Characterisation of senescence entry point using long-lived ...

HAL Id: tel-03398723https://tel.archives-ouvertes.fr/tel-03398723

Submitted on 23 Oct 2021

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Characterisation of senescence entry point usinglong-lived mutants and intervention related to ageing

factors ERC and ROSJia Song

To cite this version:Jia Song. Characterisation of senescence entry point using long-lived mutants and intervention re-lated to ageing factors ERC and ROS. Biophysics. Université de Strasbourg, 2020. English. �NNT :2020STRAJ008�. �tel-03398723�

https://tel.archives-ouvertes.fr/tel-03398723

https://hal.archives-ouvertes.fr

UNIVERSITÉ DE STRASBOURG

ÉCOLE DOCTORALE SCIENCES DE LA VIE ET DE LA SANTE

[ Institut de Génétique et de Biologie Moléculaire et Cellulaire ]

THÈSE présentée par :

[ Jia SONG ]soutenue le : 26 Février 2020

pour obtenir le grade de : Docteur de l’université de Strasbourg

Discipline/ Spécialité : Biophysique

THÈSE dirigée par :[ M. Gilles CHARVIN ] Directeur de recherche, université de Strasbourg

RAPPORTEURS :[ Mme. P-Y.J. WU ] Directeur de recherche, Université de Rennes 1 [ M. Z. XU ] Chargé de recherches, Université de Sorbonne

AUTRES MEMBRES DU JURY :[ M.M. MENDOZA ] Chargé de recherches, IGBMC

Caractérisation de l'entrée en sénescence en utilisant les mutant de longévité et

l'intervention concernant les facteurs de vieillissement ERC et ROS

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)/*)% *"'% =KB% -/+% '1'/*2-..5% .'-+% *)% &=KB% &'('*#*#1'% 3'/'% 'T,#$#)/%8"'/% =KB% ,)..#$#)/%

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*"-*%J;G%8#..%9'%&'(.#,-*'+%7)&%'-,"%,'..%,5,.'%9',-2$'%)7%#*$%;B>%&'(.#,-*#1'%)&#3#/:%!"'&'7)&'6%

*"'%'T,#$'+%J;G%4-5%9'%-,,242.-*'+%'T()/'/*#-..5%-7*'&%'-,"%,'..%,5,.'% YD#32&'%S:RZ:%!"'%

4-#/$*&'-4%"5()*"'$#$%)7%J;GX#/+2,'+% *)T#,#*5%("'/)4'/)/% #$%9-$'+%)/% *"'% *#*&-*#)/%)7%

*&-/$,&#(*#)/-.%7-,*)&$%8"#,"%$(-&'%'T,'$$#1'%&'32.-*)&5%(&)*'#/$%)/%J;G%-/+%'1'/*2-..5%#*%

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regulation and its known to induce DSB and ERC formation. It has been reported that fob1Δ

42*-/*%$")8%.'$$%J;G%-,,242.-*#)/%+2'%*)%.'$$%J;G%7)&4-*#)/%-/+%.)/3'&%;A>%,)4(-&'%*)%

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!"'&'%-&'%-9)2*%QW_XR__%*-/+'4%,)(#'$%)7%&=KB%3'/'$6%5'*%/)*%-..%)7%*"'4%-&'%-,,'$$#9.'%7)&%

*&-/$,&#(*#)/:% !"#$% -,,'$$#9#.#*5% #$% 2/+'&% *"'% &'32.-*#)/% )7% ,"&)4)$)4'% $*&2,*2&'% -/+%

72/,*#)/6%4)&'%(&',#$'.5%*"'%'(#3'/'*#,%"#$*)/'%()$*X*&-/$.-*#)/-.%4)+#7#,-*#)/$6%$2,"%-$C%95%

,)1-.'/*%4)+#7#,-*#)/%)/%"#$*)/'%&'$#+2'$%$2,"%-$%-,'*5.-*#)/%)&%+'-,'*5.-*#)/%)/%.5$#/'%'*,:%

!"#$%&'?)<'K=-&9+1"='[SE'9.0&=$0&'"2':.1-'4+$#-1&%'+24'+#&4'9.1-&%'=&00;'

!"'%$,"'4-*#,%&=KB%-/+%J;G%.),-.#U-*#)/%7#32&'%#/%+-23"*'&%,'..%-/+%-3'+%4)*"'&%,'..:%

!"'%9.2'%9.),<%#/+#,-*'$%*"'%&#9)$)4-.%,)+#/3%3'/':%!"'%&'('*#*#1'%&=KB%.),#%.),-*'%#/%

*"'%/2,.').2$%&'3#)/:%B$%*"'%4)*"'&%-3'$6%4)&'%-/+%4)&'%J;G$%-&'%7)&4'+%-/+%4-5%

#/+2,'%+'-*"%'1'/*2-..5:%

,+

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,"&)4)$)4'%&'4)+'.#/3%Y>&#1-$*-1-6%>&#1-$*-1-%'*%-.:%R_Q[Z:

