Structural and functional analyses of microtubule nucleation:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Heidelberg
2020
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | IX, 108 Seiten Illustrationen 30 cm |
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| 100 | 1 | |a Liu, Peng |d 1988- |e Verfasser |0 (DE-588)1196704945 |4 aut | |
| 245 | 1 | 0 | |a Structural and functional analyses of microtubule nucleation |c Peng Liu |
| 264 | 1 | |a Heidelberg |c 2020 | |
| 300 | |a IX, 108 Seiten |b Illustrationen |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität Heidelberg |d 2020 | ||
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| adam_text | TABLE
OF
CONTENTS
SUMMARY
....................................................................................................................
IV
ZUSAMMENFASSUNG
......................................................................................................
V
ABBREVIATIONS
............................................................................................................
VII
1.
INTRODUCTION
............................................................................................................
1
1.1
CYTOSKELETON
......................................................................................................
1
1.2
MICROTUBULES
......................................................................................................
2
1.2.1
THE
DISCOVERY
OF
MICROTUBULES.....................................................................
2
1.2.2
THE
CHARACTERISTICS
OF
MICROTUBULES
.............................................................
2
1.2.3
MICROTUBULE
DYNAMICS
.................................................................................
2
1.3
MAPS
.................................................................................................................
4
1.4
TUBULIN
POST-TRANSLATIONAL
MODIFICATIONS
.............................................................
5
1.5
MTOCS
..............................................................................................................
5
1.6
MICROTUBULE
NUCLEATION
AND
RELATED
PROTEINS
.....................................................
6
1.6.1
Y-TUBULIN
.......................................................................................................
7
1.6.2
Y-TUCS
.........................................................................................................
8
1.6.3
Y-TURC-ASSOCIATED
PROTEINS
........................................................................
9
1.6.3.1
NEDD1
...............................................................................................
10
1.6.3.2
CDK5RAP2
.........................................................................................
11
1.6.3.3
NME7..................................................................................................
13
1.6.3.4
MOZART
1
..........................................................................................
14
1.6.3.5
MOZART2
..........................................................................................
14
1.6.3.6
RANGTP
AND
NUPS
............................................................................
16
1.6.3.7
TPX2
...................................................................................................
17
1.6.3.8
THE
AUGMIN
COMPLEX
..........................................................................
19
1.6.3.9
BRANCHING
MICROTUBULE
NUCLEATION........................................................
20
1.6.3.10
XMAP215
.........................................................................................
22
1.6.3.11
MICROTUBULES
AT
THE
GOLGI
APPARATUS
................................................
23
1.7
STRUCTURAL
ANALYSES
OF
Y-TUCS
..........................................................................
24
1.7.1
THE
STRUCTURE
OF
S.
CEREVISIAE
Y-TUSC
......................................................
24
1.7.2
THE
STRUCTURE
OF
S.
CEREVISIAE
Y-TUSC
RING...............................................
25
1.7.3
Y-TUCS
IN
S.
POMBE
..................................................................................
26
1.7.4
THE
STRUCTURE
OF
CANDIDA
ALBICANS
Y-TUSC
................................................
27
1.7.5
STRUCTURAL
ANALYSIS
OF
D.
MELANOGASTER
Y-TURC
.......................................
28
1.7.6
STRUCTURAL
ANALYSES
OF
Y-TURCS
FROM
VERTEBRATES
.....................................
29
1.7.7
Y-TUC-CAPPED
MICROTUBULES
......................................................................
31
1.8
STOICHIOMETRIES
OF
VERTEBRATE
Y-TURC
COMPONENTS
........................................
32
1.9
MICROTUBULE
NUCLEATING
MODELS
........................................................................
32
1.10
KEY
MATERIALS
AND
TOOLS
IN
THIS
STUDY
..............................................................
33
1.10.1
X.
LAEVIS
EGG
EXTRACTS
..............................................................................
33
1.10.2
ELECTRON
MICROSCOPY
................................................................................
35
1.11
AIM
OF
THIS
STUDY
...........................................................................................
36
1.12
BIOLOGICAL
AND
TRANSLATIONAL
SIGNIFICANCE
.......................................................
36
2.
RESULTS
.................................................................................................................
38
2.1.
Y-TURC
PURIFICATION
FROM
X.
LAEVIS
EGG
EXTRACTS
.............................................
38
2.2
QUALITY
CONTROL
OF
PURIFIED
Y-TURCS
....................
39
2.2.1
MASS
SPECTROMETRY
ANALYSIS
OF
PURIFIED
Y-TURCS
.....................................
39
2.2.2
ABANDUNCE
OF
Y-TURC
COMPONENTS
.........................................................
41
2.2.3
NEGATIVE
STAIN
EM
ANALYSIS
OF
PURIFIED
Y-TURCS
......................................
42
2.2.4
PURIFIED
Y-TURCS
SHOW
BASAL
NUCLEATION
ACTIVITY
......................................
