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

Julia Dalrymple

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait







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