D)&% &=KB% .),#6% #*$%/2,.')$)4'%,-/%9'% &'4)+'.'+%2/+'&% *"'%,)/*&).%)7%'/U54'%'T"#9#*#/3%

"#$*)/'%+'-,'*5.-$'%-,*#1#*5%1#-%>#&R%YE2,<6%>-/+4'#'&%'*%-.:%R__R6%O2-/3%-/+%?)-U'+%R__SZ:%

='.'*#/3%>#&R%3'/'%,-/%&'+2,'%$#.'/,#/3%*"'&'7)&'%#/,&'-$'%*"'%*&-/$,&#(*#)/-.%-,,'$$#9#.#*5%

-&)2/+% *"'% &'('*#*#1'% &=KB% &'3#)/:% !"#$% 'T()$2&'%4-5% 72&*"'&% -77',*% *"'% J;G% 7)&4-*#)/%

2/+'&%*"'%#/7.2'/,'%)7%&=KB%$*-9#.#*5:%0/+''+6%#/%$#&RΔ6%*"'%,'..%'T"#9#*$ "#3"%&=KB%#/$*-9#.#*5%

-/+%&-(#+.5%7)&4%.-&3'%-4)2/*%)7%J;G%Yl)9-5-$"#6%O)&#2,"#%'*%-.:%R__V6%l)9-5-$"#%-/+%>-$-<#%

R_Q\Z:%!"#$%42*-/*%$")8%$#3/#7#,-/*.5%$")&*'/'+%;A>:

Q:Q:S%!"'%&'.-*#)/$"#(%9'*8''/%&=KB`J;G%-/+%>JM%Y8)&<%#/%Y?)&.)*6%>)/3%'*%-.:%R_Q^Z

M&'1#)2$.5%#/%*"'%.-96%*"'%8)&<%)7%=&:%?)&.)*%Y?)&.)*%'*%-.:6%R_Q^Z "-$%&'1'-.'+%*"'%+#&',*%

&'.-*#)/$"#(%9'*8''/%>JM%-/+%*"'%-3'#/3%7-,*)&%J;G:%E5%2$#/3%-%&=KB`J;G%4-&<'&6%$"'%7)2/+%

*"-*%>JM%4-5%9'%-%,)/$'b2'/,'%)7%J;G%-,,242.-*#)/%YD#32&'%S:SZ:

>"'%"-$%2$'+% *"'% &'()&*'&% $*&-#/%)7% J;G`&=KB% *)%4)/#*)&% #*$% ,)(5%/249'&%+2&#/3%-3'#/3%

YD#32&'%S:SZ:%J-,"%&=KB%3'/'%#$%#/$'&*'+%8#*"%&'('*#*#1'%A-,@%$'b2'/,'%$)%*"-*%A-,0XNDM%,-/%

9#/+%*)%*"'%&=KB`J;G%4).',2.'%-/+%&'1'-.%3&''/%7.2)&'$,'/,'%$#3/-.:%!"'%J;G%-,,242.-*#)/%

!"#$%&' ?)?' K[\' ";' +' =.2;&]$&2=&' ./' [SE' +==$9$0+1".2' %&7&+0&4' :A' [SEPC+=`' a' *!\PC+=U'

%&,.%1&%';1%+"2'

B%$,"'4-*#,%7#32&'%)7%J;G%-,,242.-*#)/%(&#)&%*)%*"'%>JM%)1'&%*"'%,)2&$'%)7%-3'#/3:%!"'%3&''/%

U)/'%&'(&'$'/*$%/2,.').-&%&'3#)/%)7%/2,.'2$q%*"'%9.-,<%+)*%#/+#,-*'$%*"'%J;G%4).',2.':%!"'%

&'()&*'&%$*&-#/%2$'+%7)&%&=KB`J;G%#$%$")8/%)/%*"'%7#32&':%B$%*"'%J;G%,)(5%/249'&%&'-,"'$%

-%,'&*-#/%*"&'$").+6%*"'%>JM%"-(('/$%-/+%'1'/*2-..5%*"'%,'..%+'-*":%

,,"

$*-&*$%$")&*.5%-7*'&%9#&*":%c"'/%*"'%b2-/*#*5%)7%J;G%&'-,"'$%-%*"&'$").+6%>JM%#$%*&#33'&'+:%0/%

-++#*#)/6%*"'%$*-*#$*#,-.%'$*#4-*#)/%)7%7.2)&'$,'/,'%$#3/-.%#/+#,-*'$%-/%'T()/'/*#-.%#/,&'-$'%

8#*"#/%'-,"%+#1#$#)/:%!"#$%)9$'&1-*#)/%$233'$*$%*"-*%7.2)&'$,'/,'%$#3/-.%#/,&'4'/*%#$%#/+''+%

#$$2'% 7&)4% *"'% 'T*&-,"&)4)$)4-.% J;G% 3'/'% ,)(#'$:% !"'&'7)&'6% )/,'% J;G% #$% 'T,#$'+6% *"'%

+2(.#,-*#)/%$*-&*$%-$%,'..%-3'$:%J1'/*2-..56%-*%*"'%'/+%)7% .#7'$(-/6%*"'%b2-/*#*5%)7%J;G%,-/%

&'-,"%-%$#U'%)7%Q:[X7).+%)7%5'-$*%3'/)4':%

@/'%)7%*"'%42*-/*$%$")8#/3%.'$$%J;G%-,,242.-*#)/%#$%*"'%.)/3Xlived fob1Δ mutant. As the

