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Uni e si y o Copenhagen
Bachelo Thesis
S udies o Dynamin 2 in
Megaka yocy es
Amalie Dahl Haue
Niels Bo egaa d
Supe iso
Uni e si y o Copenhagen
He ´e Fale
Supe iso
Ha a d Medical School
Decembe 3, 2013
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P eamble
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The p esen hesis is based on expe imen s conduc ed a Di ision o T ansla ional Medicine,
B igham & Women's Hospi al, Ha a d Medical School in Bos on. The expe imen s we e
designed by associa e biologis He é Fale and conduc ed unde supe ision o pos doc Ma kus
Bende . The hesis was w i en unde supe ision o p o esso Niels Bo egaa d, Uni e si y o
Copenhagen.
The hesis has he ollowing s uc u e. The in oduc ion includes an accoun o ele an
backg ound knowledge, including an expe imen al mo i a ion, a b ie desc ip ion o molecula
s uc u es and mechanisms cen al o he expe imen s and a p esen a ion o he hypo hesis.
Ma e ials and me hods p esen s a desc ip ion o he expe imen s conduc ed - a me hodological
iad comp ising mu agenesis, e o i al p oduc ion and i al ans ec ion. The esul sec ion
p esen s sequencing esul s ollowing mu agenesis and wo se s o pic u es, one o e o i al
p oduc ion and one o i al ans ec ion. The discussion includes a c i ical e alua ion o he da a
and he expe imen al se up.
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Resumé
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Fo måle med næ æ ende opga e a a unde søge dynamin 2’s be ydning o dannelse og
unk ion a blodplade . Kondi ione e knockou a dynamin 2 i megaka yocy e hos mus e
å sag il h ombocy openi med mac o- h ombocy e , splenomegali og megaka yocy
hype plasi. I menneske e mu a ione i gene de kode o dynamin 2 å sag il Cha co -Ma ie
Too h sygdom. Med henblik på a unde søge unk ionen a dynamin 2 i megaka yocy e , ble
en ække o søg ud ø . Disse al e e, nemlig mu agenese, e o i al p oduk ion og e o i al
ans ek ion. De ekspe imen elle mål a (i) a lokalise e dynamin 2 i megaka yocy e og (ii)
a e e e e blodpladede ek en i knockou mus ed a ans ek e e dymanin 2 ind
megaka yocy e a disse. Da de ikke e an is o e ilgængelige mod dynamin 2 ble
lokalisa ionen o søg gennem ø ed a unde søge lokalisa ionen a e usionsp o ein bes ående
a dynamin 2 og e luo esce ende p o ein. Dog lykkedes de h e ken a lokalise e dynamin 2,
elle a opnå ils ækkelig ans ek ionse ek i i e a dynamin 2 i megaka yocy e , h o o
spø gsmåle o sa e ubes a e .
I o længelse a de ekspe imen elle esul a e ølge en e alue ing a ekspe imen e ne. De
konklude es, a esul a e ne a inkonklusi e g unde beg ænse e a ing og id. De med e
p ojek e s egen lige konklusion ikke a idenskabelig ka ak e , men bes å i e e h e e
kompe encesæ . He ed o s ås, a jeg i o bindelse med uda bejdelsen a opga en ha
opa bejde en beskeden labo a o iee a ing. Yde me e e jeg ble e bekend med an endelsen
a den bibliog a iske da abase Pubmed i o bindelse med a indhen e og selek e e idenskabelig
in o ma ion.
