Kuo-Lun Chang SID 22977459 The Effects of Kinesin-5 Cin8 Complementation of Temperature-Sensitive Kinesin-5 Kip1 On Mitotic Phenotype in Saccharomyces

cerevisiae Kuo-Lun Chang A stract! Various CIN8 gene mutants with truncations at C-terminus (CIN8-590, CIN8-871, CIN8955, number enote si!e) were use to estimate the essentia" "ength o# Cin$ %rotein in or er to con#er a regu"ar mitotic %henot&%e' (on-truncate CIN8 e)%ression was etermine to be crucia" in the segregation o# re%"icate genome in mitosis' (*+ , -ra #or assa& was ta.en with an un.nown %rotein against a -S, stan ar cur/e to 0uanti#& its concentration' 1utants an wi" t&%e CIN8 were am%"i#ie through 2C3, %uri#ie an image with 4t-r' 2uri#ie D(, was b& e"ectro%horesis on agarose ge" igeste using 5in III an -am5I a"ong with %"asmi use #or

trans#ormation6 %4724b-5is bacteria" shutt"e, an %Cin$-892-C-9:S(with a ;892 contro"+ #or the e)%ression in &east' Digeste %ro ucts were again %uri#ie through co"umn %uri#ication using <iagen .it' Ligation was one with %hage 74 D(, "igase' Ligate %"asmi s were chemica""& trans#orme to bacteria D55= b& heat shoc.'' 7rans#ormation e##icienc& was measure b& screening with antibiotics' 7rans#orme %"asmi s in D55= were e)tracte an se0uence ' , ma"achite green assa& was ta.en with commercia" .inensin to estimate ,72ase acti/it& o# Cin$' 4)tracte %4724b %"asmi was trans#orme to E.Coli -L3 ce""s through e"ectro%oration #or #uture %rotein in uction, whi"e the C-9:S %"asmi s were trans#orme chemica""& into &east strains #or %henot&%ic ana"&sis' 9o""ow u% e)%eriments wou" inc"u e the in vivo /isua"i!ation o# sub"oca"i!ation o# mutants an wi" t&%e Cin$, the in vitro ana"&sis o# Cin$ chemistr&, the mitotic %henot&%ic ana"&sis o# Cin$ com%"ementation, an transcri%tiona" ana"&sis /ia re/erse-transcri%tion' (2+

Kuo-Lun Chang SID 22977459 "ac#$roun% an% Si$nificance! In S.cerevisiae, KIP1 an CIN8 genes enco e #unctiona""& re un ant .inesin-re"ate %roteins (.inesin-5+ Ki%* an Cin$, each ai e in the stabi"i!ation o# microtubu"es in the mitotic s%in "e' (*+' 4u.ar&otic .inesins were %roteins with ,72ase acti/it&, in which uti"i!e the energ& o# ,72 h& ro"&sis to carr& out essentia" ce""u"ar acti/ities, inc"u ing intrace""u"ar trans%ortation an mitosis' (>+ 4/er& #ami"& o# .inesins share conser/e motor omain, where con#ormationa" change was in uce b& ,72 h& ro"&sis to achie/e its s%eci#ic #unctiona"it&? in terms o# the e)%eriment, Cin$ ha a motor omain near the (-terminus o# the %rotein, there#ore truncations %er#orme at C-terminus wou" not a##ect its #unction e#inite"&'(4+ Ki%* an Cin$ were not irect"& in/o"/e in intrace""u"ar trans%ortation (a .inesin-* characteristic+, an were c"assi#ie as .inesin-5 (.inesin-re"ate %roteins+' 7he two .inesin-re"ate %roteins %artici%ate in mitosis b& cross-"in.ing between microtubu"es in the mitoric s%in "e as a tetramer? #o""owing cross-"in.ing, the ,72 h& ro"&sis ri/en anti-%ara""e" mobi"i!ation o# the motor omain cause the mitotic s%in "e to se%arate, thus achie/ing genome se%aration' (4,$+ 7he im%ortance o# research on Cin$ an Ki%* in S.cerevisiae were their in/o"/ement in eu.ar&otic mitosis, a critica" ce""u"ar acti/it& share b& e/er& mu"tice""uar eu.ar&otic organism' (*+ 3esearches ha shown that mutations an a"terations o# %roteins in/o"/e in the mitotic s%in "e were "i.e"& to cause chromosoma" instabi"it& an tumor in human'(5+ 7here#ore, stu &ing .inesin-re"ate %roteins in S.cerevisiae was an im%ortant %ioneering ste% that wou" %ro/i e resu"ts that cou" be use #or the #uture treatment o# mitosis-re"ate cancer, "eu.emia, an h&%o%"astic 1DS' (7+ In the conte)t o# this %ro@ect, the com%"ementation o# a tem%erature-sensiti/e Ki%* b& Cin$ an the #o""ow-u% %henot&%ic assessment was /er& simi"ar to gene thera%&, in which a thera%eutic D(, was inserte to restore norma" #unction o# human ce""s' Simi"ar"&, etermination o# the crucia" "ength o# Cin$ %rotein to maintain #unctiona"it& %ro/i e an insight #or the characteri!ation o# s%in "e-re"ate isease? resu"ts obtaine cou" be a%%"ie ana"ogous"& to human, gi/ing one a itiona" trace to the #actua" cause behin cancer' 7he in vivo sub"oca"i!ation o# Cin$-892 associate with microtubu"e =-tubi"in-mcherr& obser/e through #"uorescence microsco%& wou" %ro/i e a irect /isua" in#erence, a""owing inter%retation o# re"ationshi% between Cin$ mutant "ength an "oca"i!ation acti/it&'(A+ 7he in vitro %rotein ana"&sis inc"u ing o/ere)%ression b& in uction an so"ubi"it& test wou" i/ersi#& the isease in human, inc"u ing

