitochondria to c#toplasm' B. Curin this process( the hei ht and the width o% each ,C :aussian are chan ed so the volume under the ,C :aussian is kept constant( to simulate a redistri)ution o% the %luorescence over the entire cell' C. Top view o% the simulation o% the translocation o% *2 %rom mitochondria to c#toplasm' A uni%orm random noise is added to enerate the %inal simulation ima es'

Fig !: The local variance al orithm e%%icientl# detects redistri)ution )etween compartments' A. Application o% the wei hted variance map al orithm to the model

illustrated in %i ure ,' As seen in the ima es( the spatial local variance is hi h in the re ions o% hi h %luorescence and decreases over time as the %luorescence is redistri)uted over the whole ima e' B. 0rom the time course o% the chan e in variance( the time constant o% dissociation can )e measured' <ote that this time constant -9'@8 a'u'. is ver# close to the time constant used to model the translocation -G a'u'( %i ure ,.' C. The plot o% the local variance mean o)tained )# the anal#sis a ainst the true de ree o% simulated protein translocation( which reveals that the anal#sis tends to )e more sensitive to small chan es in variance when the variance is hi h( whatever the kernel si&e used %or the anal#sis' This propert# is an advanta e to detect earl# translocation'

Fig ": Ro)ustness o% the method when simulated mitochondria chan e position over time without otherwise redistri)utin their %luorescence' A. K$ample o% a se+uence o%

simulated ima es recapitulatin mitochondrial motilit# )# random assi nment o% the location o% the ,C :aussians( while the si&e and width o% the ,C :aussians are kept unchan ed over time -mitochondrial motilit# )ut no protein translocation.' B. The correspondin computed spatial local variance %or the ima es remains almost constant' C. The ro)ustness o% the method was tested on ima es simulatin ?(777 di%%erent patterns o% mitochondrial motilit# over time' 3verall( the spatial local variance measured %or all the simulations remains constant'

Fig #: *e$okinase , translocation in livin cells e$posed to ano$iaBreo$# enation' A. ;napshot o% %luorescence in C*3 cells were su)4ected to ?> min chemical ano$ia usin the o$# en scaven er dithionite -? mM.( %ollowed )# ?7 min o% reo$# enation' B.

Correspondin spatial variance ima es' C $ D. Plot o% the avera e spatial variance at the various time points( showin the time course o% the dissociation -C. and re-association -C. o% *2, with mitochondria durin the ano$iaBreo$# enation episode' <ote that the measurement time points on di%%erents cells varied( e$plainin the di%%erent num)er o% measurements %or each time point'

Fig %: Ano$ic preconditionin -APC. prevents *2II dissociation %rom mitochondria durin ano$ia in <R"M' A $ B. ;napshots o% %luoresence in <R"M durin ano$ia( illustratin rapid translocation o% *2II-L0P %rom mitochondria to c#tosol -A.( which is prevented i% <"RM are e$posed to 8 short ano$ic preconditionin e$posures -?min each. )e%ore the prolon ed ano$ia episode -B.' C $ D. Time course o% chan es in spatial variance -C. versus the ratiometric R3I method -C.' 0or the ratiometric R3I methods( a R3I with a hi h concentration o% mitochondria -?. was compared to a R3I placed into a re ion with %ew mitochondria -,. as illustrated in the series o% ima es in A U B' <ote that the measurement time points on di%%erents cells varied( e$plainin the di%%erent num)er o% measurements %or each time point'

Fig &: A. 0luorescence snapshots o% A"RM e$pressin *2I-L0P -upper. or *2II-L0P under control conditions and a%ter di%%erent periods o% ano$ia' <ote that the mitochondrial network is ver# dense and uni%orml# dispersed( makin di%%icult to de%ine appropriate R3I i% we wanted to appl# the ratiometric R3I method' B. Correspondin spatial

variance ima es' C' Avera e chan es in spatial variance durin ano$ia( con%irmin that *2,( )ut not *2?( translocates to the c#toplasm durin ano$ia' <ote that the

measurement time points on di%%erents cells varied( e$plainin the di%%erent num)er o% measurements %or each time point'

