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Identification of components of the intracellular transport machinery of acylated proteins by a genome-wide RNAi screen [Elektronische Ressource] / presented by Julia Ritzerfeld

De
154 pages



IDENTIFICATIO N
O F
CO M PO NENTS
O F
TH E
INTRACELLU LAR
TRANSPO RT
M ACH INERY
O F
ACYLATED
PRO TEINS
BY
A
GENO M E‐W IDE
RNAI
SCREEN















DISSERTATIO N






submitted
to
the
Combined
Faculties
for
the
Natural
Sciences
and
for
Mathematics 
of
the
Ruperto
Caro la
University
of
Heidelberg,
Germany 
for
the
degree
of
Doctor
of
Natural
Sciences 





Julia
Ritzerfeld 
2009















































“Ich
weiß,
dass
ich
n ichts
weiß.”
Sokra tes





DISSERTATIO N














submitted
to
the
Combined
Fac ulties
for
the
Natural
Sciences
and
for
Mathematics
of
the
Ruperto
Carola
University
of
Heidelberg,
Germany
for
the
degree
of
Doctor
of
Natural
Sciences 









presented
by
Master
of
Science
in
Molecular
Bioengineering
Julia
Ritzerfeld 
born
in
Berlin 




oral
examination:




IDENTIFICATIO N
O F
CO M PO NENTS
O F
TH E
INTRACELLU LAR
TRANSPO RT
M ACH INERY
O F
ACYLATED
PRO TEINS
BY
A
GENO M E‐W IDE
RNAI
SCREEN






























Referees:
Prof.
Dr.
Walter
Nickel
Prof.
Dr.
Thomas
Söllner 


LIST
O F
PU BLICATIO NS

To urnaviti,
S.,
Hannemann,
 S.,
Terjung,
 S.,
Kitzing,
 T.M.,
 Stegmayer,
C.,
Ritzerfeld,
 J.,
Walther,
P.,
 Grosse,
 R.,
Nickel,
W.,
 and
 Fackler,
O.T.
(2007).
Voir plus Voir moins




IDENTIFICATIO N
O F
CO M PO NENTS
O F
TH E

INTRACELLU LAR
TRANSPO RT
M ACH INERY

O F
ACYLATED
PRO TEINS
BY
A
GENO M E‐
W IDE
RNAI
SCREEN
















DISSERTATIO N







submitted
to
the

Combined
Faculties
for
the
Natural
Sciences
and
for
Mathematics 

of
the
Ruperto
Caro la
University
of
Heidelberg,
Germany 

for
the
degree
of
Doctor
of
Natural
Sciences 






Julia
Ritzerfeld 

2009

















































“Ich
weiß,
dass
ich
n ichts
weiß.”

Sokra tes







DISSERTATIO N















submitted
to
the

Combined
Fac ulties
for
the
Natural
Sciences
and
for
Mathematics

of
the
Ruperto
Carola
University
of
Heidelberg,
Germany

for
the
degree
of
Doctor
of
Natural
Sciences 










presented
by

Master
of
Science
in
Molecular
Bioengineering

Julia
Ritzerfeld 

born
in
Berlin 





oral
examination:






IDENTIFICATIO N
O F
CO M PO NENTS
O F
TH E

INTRACELLU LAR
TRANSPO RT
M ACH INERY

O F
ACYLATED
PRO TEINS
BY
A
GENO M E‐
W IDE
RNAI
SCREEN































Referees:

Prof.
Dr.
Walter
Nickel

Prof.
Dr.
Thomas
Söllner 




LIST
O F
PU BLICATIO NS


To urnaviti,
S.,
Hannemann,
 S.,
Terjung,
 S.,
Kitzing,
 T.M.,
 Stegmayer,
C.,

Ritzerfeld,
 J.,
Walther,
P.,
 Grosse,
 R.,
Nickel,
W.,
 and
 Fackler,
O.T.
(2007).