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Abs ac
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The aim o he p esen hesis was o ob ain an in-dep h desc ip ion o he ole o dynamin 2 in
pla ele o ma ion and ac i a ion o pla ele s. In mice, dynamin 2 knockou in he
megaka yocy e lineage causes h ombocy openia wi h mac o- h ombocy es, splenomegaly and
megaka yocy e hype plasia. In humans, Cha co -Ma ie-Too h disease is caused by dynamin 2
mu a ions. On ha accoun , a se ies o expe imen s we e designed o s udy megaka yocy es
om dynamin 2 knockou mice. The expe imen s included mu agenesis, e o i al p oduc ion
and i al in ec ion. The aim was o (i) loca e dynamin 2 in megaka yocy es and (ii) escue
pla ele de iciency in knockou megaka yocy es by ans ec ing dynamin 2 in o he
megaka yocy es. Since he e is no an ibody agains dynamin 2, a usion p o ein comp ising o
dynamin 2 and a luo escen p o ein was cons uc ed. Theo e ically, luo escence should hen
be used o loca e dynamin 2. Howe e , loca ing dynamin 2 ailed. Consequen ly, he aim
emains open o examina ion.
Subsequen o a p esen a ion o he expe imen al esul s, he hesis commen s on he
expe imen al esul s and e alua es he expe imen al se up. I concludes ha he esul s we e
inconclusi e due o limi ed expe ise and ime. Hence, he main conclusion is no s ic ly
scien i ic. Ra he , i consis s in he ac , ha I gained modes labo a o y expe ience in collec ing
da a. Fu he , I became amilia wi h he bibliog aphic da abase Pubmed and lea ned how o
use he ool in means o inding and selec ing in o ma ion.
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1
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Table o Con en s
INTRODUCTION 2!
EXPERIMENTAL MOTIVATION! 2!
THEORETICAL COMPONENTS! 3!
DYNAMIN! 3!
THROMBOPOIESIS! 5!
CHARCOT-MARIE-TOOTH DISEASE! 6!
EXPERIMENTAL APPROACH! 7!
EXPERIMENTAL MODEL! 7!
HYPOTHESIS! 8!
EXPERIMENTAL OBJECT! 9!
MATERIALS AND METHODS 9!
PLASMID CONSTRUCT! 9!
MUTAGENESIS! 9!
DNA PURIFICATION!10!
RETROVIRAL PRODUCTION!11!
MEGAKARYOCYTE TREATMENT!12!
ISOLATION!12!
TRANSFECTION!12!
METHODOLOGICAL ACHIEVEMENTS!12!
RESULTS 13!
MUTAGENESIS!13!
RETROVIRAL PRODUCTION!14!
MEGAKARYOCYTE TRANSFECTION!15!
DISCUSSION 16!
DESCRIPTIVE SUMMARY!16!
EXPERIMENTAL EVALUATION!17!
EXPERIMENTAL LIMITS!17!
CONCLUSION!18!
REFERENCES 19!
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F on -page illus a ion om: h p://www.abo e opsec e .com/ o um/ h ead682667/pg2
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In oduc ion
Expe imen al Mo i a ion
Pla ele s, he smalles membe o he blood co puscle, a e unique in wo aspec s. S uc u ally,
hey a e anuclea e. Func ionally, hey a e he cells egula ing hemos asis. These s uc u al and
unc ional cha ac e is ics make hem a p e alen objec o scien i ic in es iga ions(1).
In 1882 Giulio Bizzoze o, he man who came o be known as he disco e e o pla ele s,
desc ibed he exis ence o ‘cons an blood pa icles, di e ing om ed and whi e blood cells
(…)’(2). E e since, pla ele s ha e been s udied in ensely esul ing in a deepe cellula
unde s anding, which has o med he basis o a ious ea men egimes. In spi e o many
pha macological con ibu ions al eady, he ield is s ill e ol ing(3). Despi e subjec o scien i ic
in es iga ion o cen u ies, much emains o be e ealed abou he mechanisms unde lying
no mal pla ele unc ion. In pa icula he desc ip ion o pla ele o ma ion and ac i a ion
con inually gene a es mo e ques ions han answe s(1). Wi h ha in mind, a se ies o
expe imen s we e designed o s udy unc ional aspec s o h ombopoiesis, he o ma ion o
pla ele s. The s udy included pa ien s su e ing om Cha co -Ma ie Too h disease and a
condi ional dynamin 2 knockou mice. Cha co -Ma ie Too h disease is an au osomal dominan
neu omuscula diso de , which is caused by mu a ions in DNM2, he gene coding o dynamin
2. Speci ic DMN2 mu a ions lead o h ombocy openia in Cha co -Ma ie Too h disease
pa ien s(4) and he condi ional megaka yocy e lineage speci ic dynamin 2 knockou mice su e
om h ombocy openia (unpublished da a, Fale , H.). Hence, Cha co -Ma ie Too h pa ien s
and condi ional dynamin 2 knockou mice sha e a ec ion si e and hei pheno ypes o e lap.