Fig (: Calcein-)ased assessment o% the mitochondrial permea)ilit# transition -MPT. in live adult cardiac m#oc#tes' A. The mitochondrial network in A"RM was pre-loaded with Calcein-AM and then super%used with TMRM to record mitochondrial mem)rane potential' Upon e$posure to 7'?mM Phen#larsine 3$ide -PA3. to induce MTP( calcein %luorescence redistri)utes within ?7 min to the c#toplasm indicatin MPT -upper series o% ima es.( and TMRM %luorescence concomitantl# decreases dramaticall# indicatin mitochondrial depolari&ation' Under each ima e( the correspondin spatial variance

ima e is reported' B'' Cespite the di%%erences in total %luorescence %or the calcein and TMRM ima es( the correspondin spatial variance ima es predict similar times course o% redistri)ution'

Fig )1: Implementation o% a :aussian window -wei hted kernel. preserves ed es )etter compared to a uni%orml# wei hted windows -unit# kernel.( especiall# %or lar e window si&es' !indow si&es %rom -8$8 pi$els. to -,@$,@ pi$els. were tested'

Fig ) : 0luorescence )leachin correction' A. Linear )leachin over time was simulated on our model )# dividin the re%erence ima e with increasin %actors -# V 7',>$ I ?.' B* D. !ithout normali&in to the total %luorescence o% the ima e( the intensit# o% the

variance map ima es decreases over time -B.( with the values reported in C' C* +. <ormali&in to the total %luorescence o% the ima e allows to correct %or )leachin in the

variance map ima es -C.( as seen in the values -K.'

Fig )!: Comparison spatial variance and ratiometric R3I methods' A. R3I chosen %or the c#toplasmic -?. and mitochondrial -,. area' B. Cetection o% he$okinase -*2. dissociation with the variance -)lack. and ratiometric R3I -)lue. ima e processin methods' C. The spatial variance method is insensitive to mitochondrial movement whereas the ratiometric R3I method is stron l# distorted )# mitochondrial movement'

Fig )": Pair-wise comparison o% the variance mean computed with a :aussian-wei thed -:. and a unit# -U. kernel %rom ima es simulatin translocation o% a protein %rom %i$ed mitochondria to the c#tosol' A. The dashed red line is the line o% e+ualit# on which all points would lie i% the two kernels ave e$actl# the same variance mean %or each ima e' As shown )# data( the unit# and :aussian kernel ive similar results in this scenario where the mitochondria are %i$ed' B. :raphical depiction o% di%%erences )etween paired o)servations %rom the two methods versus their avera e -le%t. and histo ram o% those di%%erences -ri ht.' The mean o% the di%%erences -oran e s+uare and oran e dashed line. is not statisticall# di%%erent %rom &ero -red dashed line.( revealin that there is no constant )ias when usin the unit# kernel to calculate the variance compared to the :aussian kernel' The slope o% the re ression o% di%%erences on means - ra# line. is also non di%%erent %rom &ero( indicatin an a)sence o% proportional )iais' These results su est that

there is a ver# hi h de ree o% a reement )etween the unit# and the :aussian kernel variance computation when measurin the translocation o% %luorophoreBd#es %rom non motile compartments'

Fig )#: Pair-wise comparison o% the variance mean computed with a :aussian-wei thed -:. and a unit# -U. kernel %rom ima es simulatin translocation o% a protein %rom motile mitochondria to the c#tosol' A. The dashed red line is the line o% e+ualit# on which all points would lie i% the two kernels ave e$actl# the same variance mean %or each ima e' As shown )# data( the unit# kernel tends to overestimate the variance compared to the :aussian kernel especiall# %or hi h values o% variance' B. :raphical depiction o% di%%erences )etween paired o)servations %rom the two methods versus their avera e -le%t. and histo ram o% those di%%erences -ri ht.' As shown )# the slope o% the re ression line - ra#. that di%%ers si ni%icantl# %rom &ero -pN7'7>.( usin a unit# kernel instead o% a aussian-wei hted kernel ives a proportional )ias on the measure o% the variance when compartments are motiles' In addition( the mean value %or the di%%erences -oran e s+uare and dashed line. also di%%ers si ni%icantl# %rom 7 -pN7'7>.( revealin a %i$ed -or

Wrelative/. )ias' Those )iases mi ht )e the conse+uence o% the e$cessive sensitivit# o% the unit# kernel to ima e points near the ed e o% the unit# window ivin ver# hi h values when the simulated movin mitochondria suddenl# enter the window'