SH4‐domain‐induced
 plasma
 membrane
 dynamization
promotes
 bleb‐
associated
cell
motility.
J
Cell
Sci 
120,
3820 ‐3829.

Remmele,
S.,
Ritzerfeld,
J .,
Nickel,
W.
and
Hesser,
J.
(2008):
Automated
cell

analysis
tool
for
a
genome ‐wide
RNAi
screen.
MIAAB
(attached
in

appendix)

Tournaviti,
S.,
Pietro,
 E.S.,
 Terjung,
 S.,
Schafmeier,
 T.,
Wegehingel,
S.,
Ritzerfeld,

J.,
Schulz,
 J.,
Smith,
D.F.,
Pepperkok,
R.,
and
 Nickel,
W.
 (2009).
Reversible

phosphorylation
as
 a
 molecular
switch
to
 regulate
plasma
 membrane

targeting
of
acylated
SH4
domain
proteins.
Traffic 
10,
1047‐1060.



Table
of
Contents 

SUMMARY 
 1

ZUSAMMENFASSUNG 
 2

1
 INTRODUCTION 
 3

1.1 
 PRO TEIN
SECRETIO N
AND
ENDO CYTO SIS
 3

1 .1 .1 
 T he
Classical
Secretory
Pathw ay
 4

1 .1 .2 
 Un con ven tion al
Protein 
Secretion 
 7

1 .1 .3 
 En docytosis
 8

1.2 
 PO ST‐TRANSLATIO NAL
LIPID
M O DIFICATIO NS
AND
TH EIR
EFFECT
O N
PRO TEIN
TARGETING

 
 8

1 .2.1 
 G lycosylphosphatidylin ositol‐an chored
Protein s
 8

1 .2.2 
 Protein 
Pren ylation 
 9

1 .2.3 
 N ‐M yristoylation 
of
Protein s
 9

1 .2.4
 Protein 
Palm itoylation 
 1 1 

1.3 
 INTRACELLU LAR
TRANSPO RT
O F
ACYLATED
SH4‐DO M AIN‐CO NTAINING
PRO TEINS
 14 

1 .3.1 
 Src
Fam ily
Kin ases
 1 5

1 .3.2 
 Leishm an ia
Hydrophilic
A cylated
Surface
Protein 
B
 1 8

1.4 
 M EM BRANE
M ICRO DO M AINS
 19

1 .4.1 
 T he
Role
of
M em bran e
M icrodom ain s
in 
Sign al
T ran sduction 
 21 

1 .4.2 
 T he
Role
of
M em bran e
M icrodom ain s
in 
In tracellular
T ran sport
 22 