Combined, hese wo ea u es a e he a ional behind s udying hem join ly.!!
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Figu e 1 Expe imen al o igin
In summa y, an in-dep h desc ip ion o h ombopoiesis is he incen i e behind he expe imen s
ha he p esen hesis p esen s. The hypo hesis is ha dynamin 2 is esponsible o he
agmen a ion o pla ele s om hei p ecu so s and he eby he pla ele syn hesis. The
hypo hesis is g ounded in wo ea u es. One is a clinical obse a ion and one is heo e ical hook
de i ed om well-s udied unc ions o dynamin iso o ms elsewhe e in he o ganism. The
clinical obse a ion is ha Cha co -Ma ie Too h disease is caused by DNM2!mu a ions and ha
hey su e om h ombocy openia(4). The heo e ical hook is ha he p ime unc ions o
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dynamin a e ob ained h ough in e ac ion wi h he plasma memb ane(5).
Th ee me hods cons i u e he expe imen al app oach. One is mu agenesis, one is e o i al
p oduc ion and one is e o i al ans ec ion as p esen ed in igu e 2. The expe imen al subjec s
comp ise a plasmid cons uc and megaka yocy es isola ed om mouse emb yos. The plasmid
was cons uc ed in such a way ha dynamin 2 is used wi h Dend a2. Dend a2 is a luo escen
p o ein, which has been modi ied om Dend a, a luo escen p o ein de i ed om oc oco al
Dend oneph hya sp(6, 7).
The mu agenesis was pe o med o acqui e he co ec DNA sequence in he plasmid used o
ans ec ion. Re o i al p oduc ion was pe o med o allow ans ec ion o he plasmid in o he
megaka yocy es. Finally, e o i al ans ec ion was pe o med o induce he exp ession o
Dend a2-dynamin 2 in megaka yocy es, and hus loca e dynamin 2.
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Figu e 2 Expe imen al lowcha
Theo e ical Componen s
Dynamin
Dynamins a e la ge GTPases in ol ed in a wide ange o cellula unc ions, including
memb ane emodeling, endocy osis, in acellula a icking and in e ac ion wi h he ac in and
mic o ubule cy oskele al ne wo ks. The mammalian genome encodes h ee dynamin iso o ms.
They sha e cha ac e is ics, in ha hey ha e GTPase ac i i y, ha e a ini y o a ious
in acellula p o eins, abili y o oligome ize and a e capable o binding lipid. Toge he , hese
cha ac e is ics enable dynamins o induce con o ma ional changes, in e ac wi h a a ie y o
o he p o eins and induce s uc u al changes in he cellula memb anes, esul ing in memb ane
scission(5).
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Ini ially, dynamin was iden i ied as a p o ein associa ed wi h mic o ubules (8). Subsequen ly,
a ious dynamin iso o ms ha e been iden i ied. Dynamin 1 is exp essed mainly in neu ons(9),
dynamin 2 is ubiqui ously exp essed(10, 11) and dynamin 3 was o iginally pu i ied om
es is(12).
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Figu e 3 Th ee-dimensional s uc u e o dynamin, PH: plecks in homology, GED: GTPase e ec o domain,
PRD: p oline ich domain(13)
Fi e domains make up he p ima y s uc u e o a dynamin monome , as illus a ed in igu e 3.