Kuo-Lun Chang SID 22977459 un erstan ing o# .inesin-re"ate %roteins, an their %ossib"e regu"ations an acti/ities in vivo' Last"&, the stu &ing o# the gene e)%ression o# the gene b& re/erse-transcri%tase 2C3 wou" gi/e in#ormation to transcri%tion o# CIN8 genes un er /arious con ition' (2+ :%on com%"etion o# this %ro@ect, the critica" "ength o# Cin$ to retain its #unctiona"it& in mitosis wou" be etermine ? the e##ects o# Cin$ mutants com%"ementation to ts-Ki%* with res%ect to i##erent a iti/es on mitotic %henot&%es wou" be c"ari#ie ? an the e)act acti/it& o# Cin$ sub"oca"i!ation in association with =-tubi"in wou" be obser/e ' 7hese ata, a"ong with the regu"ation,e)%ression, an transcri%tion characteristic o# CIN8, wou" %ro/i e a com%rehensi/e ma% o# how a .inesin-re"ate %rotein cou" be a"tere or com%"emente , to a##ect mitotic %henot&%e o# S.cerevisiae. 7he goa" o# this %ro@ect was to she "ight to #uture research in mitotic s%in "e-re"ate isease an %athogenesis %athwa&'

Kuo-Lun Chang SID 22977459 "ioinformatics an% $ene e&pression! Kinesins in eu.ar&otic organisms were c"assi#ie into se/era" #ami"ies, each containing the conser/e motor omain an i##erent mo i#ications outsi e the cata"&tic core' 7he i/ersit& o# .inesins might arise #rom its i##erence #rom &nein, another motor %rotein in eu.ar&otic ce""s' D&neins cou" mo/e to either irection on the associate microtubu"e? .inesins, howe/er, ha sing"e irectiona"it& eci e b& the sta". an tai" region outsi e motor omain'(9+ Since the ce""u"ar acti/ities .inesins %artici%ate were not a"wa&s one- irectiona", i##erent t&%es o# .inesins must e)ist to carr& out the s%eci#ic #unctions'(9+ , itiona""&, .inesins were essentia" #or intrace""u"ar trans%ortation, there#ore the tai" omain (cargo bin ing+ must be mo i#ie to ha/e i##erent a##init& to /arious t&%es o# cargos' 5owe/er, a"though .inesins must ha/e i/ersit& in structure to %er#orm i##erent tas., .inesins between i##erent organisms were "i.e"& high"& conser/e , because most eu.ar&otic ce""s %er#orm simi"ar ce""u"ar acti/ities an cou" not to"erate "arge mutations that change the o/era"" #unctiona"it& o# the .inesins' (2+ 9rom the gene e)%ression ata o# .inesin-5 Cin$ an Ki%* in &east, one cou" easi"& /isua"i!e the transcri%tion acti/it& o# these %roteins %ea.e at the same time o# ce"" i/ision, more accurate"&, the ana%hase o# mitosis'(*+7his e)%ression %attern agree with the theoretica" ro"e o# Cin$ an Ki%* in mitosis, the stabi"i!ation o# microtubu"es an cause genome se%aration'