>JM%#$%&'.-*'+%*)%J;G%-,,242.-*#)/6%8"'*"'&%>JM%#$%+'.-5'+%-/+%")8%*"'%>JM%9',)4'%#/%

fob1Δ mutant can be asked.%

Q:R%;'$2.*%

Q:R:Q%D)9Q%42*-/*%$")8$%(&).)/3'+%;A>%#/%4#,&)7.2#+#,%$5$*'4%

='.'*#/3%D)9Q%(&)*'#/%.'-+$%*)%;A>%'T*'/$#)/6%*"#$%"-$%9''/%&'()&*'+%#/%*"'%.#*'&-*2&'$%$#/,'%

1999 (Defossez, Prusty et al. 1999, Kobayashi 2003). Fob1Δ mutants with and without

"#$*)/'% /2,.'-&% 4-&<'&% YO!ERXNDMZ% -&'% 2$'+% 7)&% *#4'% .-($'% -3'#/3% 'T('&#4'/*:% c'%

confirmed that fob1Δ mutant is longX.#1'+%#/%)2&%4#,&)7.2#+#,%$5$*'4%YD#32&'%S:VZ:%

,&

Q:R:R%M&)9-9#.#$*#,%>JM%'1'/*%95%.#4#*#/3%J;G%'T,#$#)/%#/%7)9QΔ42*-/*

>2&(&#$#/3.56%8'%)9$'&1'%*8)%+#$*#/ct cell fate in fob1Δ mutant, there are cells die with and

8#*")2*%>JM%YD#32&'%S:WZ:

0/%c!%()(2.-*#)/6%*"'%>JM%#$%*&#33'&'+%#/%4)$*%)7%*"' 4)*"'&%5'-$*6%-9)2*%]Wh%)7%,'..$%+#'%

8#*"%>JM%YKn^_6%8#*"X>JMn\\6%/)XSEP=13) (Figure 3.6). Yet in fob1Δ mutant, up to 63% of

,'..$%YKnW[6%8#*"X>JMnRQ6%/)X>JMnSWZ%+#'%8#*")2*%>JM%*&-/$#*#)/6%*"'$'%,'..$%-&'%*"2$%+'7#/'+%

-$%/)X>JM%$29()(2.-*#)/:%!"'5%$")8%$#3/#7#,-/*.5%.)/3'&%;A>%YVWZ%,)4(-&'%*)%c!%YR\:WZ:%0/%

this fob1Δ noX>JM% $29()(2.-*#)/6% *"'%/2,.'-&% $#U'% #$% &'.-*#1'.5% ,)/$*-/*%-/+%+)%/)*% $")8%

!"#$%&'?)B'!.:( Δ 9$1+21'"24&&4';-.>'0.2#&%'SCK'1-+2'_O'

!"'%&'(&'$'/*-*#1'%-3'#/3%*&-a',*)&5%)7%7)9QΔ%42*-/*%8#*"%/2,.'-&%4-&<'&%#$%$")8/%#/%*"'%

.'7*% (-/'.:% !"'% 4)*"'&% &'(.#,-*#1'% .#7'$(-/% #$% 4'-$2&'+% -/+% ,)4(-&'% *)%c!:% >#3/#7#,-/*%

increase of RLS can be observed (WT (27.5) N=130; fob1Δ (39) N=63).

!"#$%&'?)F'!.:(Δ 9$1+21';-.>'0.2#P0"7&4'9.1-&%'=&00'+#&'>"1-.$1'K[\

;'(&'$'/*-*#1'%7)9QΔ mutant show noX>JM%-3'#/3%(-**'&/:%E)*"%$'b2'/*#-.%#4-3'$%-/+%,'..%

,5,.'%+2&-*#)/%*&-a',*)&5%#$%$")8/%9'.)8:%!"'%-1'&-3'%,'..%,5,.'%+2&-*#)/%#$%�]_%4#/2*'$%

('&%+#1#$#)/%*"&)23")2*%*"'%8").'%.#7'$(-/:%

&("

'/.-&3'4'/*%9'7)&'%,'..%+'-*"%YD#32&'%S:WZ:%!"#$%$233'$*$%*"-*%J;G%,)(5%/249'&$%)7%/)X>JM%

,'..$%4-5%/)*%9'%#/,&'-$'+%-$%#/%8#*"X>JM%()(2.-*#)/:%!"'%4)*"'&%5'-$*%4-5%9'%+#'+%#/%-/%

J;GXfree status. This hypothesis is indeed confirmed by Dr. Morlot’s complementary work

95%+#&',*.5%2$#/3%rDNA/ERC reporter in a fob1Δ mutant. When monitoring the ERC signal

*"&)23")2*%*"'%8").'%.#7'$(-/%#/%/)X>JM%,'..$6%*"'%7.2)&'$,'/,'%$#3/-.%$")8$%-%,)/$*-/*%.'1'.:%

For fob1Δ withX>JM%$29()(2.-*#)/6%*"'&'%#$%S\h%)7%4)*"'&%5'-$*%2/+'&3)%>JM%*&-/$#*#)/%-/+%

they show slightly longer RLS than the WT. I then asked whether the SEP in both fob1Δ and

c!%#$%"-(('/#/3%-*%*"'%$-4'%*#4':%>*-*#$*#,-.%?-//–c"#*/'5–c#.,)T)/%*'$*%7)&%9)*"%72..%;A>%

and SEP are compared between WT and fob1Δ withX>JM%$29()(2.-*#)/%YD#32&'%S:\Z:%

!"#$%&'?)5'Y";1%":$1".2'./'>"1-PK[\'+24'2.PK[\',.,$0+1".2'

!"'%"#$*)3&-4%+#$*&#92*#)/%)7%8#*"X>JM%-/+%/)XSEP population in both WT and fob1Δ mutant.