43
2.2.5
FURTHER
PURIFICATION
OF
THE
Y-TURCS
WITH
GEL
FILTRATION
CHROMATOGRAPHY....
44
2.2.6
CROSS-LINKING
OF
PURIFIED
Y-TURCS
.............................................................
45
2.2.7
FURTHER
PURIFICATION
OF
THE
Y-TURCS
WITH
SUCROSE
GRADIENT
.......................
46
2.3
CRYO-EM
ANALYSES
OF
THE
Y-TURC
...................................................................
47
2.3.1
STARTING
MODELS
OF
THE
Y-TURC
................................................................
47
2.3.2
THE
Y-TURC
CRYO-EM
MODEL
WITH
AN
ANISOTROPIC
RESOLUTION
.....................
48
2.3.3
GRIDS
OPTIMIZATION
FOR
CRYO-EM
ANALYSIS
...................................................
49
2.3.4
CRYO-EM
STRUCTURE
OF
THE
Y-TURC
.............................................................
51
2.4
FUNCTIONAL
ANALYSES
OF
PURIFIED
Y-TURCS
........................................................
53
2.4.1
BIOCHEMICAL
ANALYSIS
OF
THE
ACTIN
IN
THE
Y-TURC
......................................
53
2.4.2
FUNCTIONAL
ANALYSIS
OF
GCP6-ID0
.............................................................
53
2.4.2.1
DOMAIN
ORGANIZATION
OF
GCP6-ID0
..................................................
54
2
4.2.2
GCP6-ID0
BINDS
TO
GCP2
AND
GCP5
................................................
56
2.5
ASSEMBLY
OF
THE
Y-TURC
.................................................................................
57
2.6
THE
MAKING
OF
*
ACTIVE
*
Y-TURCS
.....................................................................
59
2.6.1
MISMATCHES
BETWEEN
THE
Y-TURC
AND
MICROTUBULES
................................
59
2.6.2
CEP215
DOES
NOT
AFFECT
THE
CONFORMATION
OF
PURIFIED
Y-TURCS
...............
59
2.6.3
CEP215
REQUIRES
CO-FACTORS
TO
PROMOTE
MICROTUBULE
NUCLEATION
.............
60
2.7
MOZART
1
IS
INDISPENSABLE
FOR
THE
ACTIVITY
Y-TURC
........................................
62
2.8
NEDD1
DEPLETION
DOES
NOT
AFFECT
THE
INTEGRITY
AND
ACTIVITY
OF
Y-TURC
...........
63
3.
DISCUSSION
...........................................................................................................
65
3.1
THE
Y-TURC
CRYO-EM
STRUCTURE
......................................................................
65
3.1.1
LOCATION
OF
GCP4-5-4-6
WITHIN
THE
Y-TURC
..............................................
65
3.1.2
POTENTIAL
FUNCTIONS
OF
GCP4
AND
GCP5
..................................................
65
3.1.3
GCP6-ID0:
*
SNOW
WHITE
AND
THE
SEVEN
DWARFS
*
.....................................
66
3.1.4
TO
*
LOCK
*
THE
Y-TURC
................................................................................
67
3.2
ASSEMBLY
PROCESS
OF
THE
Y-TURC
...................................................................
67
3.3
ACTIN
IN
THE
Y-TURC........................................................................................
68
3.4
Y-TUBULIN:
A
*
GTPASE
*
?
...................................................................................
69
3.5
Y-TUBULIN
AS
THE
CENTRAL
PLAYER
IN
MICROTUBULE
NUCLEATION
................................
69
3.6
Y-TURC-ASSOCIATED
FACTORS
.............................................................................
70
3.6.1
MISSING
FACTORS
IN
THE
CRYO-EM
RECONSTRUCTION.........................................
70
3.6.2
MISSING
FACTORS
IN
MASS
SPECTROMETRY:
*
TO
BIND
OR
NOT
TO
BIND,
THIS
IS
A
QUESTION
*
...........................................................................................................
70
II
3.6.3
CDK5RAP2
IN
X.
LAEVIS
EGG
EXTRACTS
.......................................................
71
3.6.4
TWO
CLASSES
OF
NEDD1
.............................................................................
71
3.7
ADDITIONAL
FUNCTIONS
OF
Y-TURC
........................................................................
72
3.8
HYPOTHESIS
FOR
MICROTUBULE
NUCLEATION
.............................................................
72
3.9
MICROTUBULE
NUCLEATION:
S.
CEREVISIAE
VS.
VERTEBRATES
.....................................
72
3.10
CLOSING
REMARKS
AND
PERSPECTIVES
.................................................................
73
4.
MATERIALS
AND
METHODS
..........................................................................................
74
4.1
PLASMIDS
..........................................................................................................
74
4.2
ANTIBODIES
........................................................................................................
74
4.3
Y-TURC
PURIFICATION
..........................................................................................
76
4.4
Y-TURC
SALT
TREATMENT
.....................................................................................
77
4.5
IMMUNOPRECIPITATION
........................................................................................