Kuo-Lun Chang SID 22977459 Preliminary 'esults! Assays! 2rotein an en!&me assa&s were /ersati"e "aborator& techni0ues to ins%ect i##erent %ro%erties o# %rotein' 2rotein assa& etermine the 0uantit& (concentration+ o# a %rotein in so"ution through i##erent ana"&tica" techni0ues? en!&me assa&, on the other han was use to 0uanti#& the acti/it& o# the %rotein as an en!&me interacting with substrate' (*B+ 7he -ra #or assa& use in this %ro@ect was a %rotein assa& that etermine the concentration o# an un.nown %rotein against an stan ar cur/e estab"ishe with .nown concentration o# -S, %roteins? the ma"achite green ,72ase assa& was an en!&me assa& that a%%ro)imate the ,72ase acti/it& o# Cin$ with a commercia" .inesin' 7he %ur%ose o# %rotein assa& in the e)%eriments was to 0uanti#& the amount o# Cin$ mutant %roteins co""ecte #rom o/ere)%ression to assess the e##ect o# truncations on in uction? the .inesin ,72ase assa& was necessar& to estab"ish a stan ar %rotoco" #or #uture acti/it& an "inearit& assa& o# the actua" Cin$ %roteins, in which the e##ects o# truncations an in uction on en!&matic acti/it& wou" be etermine ' (2+ 7he -ra #or %rotein assa& was an co"ormetric assa& that wor.e co""aborati/e"& with s%ectrosco%ic ana"&sis' 7he -ra #or reagent containe Coomassie -ri""iant -"ue that cou" bin to aromatic amino aci s as we"" as histi ine an arginine? %rotein boun Coomassie &e ha an absor%tion %ea. at 595 nm, the increase in absorbance was %ro%ortiona" to the concentration o# boun %rotein o/er a "inear range (B to 2BBB CgDm"'+ (**+ ,"though 0uic. an sensiti/e, the -ra #or reagents %ermanent"& boun the %rotein thus %re/ent #urther ana"&sis, an the "inear range o# etection was rather narrow an o#ten re0uire i"ution o# sam%"es' ,ssuming -S, (bo/ine serum a"bumin+ boun to Coomassie at same a##init& as the un.nown %rotein, the concentration o# the un.nown %rotein cou" be etermine b& -eer-LambertEs "aw, , F eGcG", in which the absorbance (,+ was %ro%ortiona" to the concentration (c+, e)tinction coe##icient (e+, an %ath "ength ("+' In this e)%eriment, i##erent amount o# *mgDm" -S, %rotein was a e to a constant amount o# -ra #or reagent an i"ute to a #ina" /o"ume o# * m", /arious amount o# un.nown %rotein were a"so a e to the same setu%' 7he sam%"es were incubate at room tem%erature #or #i/e minutes, then ana"&!e b& s%ectro%hotometer at 595 nm' 7hree tria"s un er same e)%erimenta" setu% were %er#orme #or error ana"&sis' 7he %arameters o# absorbance ata were shown in 7ab"e *'

Kuo-Lun Chang SID 22977459 -S, ,bsorbance Data 3un 5 ug *B ug *5 ug * B'*95 B'429 B'A45 2 B'>2B B'547 B'744 > B'29B B'52B B'A29 ,/erage B'2A$ B'499 B'A7> StD B'BA5 B'BA2 B'BA2 7ab"e *' -ra #or ,ssa& 2arameters' 2B ug B'A97 B'7>7 B'7A* B'7>2 B'B>2 :n.nown ,bsorbance Data 3un * u" > u" * B'*9* B'559 2 B'>*> B'5*> > B'25* B'5B* ,/erage B'252 B'524 StD B'BA* B'B>* 5u" B'7$B B'72> B'AAA B'72> B'B57

9rom the measurements obtaine , a stan ar cur/e was estab"ishe to etermine the un.nown %rotein concentration #rom "inear #itting, which cou" be seen in 9igure *'

"SA Stan%ar% Curve for "ar%for% Assay

B'$

A sor ance (A+*

B'A y = 0.1564x + 0.1518 R2 = 0.9421 B'4

B'2 5 10 15 20 "SA Concentration (m$)ml* 3un * 3un 2 3un > ,/erage Linear 9it (,/erage +

9igure *' 7he -S, Stan ar Cur/e #or -ra #or ,ssa& 7he inter%retation o# the "inear #it a""owe #or the ca"cu"ation o# the un.nown concentration, which was "iste in 7ab"e 2', notice the stan ar :n.nown Concentration * u" > u" >'*9* ug **'9 ug e/iation was re"ati/e"& insigni#icant com%aring to the a/erage concentration' 1u"ti%"e tria"s ha success#u""& increase the accurac& o# the resu"t' Vo"ume 7ota" ,mount 5u" *$'2 ug