!"'% .'3'/+% #/+#,-*'$% *"'% ('&,'/*-3'%(&)()&*#)/% )7% *"'% *8)%$29()(2.-*#)/$:%!"'% 9)T(.)*%

7#32&'% #/+#,-*'$% '-,"% $29()(2.-*#)/% &'(.#,-*#1'% .#7'$(-/:% % !"'% .'3'/+% #/+#,-*'$% *"'%4'-/%

|$*-/+-&+%'&&)&%)/%4'-/:%YK}[[Z%

&'"

It seems that SEP is delayed in fob1Δ mutant and the extended SEP mainly contribute to the

.)/3'1#*5%'T*'/$#)/%#/%8#*"X>JM%$29()(2.-*#)/:%!"'%()$*XSEP period of both fob1Δ and WT

+)%/)*%$")8%+#$*#/,*%.#7'$(-/:%D&)4%*"#$%result, two factors contribute the fob1Δ longevity

'T*'/$#)/:%D#&$*.56%*"'%/)X>JM%,'..$%8"#,"%#$%4)$*%)7%*"'%8").'%()(2.-*#)/6%.#1'%$#3/#7#,-/*.5%

.)/3'&q%$',)/+.56%*"'%8#*"X>JM%,'..$%$")8%+'.-5'+%>JM%-.$)%'T*'/+%;A>:%!-<#/3%*)3'*"'&6%#*%

seems that fob1Δ mu*-/*%95%.#4#*#/3%*"'%J;G%7)&4-*#)/%.-&3'.5%&'+2,'%*"'%>JM%(&)9-9#.#*5%

in the population. Two distinct cell fate of fob1Δ population, suggests that ERC excision

&'32.-*'+%95%D)9Q%(&)*'#/%4-5%9'%-%(&)9-9#.#$*#,%'1'/*%+2&#/3%*"'%-3'#/3%(&),'$$:%

!"#$%&'?)J'E.9,+%";.2'./'K[\'.2;&1'+24'SCK'./';$:,.,$0+1".2'

BC%!"'%9)T(.)*%3&-("%)7%8#*"XSEP subpopulation in both fob1Δ and WT. The withX>JM%,'..$%)7%

fob1Δ strain shows slightly longevity extension. !"'%.'3'/+%#/+#,-*'$%*"'%4'-/%|$*-/+-&+%

'&&)&%)/%4'-/:%YK}R_Z%

EC% %The histogram graph of SEP comparison in both fob1Δ and WT. The SEP of fob1Δ

42*-/*%YRVZ%#$%(&).)/3'+%,)4(-&'%*)%c!%YRQZ:%!"'%()$*X>JM%.#7'$(-/%#$%#+'/*#,-.%#/%9)*"%

9-,<3&)2/+$:%%!"'%.'3'/+%#/+#,-*'$%*"'%4'-/%|$*-/+-&+%'&&)&%)/%4'-/:%

&!"

Q:R:S%>JM%,"-&-,*'&#$-*#)/%#/%8#*"X>JM%$29()(2.-*#)/%)7%c!%-/+%7)9QΔ%

!)%72&*"'&%,)4(-&'%*"'%>JM%9'*8''/%c!%-/+%7)9Q%8#*"X>JM%$29()(2.-*#)/:%E)*"%,'..%,5,.'%

+2&-*#)/6% "#$*)/'% 7.2)&'$,'/,'% #/,&'4'/*% -/+% /2,.'-&% ,5*)(.-$4#,% &-*#)% -&'% ,)4(-&'+%

YD#32&'% S:]Z:% 0*% seems that SEP exhibit identical characteristics between WT and fob1Δ

42*-/*:%

!"#$%)9$'&1-*#)/%1-.#+-*'% *"-*%>JM%)9$'&1'+% #/% 7)9QΔ%8#*"X>JM%()(2.-*#)/% #$% .#<'% *"'%>JM%

)9$'&1'+%#/%c!%()(2.-*#)/:%J1'/%*")23"%>JM%#$%$#4#.-&6%*"'%,'..%,5,.'%+2&-*#)/%)7%7)9QΔ%Y]VZ%

#$%.)/3'&%*"-/%c!%Y\[Z%7)&%5)2/3%(&'X>JM%,'..$:%!"#$%+#77'&'/,'%42$*%9'%,&2,#-.%7)&%$2&1#1)&$"#(%

)7%c!%#/%$-1-3'%'/1#&)/4'/*:%0*%"-$%9''/%(&)()$'+%#/%*"'%$*2+5%)7%4#,&))&3-/#$4%*"-*%7-$*%

3&)8*"%)7%*"'%()(2.-*#)/%#$%,&2,#-.%7)&%/2*&#'/*%-/+%$(-,'%),,2(-*#)/%-/+%,)4('*#*#)/:%!"#$%

4-59'%*"'%)/'%)7%*"'%&'-$)/$%*"-*%.)/3X.#1'+%7)9QΔ%42*-/*%,-//)*%)1'&&#+'%c!%$*&-#/:%

