Localization and functional role of RIM3gamma and RIM4gamma, the small members of the RIM protein family [Elektronische Ressource] / Elena Álvarez-Barón Fuentes

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2010
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Localization and functional role of RIM3γ
and RIM4γ, the small members of the RIM
protein family


Dissertation


zur


Erlangung
des
Doktorgrades
(Dr.
rer.nat.)



Mathematisch-Naturwissenschaftlichen
Fakultät



Rheinischen
Friedrich-Wilhelms-Universität
Bonn





vorgelegt
von

Elena
Álvarez-Barón
Fuentes



Palencia,
Spain



Bonn,
2010



aus
der
derAngefertigt
 im
 Institut
 für
 Neuropathologie
 am
 Universitätsklinikum

Bonn
mit
der
Genehmigung
der
Mathematisch -Naturwissenschaftlichen

Fakultät
der
Rheinischen
Friedrich-Wilhelms-Universität
Bonn.



























1.
Referent:
Prof.
Dr.
Susanne
Schoch


2.
Referent:
Prof.
Dr.
Albert
Haas











Tag
der
mündlichen
Prüfung:
25.
März
2010


Erscheinungsjahr:
2010










Diese
Dissertation
ist
auf
dem
Hochschulschriftenserver
der
ULB
Bonn

http://hss.ulb.-ubnoinn.de/diss_online
elektronisch
publizier
t.

ERKLÄRUNG 





Diese
 Dissertation
 wurde
 im
 Sinne
 von
 §
 4
 der
 Promotionsordnung
 vom

7.1.2004
am
Institut
für
Neuropathologie
und
Klinik
für
Epilepsie
der
Universität

Bonn
unter
der
Leitung
von
Frau
Prof.
S.
Schoch
angefertigt. 




Hiermit
versichere
ich,
dass
ich
die
vorliegende
Arbeit
selbständig
angefertigt

habe
und
keine
weiteren
als
die
angegebenen
Hilfsmitt el
und
Quelle
verwendet

habe,
die
gemäß
§
6
der
Promotionsordnung
kenntlich
gemacht
sind.
 























