Kuo-Lun Chang SID 22977459 Conc' >'*9* mgDm" >'97 mgDm" ,/erage >'AB mgDm" St B'*B2 mgDm" 7ab"e 2' Concentration o# :n.nown 2rotein >'A5 mgDm"

7he ma"achite green ,72ase assa&, on the other han , was a"so an co"ormetric assa& that in/o"/e a %hos%homo"&b ate com%"e) with ma"achite green &e' (2+ It 0uanti#ie en!&me acti/it& b& etecting #ree inorganic %hos%hate with mo"&b ate? un er aci ic con ition, mo"&b ate wou" #orm com%"e) with %hos%hate an boun ma"achite green, the %ro uct wou" ha/e a absor%tion ma)imum at A5B nm'(2+ ,"though high"& sensiti/e an a##or ab"e, this en!&me assa& ha an e/en narraower "inear etection range (2BB H *5BB ugDm"+, re0uire aci ic con ition, an a "arge e)cess o# mo"&b ate to ensure the com%"e)ation o# most #ree %hos%hate in so"ution' In this %roce ure, an stan ar cur/e was estab"ishe using .nown concentrations o# #ree %hos%hates' 7he e)%erimenta" sam%"es containe commercia" .inesin, ,72, an microtubu"es(17+, whi"e the contro" sam%"es i not contain a"" three? this setu% was use to imitate the sub"oca"i!ation en/ironment o# actua" Cin$, as we"" as testing the in uction e##ect o# microtubu"es on .inesins' 4ach sam%"e was #irst ma e to the initia" reaction with r)n bu##er (containing 1gC"2+, .inesin, 17, ,72, an i"ute to stan ar /o"ume' ,#ter twent& minutes o# incubation, ma"achite green an mo"&b ate mi) was a e in e)cess #or com%"e)ation #or #i/e minutes' So ium citrate was a e "ast to stabi"i!e the co"or #or the s%ectro%hotometer measurement at A5B nm' 7he stan ar ,72ase assa& was tabu"ate in 7ab"e' >, with corres%on ing stan ar cur/e e%icte in 9igure 2'

2'$5 *'4> 7 B'B* B'B2 ,bsorbance $ $ 7ab"e >' Stan ar ,72ase assa&

I%hos%hateJ (u1+

Stan ar ,72ase ,ssa& *4'2 2$'5 42'$ 57'* 5'7* $ 7 5 4 B'B9 B'24 *'*2 4 4 B'72 2 *'25

7*'4 > *'42 2

$5'7 *'77 7

**4'2 $ 2'>29

*42'55 2'A2$

Kuo-Lun Chang SID 22977459

ATPase Stan%ar% Curve
3 2.5

A sor ance (A+*

2 1.5 1 0.5 0 0 20 40 60 80 100 120

y = 0.014x R2 = 0.9829

140

160

,-nor$anic Phosphate. (uM*

,72ase Stan ar Cur/e Linear 9it

9igure 2' ,72ase Stan ar Cur/e' 7he stan ar cur/e suggeste a e)tinction coe##icient o# ma"achite green%hos%homo"&b ate com%"e) was B'B*4 u1-*cm-*, or **4 m1-*cm-*' 7he e)tinction coe##icient %resente here (**4 m1-*cm-*+ was agreeab"e with the resu"ts #rom co""aborators (**$ m1-*cm-*, St 5'$ m1-*cm-*+' 7he #ree %hos%hate concentration an absorbance obtaine #rom the actua" assa& was ca"cu"ate with the abo/e e)tinction coe##icient #rom absorbance "iste in 7ab"e 4' ATPase Assay 3eaction ,bsorbance -"an. B 17 B'BB> ,72 B'B42 Kinesin B 17K,72 B'BA> KinesinK17 B'BB2 KinesinK,72 B'B27 KinesinK,72K17 *'957 7ab"e4' 1a"achite greenD%hos%homo"&b ate ,72ase assa&' I2iJ (u1+ B B'B2A B'>A$ B B'55> B'B*$ B'2>7 *7'*7