The G domain, loca ed a he e y amino- e minus, is he si e o GTPase ac i i y. The middle
domain and GTPase e ec o domain acili a e dime iza ion o dynamin(14). The plecks in
homology domain is capable o binding o nega i ely cha ged memb ane bound phospholipids,
p e e en ially phospha idylinosi ol-4,5-bisphopha e. The binding o he plecks in homology
domain enhances GTPase ac i i y(15). The ca boxy- e minus pa o he s alk, also e med
GTPase e ec o domain, is unc ionally associa ed wi h he G domain as well and egula es he
GTPase ac i i y. Hence, i d i es he dynamics o dynamin(16). The p oline ich domain is
capable o binding o a ious in acellula p o eins. The in e ac ion be ween dynamin and o he
in acellula p o eins is in ol ed in guiding dynamin molecules o he plasma memb ane (17,
18). Fu he , he p oline ich domain has a ious phospho yla ion si es. Thus, i se es as a
co alen modi ica ion si e and acili a es he in e ac ion be ween dynamin and a a ie y o o he
in acellula p o eins(18).
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A h ee-dimensional econs uc ion o dynamin implies a ‘T’ shape cha ac e ized by h ee
densi ies. They ha e been e med head, s alk and oo . Figu e 3 p esen s he ela ionship
be ween he p ima y s uc u e and he h ee-dimensional s uc u e o dynamin. The GTPase
domain o ms he head, he middle domain and GED o m he s alk and he PH domain o ms
he oo (13).
The unc ion o dynamin has been desc ibed in e ms o a co ksc ew model. Acco ding o he
co ksc ew model, dynamin unc ion esul s om a con o ma ional change in he head and s alk
ha collec i ely gene a es a wis ing mo ion ex ending om he dynamin assembly. I has been
assumed ha he wis ing mo ion is he sum o wo o ces. One esul s om a o ce pa allel
wi h he plasma memb ane and one causes a o ce o hogonal o he plasma memb ane (19,
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Figu e 4 S uc u al and unc ional o e iew o DNM(21)
Th ombopoiesis
Th ombopoiesis, he o ma ion o blood pla ele s, akes place in he bone ma ow. I is a highly
egula ed p ocess and is essen ial o hemos asis. In humans, he daily pla ele p oduc ion
amoun s o app oxima ely 1 x 1011, and he ci cula o y li e span is oughly 10 days(22).
Megaka yocy es a e he myeloid cells ha gi e ise o pla ele s(23) ollowing a ma u a ion
p ocess ha empo ally anges om 4 o 7 days, o igina ing a he megaka yoblas and
ul ima ely leading o he o ma ion o megaka yocy es(24). Megaka yocy es send long
p o usions om he sinusoids o he bone ma ow o he ci cula ion, whe e hese p o usions
also called p o-pla ele s unde go agmen a ion o o m pla ele s. I has been es ima ed ha one
megaka yocy e gi es ise o 1000 o 3000 pla ele s(22). P o-pla ele s con ain a cha ac e is ic
dis ibu ion o mic o ubulin sugges ing ha i is in ol ed in he agmen a ion o pla ele s(25).
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Megaka yocy e T ea men
Isola ion
A day 0, e al li e cells om mouse emb yos aged 13.5-14.5 days we e collec ed in DMEM
wi h 10% e al-bo ine-se um and 1% Penicillin/S ep ococcus. Tissue was sa ed o
geno yping. Cells we e passed wi h 1 mL pipe , 18G, 22G and 25G needles. Then, cells we e
ans e ed o a BD 40µm nylon mois en cell s aine (BD Re . 352340). Cells we e spun a 200g
o 5 minu es a oom empe a u e and he pelle s we e esuspended in medium wi h 50ng/mL
h ombopoie in and incuba ed a 37°C.