1.5 
 RNA 
INTERFERENCE
 25 

1 .5.1 
 M olecular
M echan ism 
of
RN A 
In terferen ce
 25

1 .5.2 
 RN A i‐based
Screen in g
A pproaches
 27

1.6 
 AIM 
O F
TH IS
TH ESIS
 27

2
 MATERIALS
AND
METHOD S
 29

2.1 
 M ATERIALS
 29

2.1 .1 
 Chem icals
an d
Con sum ables
 29

2.1 .2 
 En zym es
 30

2.1 .3 
 M olecular
Biological
an d
Biochem ical
Kits
 30

2.1 .4
 T echn ical
Devices
 30

2.2 
 M O LECU LAR
BIO LO GICAL
M ETH O DS
 31

2.2.1 
 Polym erase
Chain 
Reaction 
(PCR)
 31 

2.2.2 
 Restriction 
Digests
 32 

2.2.3 
 Hybridization 
of
sin gle‐stran ded
Oligon ucleotides
 32 

2.2.4
 Ligation 
of
DN A 
Fragm en ts
 33 

2.2.5
 T ran sform ation 
of
Com peten t
Bacteria
 33 

2.2.6 
 Preparation 
of
Plasm id
DN A 
from 
Bacteria
 34

2.2.7
 A garose
G el
Electrophoresis
 34

2.2.8
 Clon in g
of
DN A 
con structs
 34

i
Table
of
Contents 

2.2.9
 DN A 
sequen cin g
 35

2.3 
 CELL
CU LTU RE
TECH NIQU ES
 35 

2.3.1 
 M ain ten an ce
of
Stable
Cel
Lin es
 35

2.3.2 
 Freezin g
an d
T haw in g
of
Cels
 36 

2.3.3 
 Lipofectin ‐based
T ran sfection 
of
M am m alian 
Cels
 36 

2.3.4
 Retroviral
T ran sduction 
of
M am m alian 
Cells
 36 

2.3.5
 Fluorescen ce‐activated
Cell
Sortin g
 37

2.4 
 RNA 
TECH NIQU ES
 38

2.4.1 
 Liquid‐phase
T ran sfection 
of
M am m alian 
Cels
w ith
siRN A s
 38

2.4.2 
 Reverse
T ran sfection 
of
M am m alian 
Cels
w ith
siRN A s
 38

2.4.3 
 Preparation 
of
siRN A ‐coated
m ulti‐w ell
Plates
 38

2.4.4
 Preparation 
of
siRN A ‐coated
LabT eks
 39

2.5
 LIVE‐CELL
FLU O RESCENCE
M ICRO SCO PY
 40

2.5.1 
 W idefield
an d
Con focal
Fluorescen ce
M icroscopy
 40

2.5.2 
 A utom ated
W idefield
Fluorescen ce
M icroscopy
 40

2.6 
 M ICRO SCO PY‐BASED
RNA I
SCREENING
 41 

2.6.1 
 Prim ary
Screen in g
on 
384‐spot
siRN A 
A rrays
 41 

2.6.2 
 Validation 
Screen in g
on 
siRN A ‐coated
m ulti‐w el
Plates
 42 

2.7 
 ANALYSIS
O F
H IGH ‐CO NTENT
SCREENING
DATA
 43

2.7.1 
 Im age
A n alysis
w ith
the
A utom ated
Sin gle‐cel
A n alysis
T ool
 43 

2.7.2 
 Spreadsheet
A n alysis
of
Cell
Classification 
Results
 44

3
 RESULTS
 45

3.1 
 G ENERATIO N
AND
CH ARACTERIZATIO N
O F
H U M AN
CELL
LINES
EX PRESSING
SH4‐
DO M AIN‐CO NTAINING
R EPO RTER
PRO TEINS
 45

3.1 .1 
 G en eration 
of
SH4‐dom ain ‐con tain in g
Reporter
Con structs
 45

3.1 .2 
 G en eration 
of
Kyoto
Cel
Lin es
Expressin g
SH4‐dom ain ‐con tain in g
Reporter

Protein s
 
 49

3.1 .3 
 Characterization 
of
Kyoto
Cel
Lin es
Expressin g
SH4‐dom ain ‐con tain in g

Reporter
Protein s
by
Flow 
Cytom etry
 50

3.1 .4
 G en eration 
of
HeLa
Cell
Lin es
Expressin g
SH4‐dom ain ‐con tain in g
Reporter

Protein s
 
 52 

3.1 .5
 Characterization 
of
HeLa
Cel
Lin es
Expressin g
SH4‐dom ain ‐con tain in g

Reporter
Protein s
by
Flow 
Cytom etry
 52 

3.1 .6 
 Characterization 
of
HeLa
Cel
Lin es
Expressin g
SH4‐dom ain ‐con tain in g

Reporter
Protein s
by
Con focal
an d
W idefield
M icroscopy
 55

3.1 .7
 In hibition 
of
Palm itoylation 
in 
HeLa
Cell
Lin es
Expressin g
SH4‐dom ain ‐
con tain in g
Reporter
Protein s
 60