Kuo-Lun Chang SID 22977459 -& com%aring the #ree %hos%hate concentration between KinesinK,72 an KinesinK ,72K17 sam%"es, the a ition o# 17 cause a H72 times stimu"ation in .inesin ,72ase acti/it&' Mutant Clonin$! 7hree mutants o# CIN8 genes, CIN8-955, CIN8-$7*, an CIN8-59B, were use to etermine the corre"ation between %rotein "ength an #unctiona"it&' 7he motor omain o# Cin$ %rotein was "ocate near the (-terminus, there#ore the mutants "iste abo/e with truncations #rom C-terminus wou" not "oss their #unctiona"it& e#inite"&' 5owe/er, e"etions at sta". an tai" omain o# the %rotein wou" sti"" a##ect the in vivo sub"oca"i!ation an ,72ase acti/it& o# the mutant %roteins' In the conte)t o# the e)%eriment, CIN8-59B, with a "ength a"most ha"/e #rom the wi" t&%e Cin$ (which containe *,BBB ,',+, was not "i.e"& to %er#orm i entica" sub"oca"i!ation acti/it&? howe/er, since the motor omainDcata"&tic core was not a##ecte , it might sti"" ha/e re uce ,72ase acti/it&' CIN8-$7* an CIN8-955 were the ma@or mutant o# interest to test the crucia" "ength o# Cin$ %rotein necessar& #or in vivo sub"oca"!ation, an were "i.e"& to ha/e certain "e/e" o# ,72ase acti/it&' 7he mutants with truncations at i##erent "ength a""owe #or etermination o# the "ocation o# essentia" omains on Cin$ %rotein' 7he c"oning %rocess starte with 2C3, where a"" the mutant constructs were combine with 59 bu##er, (72s, #orwar %rimer an /arious re/erse %rimer #or each mutatnt, an 2husion D(, %o"&merase' Subse0uence 2C3 thermoc&c"es were carrie out in a %re- etermine thermoc&c"er' 4"ectro%horesis o# the genes reco/ere #rom 2C3 o/er a *L agarose ge" K 4t-r with a D(, "a er was one to %uri#& the genes an assess the resu"t #rom 2C3' , re%resentati/e #igure reconstructe #rom actua" e"ectro%horesis %icture was 9igure >' 9igure >' 8e" e"ectro%horesis resu"t 7he re%resentati/e %icture ca%ture the essence o# the resu"t, in which the contro" "ane containe CIN8-M39 gene (>BBB b%+, an the construct "ane containe CIN8-59B (*77B b%+, one o# the mutant constructs' Com%aring to the D(, "a er in the "a er "ane, the resu"t suggeste success#u" 2C3

Kuo-Lun Chang SID 22977459 o# the genes o# interest, howe/er, no conc"usion cou" be rawn at this ste% since on"& the si!e o# re%"icate genes were .nown' 7he 2C3 %ro ucts were igeste using restriction en!&mes 5in III an -am5I in *BN igestion bu##er, a"ong with the three %"asmi s %Cin$-892-C-9:S, %4724b-5is, an %Cin$-;892-C9:S, in i/i ua""&' ,n a".a"ine %hos%hatase was a e to the %"asmi igestion reaction to remo/e the 5E %hos%hate #rom the igeste /ectors to %re/ent s%ontaneous re-"igation' 7he igeste genes an /ectors were %uri#ie using <iagen .it- co"umn %uri#ication, which in/o"/e se0uentia" wash o# 2-I (stabi"i!e D(, with ions+, an 24 (4thano" containing, remo/e im%urities+ bu##ers, #o""owe b& centri#ugation' 7his %uri#ication remo/e %hos%hatase #rom /ector so"utions #or norma" "igations to %rocee ' Ligation was carrie b& -acterio%hage 74 "igase that "igate sD(, brea.age in *BN "igase bu##er' 7he "igate %"asmi sK mutant inserts were then trans#orme to chemica""&-com%etent D55= b& e)%osing to 45 secon s o# heat shoc. at 42 egree Ce"sius #o""owe b& rescue incubation in warm me ia' 7he success#u" trans#ormation o# "igate %"asmi sK insert cou" be /eri#ie b& screening with antibiotics' 7he trans#orme ce""s wou" be strea.e onto %"ates with i##erent antibiotics, %4724-5is trans#ormants wou" be screene with .anam&cin, an the C-9:S trans#ormants wou" be screene with am%hici""in' 7he number o# co"on&-#orming-units i/i e b& the ug o# D(, %"ate wou" be use as trans#ormation e##icienc&' M/era"", this screening sha"" be /er& se"ecti/e O on"& the ce""s with "igate %"asmi wou" ha/e resistance, an on"& the gene inserts containe 5E%hos%hate essentia" #or %"asmi "igation, gi/en the %hos%hatase was #u""& e##ecti/e' D55= ce""s were use #or housing an re%"ication o# the %"asmi s, e)traction o# the %"asmi s #rom these ce""s using the <iagen .it- co"umn %uri#ication an #o""owe u% ge" e"ectro%horesis o/er *L agarose4t-r ge" wou" %uri#& the %"asmi s an a""owe #or se0uencing' Se0uencing o# the %"asmi s was com%"ete outsi e o# this %ro@ect' 7he resu"ting se0uence is%"a&e minima" mutations in a"" the omains an suggeste correct 2C3 an incor%oration o# inserts' 7he %4724b-5isKinsert %"asmi was then trans#orme to e"ectro-com%etent E.Co"i -L3 ce""s through e"ectro%oration, #o""owe b& rescue incubation' Mutcome o# e"ectro%oration was /eri#ie b& antibiotics screening o# .anam&cin (%"asmi + an Ch"ore%henico" resistance(ce""s+' 7he C-9:S %"asmi s were trans#orme into 1S2922 an DDP9B4 (containe #"uorescent-"abe"e 17+ b& chemica" trans#ormation with 248 (increase D(, bin ing+, LiM,c (increase %ermeabi"it&+, an ssD(, (#"oo s sur#ace channe"s+'