T ans ec ion
A day 2, e al li e cells we e ans ec ed. They we e esuspended ca e ully, ans e ed o a
15 mL Falcon ube and spun a 200g o 5 minu es a oom empe a u e. The pelle s we e
esuspended in 1.5mL esh DMEM. Then, 1 mL i us and 2.5µL polyb ene we e added o
each well, ans e ed o a 6-well pla e and spun (So all, RT6000B) a 800g o 90 minu es a
oom empe a u e. The ea e , he ans ec ed cells we e incuba ed o 90 minu es a 37°C.
Then, cells we e ans e ed o 15 mL Falcon ubes, spun a 200g o 5 minu es and
esuspended in 2 mL DMEM wi h h ombopoie in and incuba ed o e nigh in a 6-well pla e a
37°C. A day 3, megaka yocy es we e esuspended in 1.5 mL esh medium and isola ed o e a
bo ine-se um-albumin g adien a 1.5%/3.0% and cells we e esuspended in esh medium
wi h h ombopoie in. A day 4, cells we e isualized in a luo escen -ligh -mic oscope and
pic u es we e aken.
Me hodological Achie emen s
I gained expe ience in molecula biological p ocedu es om he expe imen s conduc ed. The
molecula biological p ocedu es coun mu agenesis comp ised o polyme ase chain eac ion and
bac e ial ans o ma ion, e o i al p oduc ion and in ec ion. Hence, I became amilia wi h
wo ways in which DNA mul iplica ion may come abou , one being polyme ase chain eac ion
and one being bac e ial g ow h. Finally, I became awa e o he ac ha e o i us unc ions as a
c i ical ool in manipula ing cellula p o ein syn hesis.
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Resul s
Mu agenesis
Tes diges ions we e pe o med o con i m success ul ans o ma ion in o he XL-10
ul acompe en cells.
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Figu e 11 Tes diges ion wi h NcoI, le : 6 samples and 2 nega i e con ols on 0.8% aga ose gel a 120V o 30
minu es, igh : expec ed band pa e n
Figu e 11 demons a es ha diges ion o he pu i ied plasmid yields a band co esponding o he
expec ed band size. Fu he mo e, diges ion wi h he es ic ion enzyme BglII ha has h ee
clea age sides on he plasmid yielded he expec ed band pa e n. A pic u e o he gel is shown
in igu e 12.
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Figu e 12 Tes diges ion wi h BglII, le : 6 samples and 2 nega i e con ols on 0.8% aga ose gel a 120V o 30
minu es, igh : expec ed band pa e n
To con i m success ul mu a ion, wo samples we e sen o sequencing. Resul s a e p esen ed
in igu e 13.!
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Figu e 13 Sequencing esul s: F om le : sequence p io o cys eine inse , 2 samples a e success ul cys eine
inse . The a ows ma k si e o in e es .!
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Re o i al P oduc ion
Pic u es we e aken 48 hou s a e ans ec ion o con i m success ul ans ec ion o he HEK
293T cells. The pic u es p o ide a quali a i e es ima e o he ans ec ion e iciency and hey
demons a e ha he dis ibu ion o dynamin 2 is con ined o he cy oplasm as expec ed.
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Figu e 14 HEK 293T cells, 4X b igh ield and luo escence, 48 hou s a e ans ec ion
The es ima e o he ans ec ion e iciency does no ans e o in o ma ion on he e o i al
p oduc ion, explici ly. Hence, nei he he quali y no he quan i y o he i us was de e mined.
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Megaka yocy e T ans ec ion
Pic u es o he ans ec ed megaka yocy es a e shown in igu e 15, 16 and 17. The size o he
cells is used o iden i y he megaka yocy es. The megaka yocy e diame e in mice anges om
20-30µm(34).