3.2 
 ESTABLISH M ENT
O F
AN
AU TO M ATED
IM AGE
ANALYSIS
TO O L
 65

3.2.1 
 Requirem en ts
for
an 
A utom ated
Im age
A n alysis
T ool
 65

3.2.2 
 Softw are‐based
Cel
Iden tification ,
Segm en tation 
an d
Classification 
 69

3.2.3 
 Validation 
of
the
A utom ated
Im age
A n alysis
T ool
 72 

ii
Table
of
Contents 

3.3 
 G ENO M E‐W IDE
M ICRO SCO PY‐BASED
RNA I‐SCREENING
 76 

3.3.1 
 Experim en tal
Procedure
of
the
G en om e‐w ide
RN A i
Screen 
 76 

3.3.2 
 M icroscopy‐based
RN A i
Screen 
of
the
Hum an 
G en om e
 82 

3.3.3 
 M icroscopy‐based
RN A i
Validation 
Screen 
 86 

3.4 
 EX AM PLES
O F
SCREENING
DATA
FO R
VALIDATED
H ITS
 93

4
 DISCUSSION
 101

4.1 
 G ENERATIO N
AND
CH ARACTERIZATIO N
O F
A
H U M AN
M O DEL
CELL
LINE
FO R

M ICRO SCO PY‐BASED
RNA I
SCREENING
 101

4.2 
 ESTABLISH M ENT
O F
AN
AU TO M ATED
IM AGE
ANALYSIS
TO O L
 103

4.3 
 PERFO RM ANCE
O F
A
G ENO M E‐W IDE
M ICRO SCO PY‐BASED
RNA I
SCREEN
 104

4.3.1 
 Poten tial
Role
of
Coatom er
in 
In tracelular
T ran sport
of
A cylated
Protein s


 
 
 1 05

4.3.2 
 Poten tial
Role
of
Kin ases
an d
Phosphatases
in 
In tracelular
T ran sport
of

A cylated
Protein s
 1 05

4.3.3 
 Poten tial
Role
of
lipid‐hom eostatic
En zym es
in 
In tracelular
T ran sport
of

A cylated
Protein s
 1 07

4.3.4
 Poten tial
Role
of
N A E1 
in 
In tracelular
T ran sport
of
A cylated
Protein s
 1 1 0

4.3.5
 Poten tial
Role
of
PHF5A 
in 
In tracelular
T ran sport
of
A cylated
Protein s
 1 1 1 

4.4 
 CO NCLU SIO N
AND
FU TU RE
PERSPECTIVES
 112 

5 
 APPENDIX 
 114 

5.1 
 ABBREVIATIO NS
 114 

5.2
 CO M PREH ENSIVE
LIST
O F
CELL
LINES
G ENERATED
IN
TH IS
STU DY
 116 

5.3
 CO M PREH ENSIVE
LIST
O F
G ENES
AND
SIRNA S
VALIDATED
IN
TH E
G ENO M E‐W IDE
RNA I

SCREEN
 117 

5.4 
 CO M PREH ENSIVE
LIST
O F
SIRNA S
U SED
FO R
FU RTH ER
ANALYSES
 119 

5.5
 “A U TO M ATED
CELL
ANALYSIS
TO O L
FO R
A
GENO M E‐W IDE
RNA I
SCREEN”
(REM M ELE
ET

AL.,
2008)
 120

6 
 REFERENCES
 128

ACKNO WLEDGEMENTS 
 146 


iii
Summary 

SU M M ARY

Targeting
of
peripheral
 membrane
 proteins
to
different
cellular
 compartments 

is
often
mediated
by
post‐translational
 fatty
acylation.
 For
 example,
N ‐terminal