Kuo-Lun Chang SID 22977459 , "ist o# bio"ogica" reagents use cou" be seen in 7ab"e 5' -acteria" Strains E.Coli -L3, contains I278 in ucib"e D4> an arabinose in ucib"e 8ro4LD4S' 9or %rotein Peast Strains in uctionDo/ere)%ression' 1S2922 O containe tem%erature sensiti/e Ki%* #or %henot&%ic ana"&sis' Vectors %4724b O 5is6 bacteria" /ector with .anam&cin resistance' 7ab"e 5' -io"ogica" 3eagents use (2+ E.Coli D55=, housing gene with re%resse en onuc"ease acti/it& (4n ,+' DDP9B4 mCherr& O contains 392 tagge tubu"in #or sub"oca"i!ation /isua"i!ation' %892-C-9:S6 &east /ector with CIN8 %romoter an am%hici""in resistance %;892-C-9:S6 same as %892-C9:S, without 892'

Kuo-Lun Chang SID 22977459 'esearch /esi$n an% Metho%s! Su cellcular locali0ation! ,s iscusse ear"ier, Cin$, as a .inesin-re"ate %rotein, carrie out its s%eci#ic tas. b& tetrameri!ing an cross-"in. between microtubu"es in the mitotic s%in "e' In accor ance to its e)%ression %ro#i"e, wi" t&%e Cin$ %rotein was on"& e)%resse uring mitosis, an was on"& "i.e"& to be "ocate aroun the mitotic s%in "e o# segregating chromosomes' 1utant Cin$ %roteins that carrie truncations not a##ecting the mitotic %henot&%e o# S.cerevisiae wou" a"so be "ocate aroun the mitotic s%in "e, simi"ar to wi" t&%e? i# the mutations a##ecte the #unctiona"it& o# the mutant %rotein to tetrameri!e an associate with microtubu"es, it sha"" not be "ocate in the mitotic s%in "e an cou" be an&where' ,n e)%eriment esigne to "ocate the e)act "ocation o# Cin$ mutant %roteins wou" be /er& simi"ar to the #"uorescence microsco%& e)%eriment escribe in #uture e)%eriments? (2+ howe/er, instea o# ta.ing a sing"e %icture o# in vivo co-"oca"i!ation o# tubu"in-mCherr& an Cin$-892, a continuous /i eo sha"" be recor e un er 247 scan' (5+' :n erstan ing o# the e)act "ocation o# non-#unctiona" Cin$ mutants or #unctiona" mutants with a"tere sta". an tai" omain wou" enote the e##ects o# e#icient .inesin on ce""u"ar acti/it& an isease an #itness, asi e #rom its non-#unctiona"it&' In the "arger scheme, "ocating a e#icient #actor o# the mitotic s%in "e might "ea to the so"ution o# e)act mechanism o# s%in "e-re"ate res%ecti/e tumor %rogression' En0yme E&periment! In or er to %er#orm en!&me e)%eriments, the Cin$Dmutant Cin$ %roteins must be %uri#ie ' 7&%ica" %uri#ication %rocess o# a %rotein starte with "&!ing the housing ce""s to re"ease c&to%"asmic contents? centri#ugation wou" then remo/e most #"ui s an sma"" mo"ecu"es in the su%ernant' Se%aration o# hea/& mo"ecu"es such as other %roteins cou" be one b& si!e e)c"usion chromatogra%h&, in which %orous ge" #i"ters out "arge mo"ecu"es? %uri#ication with charge mo"ecu"es (D(,, other %rotein+ cou" be achie/e with ion-e)change chromatogra%h&, se%arating mo"ecu"es with i##erent ionic strength' 9ina""&, 52LC wou" u"tra-%uri#& the obtaine %rotein'