!"#$%&'3.8 Comparison of SEP characteristics in fob1Δ and WT'

!"'%'1).2*#)/%)7%,'..%,5,.'%+2&-*#)/6%O!ERXsfGFP and N/C ratio in fob1Δ and WT (aligned by

>JMZ:%!"'%'&&)&%9-&% #/+#,-*'$%|$*-/+-&+%'&&)&%)/%4'-/:%YKnR_%7)&%9)*"%$*&-#/$Z%!"'%>JM%#$%

#/+#,-*'+%95%-%+-$"'+%&'+%.#/'%-/+%.),-*'+%*)%*"'%U'&)%)7%TX-T':%!"'%3&'5%-&'-%#/+#,-*'$%.'$$%

'77',*#1'%-*%.-*'%.#7'%+2'%*)%4)*"'&%,'..%+'-*":%

&#"

R% G"-&-,*'&#U-*#)/% )7% >JM% #/% .)/3X.#1'+%42*-/*$% 95% ('&*2&9#/3% J;G%

4)*"'&X+-23"*'&%$'3&'3-*#)/%

R:Q%>(',#7#,%9-,<3&)2/+%

R:Q:Q% ;'32.-*)&5% '.'4'/*$% 7)&% J;G% -$544'*&#,% $'3&'3-*#)/% 9'*8''/% 4)*"'&% -/+%

+-23"*'&%,'..$%

B$%*"'%/249'&%)7%J;G%#/,&'-$'$6%#*%9',)4'$%,&2,#-.%7)&%*"'%4)*"'&%5'-$*%*)%3'/'&-*'%J;GX

7&''%+-23"*'&$%7)&%8"#,"%*"'#&%-3'$%-&'%&'$'*%*)%U'&)%Yl'//'+56%B2$*&#-,)%'*%-.:%Q^^VZ:%@/'%

)7% *"'% 4',"-/#$4$% *"-*% "-$% 9''/% '.2,#+-*'+% 8"#,"% #/1).1'+% #/% *"'% J;G% -$544'*&#,%

$'3&'3-*#)/%#$%95%*'*"'&#/3%*)%4)*"'&%/2,.'-&%()&'%,)4(.'T%YKMGZ%2/+'&%*"'%&'32.-*#)/%)7%

>BNB%,)4(.'T%Y='/)*"XA#((2/'&6%l&U5U-/)8$<#%'*%-.:%R_QVZ:%>BNB%,)4(.'T%#$%$")&*%7)&%>(*X

B+-XN,/WXB,'*5.*&-/$7'&-$'% ,)4(.'T:% 0*% #$% )/'% )7% *"'% 4-a)&% *&-/$,&#(*#)/-.% &'32.-*)&%

,)4(.'T'$%7)&%3'/'%'T(&'$$#)/:%!"'%>BNB%,)4(.'T%#$%,)/$*#*2*'+%)7%Q]%$292/#*$%-/+%&'32.-*'%

,"&)4)$)4-.%$*&2,*2&'%95%"#$*)/'%()$*X*&-/$.-*#)/-.%4)+#7#,-*#)/:% 0*$%'/U54-*#,%-,*#1#*#'$%

#/,.2+'%"#$*)/'%-,'*5.-*#)/%95%N,/W%,)4(.'T%-/+%"#$*)/'%+'X29#b2#*#/-*#)/%95%4=IE%4)+2.'%

YD#32&'%S:Q_BZ%Yf-/%-/+%c).9'&3'&%R_QWZ:%

!"#$%&'?)M'Comparison of cell cycle duration between WT and fob1Δ'

The boxplot graph compares the cell cycle duration og WT and fob1Δ mutant. (N=338)%

&$"

0*%"-$%9''/%72&*"'&%2/,)1'&'+%*"-*%#/%*"'%>BNB%42*-/*6%J;G%,-/%-9/)&4-..5%(&)(-3-*'%7&)4%

4)*"'&%*)%+-23"*'&%5'-$*%YD#32&'%S:Q_EZ%Y='/)*"XA#((2/'&6%l&U5U-/)8$<#%'*%-.:%R_QVZ:%!"'$'%

42*-/*$%Y>BNB%,)4()/'/*%+'.'*#/3%42*-/*$Z%.-,<#/3%J;G%-/,")&-3'%,-(-,#*5%'T"#9#*%.)/3'&%

;A>%*"-/%c!:%!"'%-2*")&$%(&)()$'+%*"-*%>BNB%,)4(.'T%$'&1'%*)%'/"-/,'%J;G%-/,")&-3'%)/%

KMG% -/+% &'$*&#,*% #*$% $'3&'3-*#)/% *)% +-23"*'&% ,'..:% 0% *"'/% ,")$'% *"'% >BNB%4=IE%4)+2.'%

mutants: sgf73Δ and ubp8Δ mutants which are known to be longX.#1'+% -/+% "-$% 9''/%