Bonn, den ______________________________
Elena Alvarez-Baron









































A mis padres


 V

TABLE
OF
CONTENTS 

ERKLÄRUNG ________________________________ ______________ III 

TABLE
OF
CONTENTS ________________________________ ______ V

ABSTRACT ________________________________ _______________ XI

1
 INTRODUCTION ________________________________ _________ 1

1.1
 The
synaptic
vesicle
cycle________________________________ ____ 2 

1.2 
 The
presynaptic
Active
Zone __4 

1.3 
The
molecular
machinery
mediating
synaptic
vesicle
exocytosis
at

the
AZ ______________________ 6

1.3.1 
 The
core
fusion
machinery _______________ 7

1.3.1.1 
 SNARES
and
SNARE
regulators ________________________________ ______ 7

1.3.1.2 
 Munc18 ____________________________ 9

1.3.1.3 
 Rab3 ________________________________ _____________________________ 10

1.3.2 
 Active
Zone
enriched
proteins ___________ 11

1.3.2.1 
 Bassoon
and
Piccolo/Azconin ________ 12

1.3.2.2 
 ELKS/ERC/CAST __________________ 14

1.3.2.3 
 Lipri-nα ___________________________ 16

1.3.2.4 
 Munc13 17

1.3.2.5 
 RIM ________________________________ ______________________________ 19

1.4 
RIMs:
main
compone nts
of
the
scaffolding
at
the
presynaptic
Active

Zone _______________________ 20

1.4.1 
 Genomic
sequence
organization :
splice
variants
and
structure _______________ 20

1.4.2 
 Protein
structure
and
functional
domains________________________________ __21

1.4.3 
 Interaction
partners _____________________ 25

1.4.3.1 
 Proteins
interacting
with
the-
tNerminal
zinc
finger
domai_______________n 25

1.4.3.2 
 Proteins
interacting
with
the
charged
domai___________________________n 26

1.4.3.3 
 Proteins
interacting
with
the
PDZ
domain______________________________ 27

1.4.3.4 
 Proteins
interacting
with
the
C2
domains 27

 VI

1.4.4 
 Role
of-
αRIMs
in
synaptic
function ________________________________ _______ 30

1.4.4.1 
 UNC-10
 deficienCt.
 
elegans _________ 30

1.4.4.2 
 RIM1α
KO
mice ____________________ 32 

1.4.4.3 
 RIM2α
KO
mice 34 

1.4.4.4 
 α-RIM
DKO
mice________________________________ ___________________ 35

1.4.4.5 
 Role
of
RIM1β
in
synaptic
fncution ____35

1.5
 Presynaptic
proteins
in
neurological
diseases_________________ 36

2
 AIMS
OF
THE
STUDY ________________________________ ___ 38

3
 MATERIAL ________________________________ _____________ 40

3.1 
 Equipment ________________________________ _________________ 40

3.2 
 Chemicals __________________ 41

3.3 
 Kits ________________________ 43 

3.4 
 Cell
culture
media __________ 43 

3.5
 Antibodies _________________ 44 

3.6 
 Oligonucleotide________________________________s ____________ 45

3.6.1 
 Sequencing
primer _____________________ 45

3.6.2 
 In
situ
hybridization
oligonucleotides ______ 45

3.6.3 
 shRNA
sequences 46

3.6.4 
 Cloning
primer _________________________ 47

3.7
 Vectors ____________________ 49 

3.7.1
 TOPO
cloning
vectors __________________ 49 

3.7.2 
 Plasmids
for
cell
culture
transfect________________________________ion ______ 52

3.7.3 
 Plasmids
for
lentivirus
production _________ 54

3.8 
 cDNA
and
protein
sequences 58

3.8.1 
 RIM1α________________________________ 58

3.8.1.1 
 RIM1α
cDNA
(rattus
novergicus)________________________________ _____ 58

3.8.1.2 
 RIM1α
protein
r(attus
novergicus) ____60

3.8.2 
 RIM3 γ 60

3.8.2.1 
 RIM3γ
cDNA
(rattus
novergicus) _____ 60

3.8.2.2 
 RIM3γ
protein
(rattus
novergicus) ____61

3.8.3 
 RIM4γ ________________________________ 61

 VII

3.8.3.1 
 RIM4γ
cDNA
(rattus
novergicus)________________________________ _____ 61

3.8.3.2 
 RIM4γ
protein
(rattus
novergicus) ____61

3.8.4 
 Protein
alignment ______________________ 61

3.9 
 URLs ________________________________ 64

4
 METHODS _____________ 65

4.1 
 Molecular
biological
metho________________________________ds 65

4.1.1 
 Preparation
of
competent
bacteria ________ 65

4.1.1.1 
 Electrocompetent __________________ 65

4.1.1.2 
 Chemically
competent ______________ 65

4.1.2 
 Transformation ________________________ 66

4.1.2.1 
 Chemical-transformation ____________ 66

4.1.2.2 
 Electroporation ____________________ 66

4.1.3 
 Bacteria
cultur________________________________e 66

4.1.4 
 DNA
plasmid
purification ________________ 67

4.1.5
 Restriction
digest,
dephosphorylation
and
ligat__________________________ion 67

4.1.6 
 DNA
precipitation ______________________ 68

4.1.7
 DNA
sequencing _______________________ 68

4.1.8 
 PCR
product
purification
and
gel
extract________________________________ion 68

4.1.9 
 Polymerase
chain
reaction ______________ 69

4.1.10 
 cDNA
preparation _____________________ 70

4.1.11 
 Cloning
strategies________________________________ 70

4.1.11.1 
 Overexpression
constructs
in
pcDNA3.1,
pL26
and
pLenti
LN -EGFP -EF1α

plasmids _________________________ 70

4.1.11.2 
 cloning
of
shRNA
sequences
in
the
pLVTHM
vector ____________________ 71

4.2 
 Biochemical
methods ________ 72

4.2.1 
 Generation
of
peptides
antibodies________________________________ 72

4.2.2 
 Western
blot ___________________________ 73

4.2.2.1 
 Preparation
of
protein
extracts _______ 73

4.2.2.2 
 Subcellular
fractionation
of
adulbtr
raaitn______________________________
 74

4.3 
 Cell
cultur________________________________e _________________ 75

4.3.1 
 Eukaryotic
cell
culture __________________ 75

4.3.2 
 Primary
cell
culture _____________________ 76

4.3.2.1 
 Coverslip
treatment 76

 VII
I
4.3.2.2 
 Dissection________________________________ _________________________ 76

4.3.2.3 
 Culture
preparation _________________ 76

4.3.3 
 Transient
transfection
of
mammalian
cells________________________________ _77

4.3.3.1 
 HEK
293T 77

4.3.3.2 
 PC12 ________________________________ _____________________________ 77

4.3.3.3 
 Primary
neurons ___________________ 78

4.4 
 Histological
and
immunohistochemical
method________________s 78

4.4.1 
 Animals
and
brain
sections ______________ 78

4.4.2 
 Immunohistochemical
analyses
on
paraffin
sections ________________________ 79

4.4.3 
 Immunohistochemical
analyses
on
cryosections ____________________________ 79

4.4.4 
 Immunohistochemical
analyses
on
the
Retina ______________________________ 80

4.4.5
 Free-floating
immunohistochemical
analyses_______________________________ 80

4.4.6 
 Immunocytochemistry ________________________________ __________________ 81

4.5
 Lentivirus
production ________ 81

4.5.1
 Lentivirus
based
vector
system __________ 81

4.5.2
 HEK
293T
culture
and
transfection _______ 82

4.5.3 
 Viral
particle
purific________________________________ation _________________ 82

4.5.4
 In
vivo
injection
of
lentivirus
particles ______ 83 

5
 RESULTS ________________________________ ______________ 84

5.1
 RIM3γ
and
RIM4γ ___________ 84

5.1.1
 Localization
of
RIM3γ
and
RIM4________________________________γ _________ 84

5.1.1.1
 RIM3γ
and
RIM4γ
mRNA
expression:
 In
situ
hybridizati_______________on 84

5.1.1.2
 RIM3γ
and
RIM4γ
protein
expressi on _87

5.1.1.2.1 
 Generation
and
characterization
of
isoform
specific
antib________odies 87

5.1.1.2.2 
 Tissue
distribution
of
RIM3γ
and
rim4γ
protein_____________________s 89

5.1.1.2.3 
 Expression
pa ttern
in
different
brain
regi________________________ons 89

5.1.