SH4‐domains
 containing
dual
lipid
 modific ations
mediate
reversible
attachment

to
 intracellular
 membranes
 of
 a
 variety
of
 proteins
such
 as
 the
Src
 family
of

kinases.
 M yristoylation
 and
 subsequent
 palmitoylation
 of
 the
SH4‐domain
 are

not
 only
 required
for
 stable
membrane
 anchoring,
 but
 are
 also
 essential
for

targeting
and
 transport
 to
 the
 plasma
 membrane.
 Their
 association
 with

membrane
 microdomains,
 which
are
enriched
 in
cholesterol
and
 sphingolipids 

and
 moreover
 contain
 a
specific 
set
of
proteins,
is
crucial
for
functionality
 and

provides
 a
 means
 of
 spatio‐temporal
 regulation
of
 acylated
proteins.
Even

though
 many
 SH4‐domain‐containing
 proteins
 are
 functionally 
 quite
well

characterized,
 little
 is
 known
 about
 the
 intracellular
 machinery
 mediating

transport
 to
 the
 plasma
 membrane.
 It
has
 been
 hypothesized
that
their

association
 with
membrane
 microdomains
 already
occurs
 at
 the
 level
of

intracellular
 membranes
 and
 is
a
prerequisite
 for
targeting
and
 transport.
It
was

the
aim
of
this
study
to
identify
 gene
 products
 involved
in
intracellular
 transport

of
acylated
proteins
to
the
plasma
 membrane
 employing
a
 genome ‐wide
RNAi

screen ing
approach.

For
 this
purpose,
 we
 established
a
 stable
human
 model
 cell
line,
which

simultaneously 
expresses
 two
 distinct
plasma‐membrane ‐associated
acylated 

reporter
 proteins.
Th is
 cell
line
was
 adapted
 to
 a
 high‐content
 screening

platform,
which
 is
based
 on
 reverse
 transfection
 of
 a
 genome ‐wide
 siRNA

library
and
 data
acquisition
by
 automated
widefield
 microscopy.
 To
 analyz e

imaging
data
in
an
 unbiased
and
 quantitative
 manner,
 an
 automated
single‐cell

image
analysis
tool
was
 developed.
This
 tool
identifies
 and
 compartmentalizes

individual
 cells
and
 determines
 intensity
distributions
 of
 the
two
 fluorescent

reporter
 proteins,
 thus
 identifying
 experimental
conditions
 under
 which

intracellular
retention
 of
 one
 or
 both
 reporter
 proteins
 occurs .
 Primary

screening
 followed
by
subsequent
 validation
 with
independent
 siRNAs
 resulted

in
the
identification
 of
60
gene
 products,
which
caused
 intracellular
 retention
of

one
 or
 both
acylated
reporter
 proteins.
Interestingly,
 we
 were
 able
to
identify

enzymes
 involved
 in
 lipid
 homeostasis
 and
 microdomain‐associated
proteins.

These
 findings
corroborate
 the
hypothesis
that
partitioning
 into
 membrane

microdomains
 is
a
crucial
 step
in
targeting
and
 transpo rt
of
acylated
 proteins
to

the
 plasma
 membrane. 
 Moreover,
 we
 were
 able
 to
 identify
kinases,

phosphatases
 and
 other
proteins,
which
may
 exert
regulatory
functions
 in
this

process.
 The
 exact
role
of
these
factors
in
transport
 of
SH4‐domain‐containing

proteins
to
the
plasma
membrane
will
be
elucidated
in
further
studies.

1 
Zusammenfassung 

ZU SAM M ENFASSU NG

Membranassoziation
 und
 intrazelluläre
 Sortierung
 peripherer

Membranproteine
 wird
 häufig
 durch
 die
post‐translationale
Anheftung
 von

Fettsäuren
 vermittelt.
 Ein
 Beispiel
 sind
 N ‐terminale
SH4‐Domänen,
 die
duale