#/1'$*#3-*'+%(&'1#)2$.5%Y?,G)&4#,<6%?-$)/%'*%-.:%R_QVZ:%

J;G%-$544'*&#,%$'3&'3-*#)/%,-/%-.$)%9'%&'32.-*'+%95%*"'%+#772$#)/%9-&&#'&%.),-*'+%*)%*"'%92+%

/',<% 9'*8''/%4)*"'&% -/+% +-23"*'&:% 0*% "-$% 9''/% (&)1'+% *"-*% E2+[X+'('/+'/*% +#772$#)/%

9-&&#'&% $'&1'% *)% ,)/7#/'% J;G% $'3&'3-*#)/% #/%4)*"'&% ,'..% Y>","'(&)1-6% E-.+#% '*% -.:% R__]Z:%

O'/,'6%92+[%"-$%9''/%7)2/+%*)%9'%-%.)/3X.#1'+%42*-/*%+2'%*)%J;G%92+%/',<%(&)(-3-*#)/%

7&)4%4)*"'&%*)%+-23"*'&%,'..$:%

!"#$%&'?)(6'KT*T'=.9,0&H'+24';=-&9+1"='/"#$%&'./'KT*T'9$1+21'+4+,1&4'/%.9'3Y&2.1-P

C",,$2&%D'b%WAW+2.>;N"'&1'+0)'<6(B8'

BC%4=IE%4)+2.'%)7%>BNB%,)4(.'T%Y-+-(*'+%7&)4%G-/U)/'*-%'*%-.:6%R_Q[Z:%

EC%>,"'4-*#,%7#32&'%$")8#/3%J;G%&'*'/*#)/%,-(-,#*5%#/%c!%-/+%>BNB%42*-/*$:%0/%c!6%>BNB%

,)4(.'T%"'.($%*)%*'*"'&%J;G%)/%*"'%KMG%-/+%(&'1'/*%*"'%J;G%(&)(-3-*#)/:%0/%>BNB%42*-/*6%

*"'%J;G%-&'%/)*%*'*"'&'+%*)%KMG6%*"'&'7)&'%(&)(-3-*#)/%)7%J;G%*)%*"'%+-23"*'&%,'..%#$%4)&'%

7&'b2'/*%#/%>BNB%42*-/*:%

&)"

R:R%;'$2.*%

R:R:Q%;A>%-/-.5$#$%7)&%1'&#7#,-*#)/%)7%.)/3X.#1'+%42*-/*$%

B9)1'%4'/*#)/'+%42*-/*$C%$37\SΔ,%ubp8Δ and bud6Δ are monitored for time lapse ageing

'T('&#4'/*:%Bud6Δ mutant shows a similar RLS to WT in our hand (Figure 3.1QZ:%M&)9-9#.#$*#,%

SEP is not observed in this mutant. From statistical test between the bud6Δ and WT, both

;A>%-/+%>JM%-&'%$#4#.-&:%!"#$%42*-/*%+)'$%/)*%$")8%.)/3'&%;A>% #/%4#,&)7.2#+#,%$5$*'4:% 0/%

-++#*#)/6%9)*"%,'..%,5,.'%+2&-*#)/6%>JM%(&)9-9#.#*5%Y(&)()&*#)/%)7%8#*"X>JM%-/+%/)X>JM%,'..$Z%

-&'%$#4#.-&%,)4(-&'%*)%c!:%!"'&'7)&'6%/)%72&*"'&%-/-.5$#$%8-$%('&7)&4'+%#/%*"#$%42*-/*:%

!"#$%&'?)(('RLS and cell cycle duration trajectories in bud6Δ mutant compare to WT'

B: ;A>%$2&1#1-.%,2&1'%#$%)9$'&1'+%#/%92+[Δ 42*-/*%-/+%c!:%YKnW_Z

E: G).)&Xcoded cell cycle duration trajectories of WT and bud6Δ. The probability for SEP onset

Y�]_hZ%-/+%,'..%,5,.'%+2&-*#)/%Y]_%4#/Z%+)%/)*%$")8%$#3/#7#,-/*%+#77'&'/,':

&*"

"

D)&%*"'%)*"'&%R%>BNB%42*-/*$6%;A>%'T*'/$#)/%,-/%9'%4'-$2&'+%YD#32&'%S:QRZ:%!"'%4'+#-/%;A>%

of the three strains WT, sgf73Δ and ubp8Δ are respectively 27.5 (±0.8), 34 (±1) -/+%SQ:W%

(±0.9). Using boxplot graphic to present the RLS and combining with statistical test, sgf73Δ

seems to exhibit more longevity extension compare to ubp8Δ.%

%

%

c"'/% #/1'$*#3-*#/3% *"'%>JM% #/%>BNB%42*-/*$6% 0% 7)2/+%$2&(&#$#/3.5% *"'%/)X>JM% ()(2.-*#)/%

8"#,"% #/+#,-*'% *"-*% >JM% #$% 2/+'&% *"'% &'32.-*#)/% )7% >BNB% 4=IE% 72/,*#)/% -$% 8'..:% !"'%

(&)9-9#.#$*#,% >JM% '1'/*% -4)/3% -..% *"'% 4)*"'&$% -/+% *"'% )/$'*% )7% >JM% #/% 8#*"X>JM%

$29()(2.-*#)/%-&'%-/-.5U'+%YD#32&'%S:QSZ:%

%

%

%

%

%

%

%

%

%

!"#$%&'?)(<'RLS extension observed in sgf73Δ and upb8Δ mutants'

E)T(.)*%3&-("%)7%;A>%)7%c!%-/+%>BNB%42*-/*$:%>BNB%42*-/*$%$")8%'T*'/+'+%;A>:%B1'&-3'%

RLS for sgf73Δ (N=85) is 34 and upb8Δ (N=102). !"'%.'3'/+%#/+#,-*'$%*"'%4'-/%|$*-/+-&+%