Kuo-Lun Chang SID 22977459 In terms o# en!&me inhibition an .inetics e)%eriments, Vma), the ma)imum reaction s%ee an Km, a characteristic rate constant o# en!&me, cou" be easi"& ca"cu"ate #rom the 1ichae"is-1enton e0uation, shown in 9igure 4' 2"otting *DV, the in/erse o# reaction rate against *DDS, the in/erse substrate concentration wou" gi/e a straight "ine with a s"o%e o# KmDVma)' Vma) cou" be etermine b& measuring the reaction rate when substrate concentration was huge', thus ma e KmDIsJ !ero an e0uation (*+ on the "e#t / F Vma)' (*2+ 9igure 4' 7he 1ichae"is-1enton 40uation' :n"i.e Vma) an Km, Ki, the characteristic constant measuring the %otenc& o# an inhibitor, /arie #rom com%etiti/e to non-com%etiti/e inhibitors' 5owe/er, %"otting the inhibitor concentration against *DV un er i##erent substrate wou" gi/e a %"ot with mu"ti%"e "ines? the )coor inate ( some concentration o# inhibitor+ o# the intersecting %oint o# the "ines wou" Ki' enote

Kuo-Lun Chang SID 22977459 In vivo phenotypes! 7he %henot&%ic assessmentD scoring o# Cin$Dmutants was one b& growing wi" t&%e 1S2922 strains a"ong with 1S2922 strains trans#orme with %892-C-9:S an %;892-C-9:S %"asmi s, incor%orate with Cin$-M39 or Cin$-mutant, on *2 %"ates with A i##erent a iti/es an 2 tem%eratures (25 QC,>7QC+' 7he a iti/es o# e/er& two %"ates inc"u e Cincreasin, benom&", sorbito", noco a!o"e, A-a!auraci", an ca##eine' Cincreasin was an i enti#ie inhibitor that wou" inter#ere with mitosis b& isab"ing .inases essentia" #or s%in "e chec. %oint? there#ore, %"ates with cincreasin were "i.e"& to obser/e minima" growth, regar "ess o# the e##ect o# com%"ementation'(*>+ -enom&" was a common #ungici e, .i""ing &east strains on the %"ates, which a"so %re/ent the growth o# &east strains regar "ess o# com%"ementation' (oco a!o"e was a cancer-treating rug that cause intentiona" mitotic arrest, hence staggering mitosis regar "ess o# com%"ementation' Sorbito" wou" cause an osmotic %ressure to &east, an stimu"ate %ressure a a%taing %athwa&s, this i not irect"& a##ect mitosis, the e)act e##ect was &et to be #oun ? Aa!auraci" was an substrate that "owere ce""cu"ar 872 "e/e" in &east, an cou" cause an mitotic arrest' (*4+ Ca##eine, as a %urine ana"ogue, wou" "i.e"& inhibit mitotis an initiate ce"" eath through /arious signa"ing casca es' (*5+ 7hese a iti/es were "i.e"& to be the negati/e contro" to ensure no growth wou" be obser/e on these %"ates, unre"ate to the success o# com%"ementation o# ts-Ki%* with Cin$ mutants' 7he goa" o# these a iti/es were to test the mitotic %henot&%es o# the com%"emente &east strains un er i##erent con itions' Transcription of CIN8! In or er to 0uanti#& the transcribe m3(, an obser/e transcri%tion o# 3(, at contro""e con itions, irect measurement the in vivo CIN8 m3(, "e/e" wou" not be the best choice? instea , 37-2C3 carrie out b& re/erse-transcri%tase an D(, %o"&merase wou" %ro uce cD(,s with e)%erimenta" /a"ue' 37-2C3 cou" be one using 5N <iagen MneSte% 37-2C3 bu##er combining with (72s, %rimers, an tem%"ate 3(,' 7hermoc&c"es simi"ar to regu"ar 2C3 with D(, %o"&merase wou" then %ro uce cD(,s'(2+ 7he 3(, "e/e" o# each CIN8 mutant cou" be etermine b& the ban intensit& generate #or ge" e"ectro%horesis o# the obtaine cD(,' 7he im%ortance o# this ana"&sis was to etermine the e##ects o# truncations in genes ha on gene transcri%tion? since eu.ar&otic D(, containe introns an e)ons, e/er& a"teration o# a gene cou" "ea to i##erentiation in transcri%tion acti/it& an %attern' 7his

Kuo-Lun Chang SID 22977459 e)%eriment was %er#orme to test whether the change in mitotic %henot&%e originate #rom the "oss o# #unctiona"it& o# Cin$ mutants, or higher "e/e" transcri%tiona" contro"s were in/o"/e '