Lipidmodifikationen
 enthalten
und
 die
reversible
Membranassoziation,
 zum

Beispiel
von
 Mitgliedern
der
 Familie
der
 Src
 Kinasen ,
 vermitteln.
Eine

Myristoylierung
und
 darauffolgende
Palmitoylierung
 der
SH4‐Domäne
 ist
dabei

nicht
nur
 für
 eine
stabile
 Membranverankerung
 notwendig,
sondern
 ist
zudem

essentiell
für
 den
 intrazellulären
Transport
 zur
 Plasmamembran.
 Ihre

Assoziation
mit
Membranmikrodomänen,
 die
Cholesterin,
Sphingomyelin
und

spezifische
 Proteine
selektiv
anreichern,
ist
 für
 die
Funktion
 von
 Src
 Kinasen

essentiell.
 Während
 die
Funktionen
 der
Src
Kinasen
 detailliert
 erforscht
wurden,

ist
über
 die
intrazelluläre
 Maschinerie,
die
ihren
Transport
 zur
Plasmamembran

vermittelt,
 nur
 wenig
 bekannt.
 Es
 wurde
 die
Hypothese
aufgestellt,
 dass
 die

Assoziation
mit
Mikrodomänen
 bereits
an
 intrazellulären
 Membranen
 erfolgt

und
 eine
Voraussetzung
 für
 den
 Transport
 zur
Plasmamembran
 ist.
Das
 Ziel
 der

vorliegenden
 Arbeit
 war
 es,
 durch
 eine
 auf
 RNA
 Interferenz
basierten,

genomweiten
 Analyse
 Genprodukte
 zu
identifizieren,
 die
am
 intrazellulären

Transport
von
acylierten
Proteinen
zur
Plasmamembran
beteiligt
sind. 

Zu
 diesem
 Zweck
 wurde
 ein
 stabiles
humanes
 Zellsystem
etabliert,
 in
 dem

simultan
 zwei
 unterschiedliche
 plasmamembranassoziierte
 acylierte

Reporterproteine
exprimiert
 werden
 können.
 Diese
Zelllinie
 wurde
 an
 eine
High

Content
 Screening 
Plattform
 adaptiert,
 die
auf
reverser
 Transfektion
 mit
einer

genomweiten
 siRNA ‐Bibliothek
 und
 anschließender
 automatischer

Weitfeldmikroskopie
beruht.
Um
 Bilddaten
objektiv
und
 quantitativ
 auswerten

zu
 können,
 wurde
 ein
 automatisiertes
Bildanalyseprogramm
 entwickelt,

welches
 einzelne
Zellen
erkennen
 und
 kompartimentalisieren
kann.
 Über
 eine

Bestimmung
 der
 intrazellulären
 Intensitätsverteilung
konnten
 experimentelle

Bedingungen
 identifiziert
 werden,
 die
durch
 die
intrazelluläre
 Retention
eines

oder
 beider
Reporterproteine
charakterisiert
 waren.
 Die
Primäranalyse
und
 eine

anschließende
 Validierung
führten
 zur
 Identifizierung
 von
 insgesamt
 60

Genprodukten,
 deren
 RNAi ‐vermittelte
Expressionsunterdrückung
 eine

intrazelluläre
Retention
 eines
 oder
 beider
 Reporterproteine
 hervorrief.

Interessanterweise
 konnten
 wir
 dabei
Enzyme
 identifizieren,
 die
an
 der

zellulären
Lipidhomeostase
beteiligt
 sind.
 Diese
Ergebnisse
 untermauern
 die 

Hypothese,
dass
 eine
Assoziation
mit
Membranmikrodomänen
 ein
essentieller

Schritt
 im
Transport
 von
 acylierten
 Proteinen
zur
Plasmamembran
 ist.
Darüber

hinaus
 haben
 wir
 verschiedene
 Kinasen,
 Phosphatasen
 und
 andere
 Proteine

identifiziert,
 die
ei ne
 regulatorische
Funktion
 in
 diesem
 Prozess
 spielen

könnten.
 Die
 genaue
 Rolle
 dieser
 Faktoren
 im
 Transport
 von
 SH4‐
domänenhaltigen
 Proteinen
zur
Plasmamembran
 kann
 nun
 in
weiterführenden

Arbeiten
untersucht
werden. 

2


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