'&&)&%)/%4'-/:%>*-*#$*#,-.%*'$*%"-$%9''/%('&7)&4'+6%8"'/%,)4(-&#/3%8#*"%c!%;A>6%$3773Δ

shows significant increase of RLS whereas ubp8Δ shows less significant increase.%

&+"

R:R:R%B.*'&/-*#1'%>JM%+'*'&4#/-*#)/%-((&)-,"%7)&%$37\SΔ%>BNB%42*-/*%

!"'%$()/*-/')2$%.)/3%,'..%,5,.'%+2&-*#)/%&'(&'$'/*'+%#/%&'+%,-/%9'%$''/%#/%,).)&%,)+'+%,'..%

cycle duration trajectories. Comparing SAGA mutants with WT, the both ubp8Δ and sgf73Δ

$''4%*)%$")8%$()/*-/')2$%-/+%'&&-*#,%.)/3%,'..%,5,.'%+2&#/3%'-&.5%-3':%B/+%*"'%4)$*%$'1'&'%

phenotype with spontaneous long cell cycle at early stage is the sgf73Δ mutant. I then focus

on sgf73Δ mutant for further analysis solely, since the two SAGA mutants are involved i/%*"'%

$-4'%'/U54-*#,%72/,*#)/:%K)*%)/.5%,'..%,5,.'%+2&-*#)/%9',)4'%#&&'32.-&6%*"'%5)2/3%4)*"'&%

,'..% $")8$% (&).)/3'+% ,'..% ,5,.'% -$%8'..:%c"'/% -/-.5U#/3% *"'% >JM% 9-$'+% )/% *"'% ,'..% ,5,.'%

$2++'/%'T*'/$#)/6%#*%9',)4'%+#77#,2.*%-/+%#/-,,2&-*'%+2'%*)%#&&'32.-&%-/+%'T*'/+'+%(&'X>JM%

,'..%,5,.'%+2&-*#)/%YD#32&'%S:QVBZ:%

!"#$%&'?)(?'E&00'=A=0&'4$%+1".2'1%+Z&=1.%A'./'_O'+24'KT*T'9$1+21;'

!"'%&'(&'$'/*-*#1'%,).)&Xcoded cell cycle duration trajectories for WT, sgf73Δ and ubp8Δ

mutants (N>36). Erratic and spontaneous long cell cycle in red can be observed in sgf73Δ

42*-/*:%

&,"

0*% -.$)% 8)&*"% /)*#/3% *"-*% 9)*"% ,'..% ,5,.'% +2&-*#)/% -/+% "#$*)/'% (&)*'#/% 'T(&'$$#)/% .'1'.%

#/,&'-$'%2()/%>JM:%c"'/%(.)**#/3% *"'%OR9%"#$*)/'%(&)*'#/% 7.2)&'$,'/,'%'1).2*#)/%)7% *"'%

$-4'%,'..%YD#32&'%S:QVEZ6%*"'%,'..%$")8$%7.2)&'$,'/,'%.'1'.%#/,&'-$'%-$%8'..:%!"'&'7)&'6%9-$'+%

)/%*"'%.#/'-&%&'3&'$$#)/%(&#/,#(.'%)7%>JM%+'*'&4#/-*#)/6%0%+'1'.)('+%-/%-.*'&/-*#1'%4'*")+%

)7%>JM%+'*'&4#/-*#)/%9-$'+%)/%7.2)&'$,'/,'%.'1'.%#/,&'4'/*:%>#/,'%*"'%9-$-.%7.2)&'$,'/,'%

.'1'.% #/,&'-$'%-*%>JM6%*"'%7.2)&'$,'/,'X9-$'+%>JM% #$% #/+#,-*'+%95%9.2'%+-$"'+% .#/'%YD#32&'%

S:QVEZ:%%E-$'+%)/%*"'%>JM%+'*'&4#/-*#)/6%0%()).'+%-..%*"'%(&'XSEP cells and found that sgf73Δ

show much longer cell cycle duration in sgf73Δ mutant (105 ± 15 min) compare to WT (80 ±

Q_%4#/Z:%!"'%-9/)&4-..5%.)/3%,'..%,5,.'%9'7)&'%>JM%,-/%-.$)%9'%-%&'-$)/%*"-*%9.2&%*"'%$2++'/%

,'..%,5,.'%'T*'/$#)/%-/+%#/7.2'/,'%*"'%>JM%+'*'&4#/-*#)/%95%,'..%,5,.'%+2&-*#)/:%

!"#$%&'?)(B'Determination of SEP in sgf73Δ mutant'

BC%G).)&X,)+'%,'..% ,5,.'%+2&-*#)/% *&-a',*)&5%-/+% ,)&&'$()/+#/3%,'..%+#1#+#/3% 7&'b2'/,5%-&'%

(.)**'+% )/% *"'% 7#32&':% J&&-*#,% -/+% 'T*'/+'+% .)/3% ,'..% ,5,.'% +2&-*#)/% ('&*2&9% *"'% >JM%

+'*'&4#/-*#)/% 9-$'+% )/% +#1#+#/3% 7&'b2'/,5:% !"'% &'+% +-$"'+% .#/'% #/+#,-*'$% *"'% .#/'-&%

regression using previously described ‘findSEP’ code. The SEP found at ‘0’ is indicated by bold