Kuo-Lun Chang SID 22977459 'eference! *' 3oo#, D'1', 1e"uh, 2'1', 3ose, 1'D' (*992+ Kinesin-re"ate 2roteins 3e0uire #or ,ssemb"& o# the 1itotic S%in "e' Journal o Cell !io. 118(*+, 95-*B$' 2' De%artment o# 1o"ecu"ar an Ce"" -io"og&' (2B*4+ "C!110# $eneral !ioc%e&istr' an( "olecular !iolo)' #a*orator'' :ni/erist& o# Ca"i#ornia, -er.e"e&' >' Va"e, 3'D', 3esse, 7'S', Sheet!, 1'2' (*9$5+ I enti#ication o# a no/e" #orce-generation %rotein, .inesin, in/o"/e in microtubu"e-base moti"it&' Cell 12(*+, >9-5B' 4' Va"e, 3'D' (2BB>+ 7he mo"ecu"ar motor too" bo) #or intrace""u"ar trans%ort' Cell 112(4+, 4A7-4$B' 5' 5ere ia, 9'9', De Sousa, R'C', 3ibeiro Rr', 5'L', Car/a"ho, ,'9', 1aga"haes, 1eira', 1aria, Si"/ia', 2inheiro, 3'9' (2B*4+ 2roteins re"ate to the s%in "e an chec.%oint mitotic em%hasi!e the i##erent %athogenesis o# h&%o%"astic 1DS' #eu+e&ia ,esearc% 38(2+, 2*$-224' A' 9ri man, V', 8erson-8urwit!, ,', Sha%ira, M', 1o/sho/ich, (', La.am%er, S', Schmi t, C'9', 8heber, L' (2B*>+ Kinesin-5 Ki%* is a bi- irectiona" motor that stabi"i!es microtubu"es an trac.s their %"us-en s in /i/o' J.Cell. Sci. 124, 4*47-59' 7' (an , S', 8o win, R'4' (*9$$+ 5&%o%"astic m&e"o &s%"astic s&n rome' Cancer 42(5+, 95$-A4' $' 5i" ebran t, 4'3', 8heber, L', Kingsbur&, 7', 5o&t, 1',' (2BBA+ 5omotetrameric 9orm o# Cin$%, a Sacc%aro&'ces cerevisiae Kinesin-5 1otor, Is 4ssentia" #or Its in -ivo 9ucntion' J. !io. C%e&. 281(>A+, 2ABB4-2AB*>' 9' 4n ow, S',', Ku"", 9'R', Liu, 5ong"ei' (2B*B+ Kinesins at a g"ance' J. Cell. Sci. 123, >42B->424' *B' Schne"", S', Cha%%e"", 1'R', 4/ans, ('D' 3osee", 1'3' (2BBA+ 7he mechanism istinguishabi"it& %rob"em in biochemica" .inetics6 7he sing"e-en!&me, sing"e-substrate reaction as a case stu &' Co&.tes ,en(us !iolo)ies 325 (*+, 5*-A*' **' Sor, 7', Se"inger, S' (*99A+ Lineari!ation o# the -ra #or %rotein assa& Tincreases it sensiti/it&6 theoretica" an e)%erimenta" stu iesU' /nal. !ioc%e&. 234, >B2->B$' *2' Si, D', Vang, P', 8uo, P', Vang, R', Shou, 5', Shou, P'5', Li, S'S', 9awcett, R'2' (2BB$+ 1echanism o# CP22C9 inhibition b& #"a/ones an /"a/ono"s' 0"0 36(>+, A29->4'

Kuo-Lun Chang SID 22977459 *>' Dorer, 3'K', Shong, S', 7a""arico, R',', Vong, V'5', 1itchison, 7'R', 1urra&, ,'V'' (2BB5+ , sma""-mo"ecu"e inhibitor o# 1%s* b"oc.s the s%in "e-chec.%oint res%onse to a "ac. o# tension on mitotic chromsosomes' Curr. !iol. 15(**+, *B7B-A *4' 7anse&, V'2' (2BBA+ A-,!auraci" Sensiti/it& ,ssa& #or Peast' Col( S.rin) 1ar*. Protoc' *5' Kuran a, K', Leberre, V', So.o",S', 2a"amarc!&., 8', 9rancois, R' (2BBA+ In/estigating the ca##eine e##ects in the &east Saccaro&'ces cerevisiae brings new insights into the connection between 7M3, 2KC, an 3asDc,12 signa"ing %athwa&s' "ol "icro*iol. 41(5+, **47-AA'