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Figu e 15 T ans ec ed wild ype megaka yocy e, b igh ield and luo escence, 48 hou s a e ans ec ion
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Figu e 16 T ans ec ed knockou megaka yocy es, b igh ield and luo escence, 48 hou s a e ans ec ion
Figu e 17 T ans ec ion end in knockou megaka yocy es, b igh ield luo escence and luo escence, 48
hou s a e ans ec ion
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Discussion
Desc ip i e Summa y
Figu e 15 and 16 e eal ha he ans ec ion is success ul in wild ype and knockou cells.
Howe e , he ans ec ion e iciency is low, educing he in o ma i e po en ial o he pic u es.
Fu he mo e, no p o-pla ele o ma ion occu s. Based on he expe imen al model ha shows
dec eased pla ele coun in knockou mice, spa se o ma ion o p o-pla ele s is o be expec ed.
The dis ibu ion o dynamin 2 is mo e sca e ed in megaka yocy es han in HEK 293T cells.
Ye , a clea pa e n in he loca ion o dynamin 2 canno be deduced om he pic u es. The
combina ion o ela i ely low ans ec ion e iciency and he appa en lack o speci ic loca ion o
dynamin 2 p ohibi s any conclusions o be d awn whe he escuing o he knockou
megaka yocy e has been ob ained.
A compa ison be ween he wild ype and knockou megaka yocy es ( igu e 15 and 16 b igh
ield pic u es), shows ha he wild ype cells a e mo e nume ous and bigge , sugges ing a
di e ence be ween he beha io o megaka yocy es in i o and in i o, since he expe imen al
model is cha ac e ized by megaka yocy e hype plasia. Ei he , he pic u es a e no
ep esen a i e, o he megaka yocy e p ogeni o s espond di e en ly o h ombopoie in in i o
and in i o. The esponse o h ombopoie in in i o may be in eg a ed wi h o he s imuli,
allowing he megaka yocy es o p oli e a e in he absence o dynamin 2. Al e na i ely, i can be
hypo hesized ha megaka yocy e p oli e a ion in dynamin 2 knockou mice accele a es
pos pa um o la e in he emb yonic de elopmen . Acco dingly, megaka yocy e hype plasia
does no p esen in cul u es om emb yos.
Figu e 17 illus a es he end o he ans ec ed cells, which can be ca ego ized in o h ee,
namely: un ans ec ed cells, es ing ans ec ed cells and ac i e ans ec ed cells. The e ms
es ing and ac i e a e coupled o he appea ance o he cell memb ane ac i e cells meaning cells
whe e he cell memb ane appea s dis up ed.
As opposed o igu e 15 and 16, igu e 17 indi ec ly pic u es he mul i lobula nucleus o he
megaka yocy es, making he iden i ica ion o he cell mo e eliable. An ibody s aining agains
lineage speci ic su ace molecules in he megaka yocy e lineage is equi ed o con i m he
na u e o he cells.
I seems ha he e is a di e ence be ween he loca ion o dynamin 2 in ac i e and es ing cells,
in ha he loca ion o dynamin 2 in ac i e cells is limi ed o speci ic a eas in close p oximi y o
he cell memb ane. Addi ionally, he luo escence is s onge in he es ing cells han in he
ac i e cells, sugges ing ha he exp ession o dynamin 2 a ies depending on he unc ional
iden i y o he cells.
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In summa y, he quali y o he pic u es is no good enough o allow conclusions o be d awn.
Fi s , he combina ion o low ans ec ion e iciency and no p o-pla ele o ma ion hinde s
isualiza ion o ans ec ed p o-pla ele s. Second, he unspeci ic localiza ion o dynamin 2 in
ans ec ed cells does no poin o any clea ela ion be ween h ombopoiesis and unc ion o
dynamin 2.