&'+%+-$"'+%.#/':%

EC%O!ERX$7NDM% 7.2)&'$,'/,'% $#3/-.%'1).2*#)/%+2&#/3%-3'#/3%)7% *"'% $-4'%,'..:% J-,"%-$*'&#$<%

#/+#,-*'$%-%,'..%+#1#$#)/:%!"'%9.2'%+-$"'+%.#/'%#/+#,-*'%*"'%>JM%+'*'&4#/'+%95%7.2)&'$,'/,'%

#/,&'-$'% 9&'-<#/3% ()#/*6% *"'% &'+% .#/'% #/+#,-*'% *"'% >JM% +'*'&4#/'+% 95% ,'..% ,5,.'% +2&-*#)/%

9&'-<#/3%()#/*:%

&&"

!"'%+',)2(.#/3%)7%OR9%(&)*'#/%-,,242.-*#)/%-/+%,'..%,5,.'%+2&-*#)/%2()/%>JM%4-5%9'%+2'%*)%

loss of random ERC copies in sgf73Δ mutants. It seems that ERC accumulation is more tightly

.#/<#/3%*)%*"'%"#$*)/'%(&)*'#/%.'1'.%&-*"'&%*"-/%*"'%,'..%,5,.'%+2&-*#)/:%

R:R:S%>BNB%42*-/*%'T*'/+%;A>%(-&*.5%95%(&)4)*#/3%3'/)4'%$*-9#.#*5%

0% *"'/% 7),2$% )/% $gf73Δ to investigate the SEP dynamic in withX>JM% $29()(2.-*#)/:%

Surprisingly, sgf73Δ mutants also show SEP probabilistic event .#<'%the fob1Δ strain. Both noX

>JM%-/+%8#*"X>JM%$29()(2.-*#)/$%,-/%9'%)9$'&1'+%YD#32&'%S:QWZ:%!"'&'%-&'%-9)2*%W^h%,'..$%

$")8#/3% >JM% -/+% VQh% 8#*")2*% >JM:% !"'% /)X>JM% $29()(2.-*#)/% YS[:\|Q:SZ% $")8$% $#4#.-&%

.#7'$(-/%.)/3'1#*5%*)%*"'%c!%/)X>JM%$29()(2.-*#)/%YS\|Q:\Z:%B/+%*"'%8#*">JM%$29X()(2.-*#)/%

-3-#/%$")8$%$.#3"*.5%#/,&'-$'+%.#7'$(-/%*"-/%*)%*"'%c!:%0%*"'/%-$<'+%")8%*"'%>JM%,"-/3'+%#/%

sgf73Δ mutant, whether the preX>JM%('&#)+%)&%*"'%()$*X>JM%('&#)+i%

'(("

D&)4%*"'%9)T(.)*6%*"'%&'(.#,-*#1'%.#7'$(-/%+)'$%/)*%$''4%*)%9'%'T*'/+'+%#/%-%.-&3'%$,-.':%!"'%

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When looking at the SEP onset, the SEP of sgf73Δ is .#<'%c!%YD#32&'%S:Q[Z:%D&)4%$*-*#$*#,-.%

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From previously study that sgf73Δ extend RLS via Sir2 function by reducing rDNA

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the fob1Δ mutant with very low ERC formation rate exhibiting much higher proport#)/%)7%

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whi5 into the daughter cell and undergo fast cell cycle duration for being ‘rejuvenated’.%

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A)/3'1#*5%'T*'/$#)/%)9$'&1'+%#/%J;GXrelated mutants (fob1Δ, SAGA mutants) prolong RLS by

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compare to WT yet was not ‘evolutionary selected’. It must be pointed out that two main

resources that are necessary for microorganisms’ survivorship: nutrient and space (Ghoul

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than WT. I proposed two reasons for ‘not been selected’ compare to WT. Firstly, by shorter

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(Huh et al., 2003). fob1Δ, tsa1Δ strains were purchased from Euroscarf%-/+%sgf73Δ, ubp8Δ,

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l-/G% !NB!!!!NB!NBGNBNGN!BB!% ='.'*#/3%,)..',*#)/%l-/?pV%

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fob1Δ_C% !!GG!BG!B!GBBBG!G!NNBB!GN%fob1Δ mutant verification%

fob1Δ_D% NGG!G!!N!BB!B!!N!!GBBNNBB%

sgf73ΔXD% BB!NGBBNBB!GBBB!GBBNB!BNG%sgf73Δ mutant verification%

sgf73ΔX;% !NNGB!!!B!BBBBB!!G!BGGGBB%

ubp8ΔXD% G!GG!!NNBBBN!!G!B!!N!N!N!%ubp8Δ mutant verification%

ubp8ΔX;% !BBBNBBBBBGB!NB!NNBBBGNB!%

bud6ΔXD% BNBG!!NNN!BBNNGB!!BNBBBG!%bud6Δ mutant verification%

bud6ΔX;% !B!NGBB!!!GB!!GN!!G!!BGBB%

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2013). The budding yeast with an average diameter around 4μm can be trapped at the tip of

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,)1-.'/*.5%9)2/+%to the coverslip glass., The cell trapping area presenting a height of 3μm

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