Expe imen al E alua ion
O he han he low ans ec ion e iciency, i is a majo p oblem ha no p o-pla ele s a e seen
in he pic u es. Since he e a e no subs an ial di e ences be ween p o-pla ele s o ma ion o
wild ype and knockou megaka yocy es, absence o p o-pla ele s is mos likely a consequence
o he expe imen al ci cums ances. The lack o p o-pla ele o ma ion may esul om (i) he
i al ans ec ion o (ii) he Dend a2-in e en ion. In case o (i) he i al ans ec ion, including
cen i uga ion may be oo ha sh on he cells. As a esul , he cells may be mo e suscep ible o
apop osis. Fu he in es iga ion o his possibili y equi es iden i ica ion o apop o ic bodies. In
line wi h ha , i should be no ed, ha he concen a ion o he i al supe na an used o
ans ec ion is no adjus ed. The elemen gi es ise o wo p oblems. Fi s , he cell cul u es a e
no necessa ily ea ed alike. Second, he amoun o i al supe na an used o ans ec ion may
be ei he oo li le o oo much o ob ain su icien exp ession le els.
In case o he sub le (ii), he expe imen al se up assumes ha Dend a2 does no dis up he
unc ional in eg i y o dynamin 2. To da e, success ul labeling o o he polyme izing p o eins
ha e been epo ed, among hem ano he nucleo ide hyd olyzing p o ein, namely ubulin (35).
Assuming ha dynamin 2 o ches a es pla ele agmen a ion, spa se p o-pla ele o ma ion
may esul in a d op in he unc ional in eg i y o dynamin 2 in ans ec ed cells, caused by
Dend a2. Based on ha , he p ope ies o dynamin 2 and Dend a2-dynamin 2 ough o be
s udied sepa a ely o ensu e eliable da a.
Expe imen al Limi s
F om he expe imen al model and he Cha co -Ma ie Too h disease pa ien s, i seems ha
dynamin 2 is a p o ein o g ea impo ance in mice and humans. Ne e heless he pa ien s and
he expe imen al model di e , and he di e ences a e c i ical o keep in mind in he a emp o
map h ombocy openia in Cha co -Ma ie Too h disease pa ien s and he mouse model.
Fo example, he Cha co -Ma ie Too h disease pa ien s do exp ess dynamin 2, al hough he
unc ion is al e ed due o mu a ions. In con as , he knockou o dynamin 2 in he
expe imen al model is lineage speci ic. Hence, he pa ien s and he model di e undamen ally,
aside om di e ences esul ing om he ac ha hey a e di e en animals. Possibly, gene ic
edundancy may p ese e some unc ions o dynamin 2 in he Cha co -Ma ie Too h disease
pa ien s. Tha possibili y may explain why h ombocy openia in Cha co -Ma ie Too h disease
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pa ien s is limi ed o one mu a ion si e. In addi ion, he p esence o dynamin 2 in he pa ien s,
albei mu a ed, may sus ain some dynamin 2 unc ions.
Conclusion
Expe imen ally, he si e-di ec ed mu agenesis yielded posi i e esul s, whe eas he a emp o
loca e dynamin 2 wi h Dend a2 was inconclusi e because o low ans ec ion e iciency
combined wi h spa se p o-pla ele o ma ion. Hence, he expe imen s ga e no basis o con i m
o ejec he hypo hesis, namely ha dynamin 2 acili a es he agmen a ion o pla ele s om
p o-pla ele s.
While acknowledging he ac ha he expe imen s we e conduc ed wi h limi ed expe ise and
ime, he hesis e alua es he expe imen al se up and d aws a en ion o ce ain ea u es o he
expe imen s. Speci ically, he e alua ion aises wo main ques ions ega ding he expe imen al
p ocedu es, (i) ha hey may be oo ha sh on he megaka yocy es and (ii) ha Dend a2 may
in e up he unc ional in eg i y o dynamin 2.
Pe sonally, he wo k ga e me insigh in o cen al molecula biological expe imen al
p ocedu es ha ha e a b oad applica ion in esea ch and clinically. Addi ionally, I s udied
pla ele de elopmen and unc ion and dynamin s uc u e and unc ion. The eby I lea ned o
acqui e knowledge om scien i ic a icles and communica e hese insigh s in w i ing.
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