78
4.6
SILVER
STAINING
..................................................................................................
78
4.7
IMMUNOBLOTTING
................................................................................................
79
4.8
IN
VITRO
MICROTUBULE
NUCLEATION
.........................................................................
79
4.9
DNASEL
INHIBITION
ASSAY
...................................................................................
80
4.10
SUCROSE
GRADIENT
............................................................................................
80
4.11
PROTEIN
PURIFICATION.........................................................................................
81
4.12
PULL-DOWN
ASSAY
.............................................................................................
81
4.13
CEP215N
ACTIVITY
ASSAY
IN
X.
LAEVIS
EGG
EXTRACTS
.........................................
82
4.14
NEGATIVE
STAIN
EM..........................................................................................
82
4.15
PLUNGE
FREEZING
..............................................................................................
82
4.16
MICROTUBULE
REGROWTH
ASSAY
...........................................................................
82
5.
PUBLICATIONS
AND
POSTER
PRESENTATIONS...................................................................
84
6.
REFERENCES
............................................................................................................
85
7.
ACKNOWLEDGMENT
.................................................................................................
104
ERKLARUNG
............................
108
III
|
| adam_txt |
TABLE
OF
CONTENTS
SUMMARY
.
IV
ZUSAMMENFASSUNG
.
V
ABBREVIATIONS
.
VII
1.
INTRODUCTION
.
1
1.1
CYTOSKELETON
.
1
1.2
MICROTUBULES
.
2
1.2.1
THE
DISCOVERY
OF
MICROTUBULES.
2
1.2.2
THE
CHARACTERISTICS
OF
MICROTUBULES
.
2
1.2.3
MICROTUBULE
DYNAMICS
.
2
1.3
MAPS
.
4
1.4
TUBULIN
POST-TRANSLATIONAL
MODIFICATIONS
.
5
1.5
MTOCS
.
5
1.6
MICROTUBULE
NUCLEATION
AND
RELATED
PROTEINS
.
6
1.6.1
Y-TUBULIN
.
7
1.6.2
Y-TUCS
.
8
1.6.3
Y-TURC-ASSOCIATED
PROTEINS
.
9
1.6.3.1
NEDD1
.
10
1.6.3.2
CDK5RAP2
.
11
1.6.3.3
NME7.
13
1.6.3.4
MOZART
1
.
14
1.6.3.5
MOZART2
.
14
1.6.3.6
RANGTP
AND
NUPS
.
16
1.6.3.7
TPX2
.
17
1.6.3.8
THE
AUGMIN
COMPLEX
.
19
1.6.3.9
BRANCHING
MICROTUBULE
NUCLEATION.
20
1.6.3.10
XMAP215
.
22
1.6.3.11
MICROTUBULES
AT
THE
GOLGI
APPARATUS
.
23
1.7
STRUCTURAL
ANALYSES
OF
Y-TUCS
.
24
1.7.1
THE
STRUCTURE
OF
S.
CEREVISIAE
Y-TUSC
.
24
1.7.2
THE
STRUCTURE
OF
S.
CEREVISIAE
Y-TUSC
RING.
25
1.7.3
Y-TUCS
IN
S.
POMBE
.
26
1.7.4
THE
STRUCTURE
OF
CANDIDA
ALBICANS
Y-TUSC
.
27
1.7.5
STRUCTURAL
ANALYSIS
OF
D.
MELANOGASTER
Y-TURC
.
28
1.7.6
STRUCTURAL
ANALYSES
OF
Y-TURCS
FROM
VERTEBRATES
.
29
1.7.7
Y-TUC-CAPPED
MICROTUBULES
.
31
1.8
STOICHIOMETRIES
OF
VERTEBRATE
Y-TURC
COMPONENTS
.
32
1.9
MICROTUBULE
NUCLEATING
MODELS
.
32
1.10
KEY
MATERIALS
AND
TOOLS
IN
THIS
STUDY
.
33
1.10.1
X.
LAEVIS
EGG
EXTRACTS
.
33
1.10.2
ELECTRON
MICROSCOPY
.
35
1.11
AIM
OF
THIS
STUDY
.
36
1.12
BIOLOGICAL
AND
TRANSLATIONAL
SIGNIFICANCE
.
36
2.
RESULTS
.
38
2.1.
Y-TURC
PURIFICATION
FROM
X.
LAEVIS
EGG
EXTRACTS
.
38
2.2
QUALITY
CONTROL
OF
PURIFIED
Y-TURCS
.
39
2.2.1
MASS
SPECTROMETRY
ANALYSIS
OF
PURIFIED
Y-TURCS
.
39
2.2.2
ABANDUNCE
OF
Y-TURC
COMPONENTS
.
41
2.2.3
NEGATIVE
STAIN
EM
ANALYSIS
OF
PURIFIED
Y-TURCS
.
42
2.2.4
PURIFIED
Y-TURCS
SHOW
BASAL
NUCLEATION
ACTIVITY
.
43
2.2.5
FURTHER
PURIFICATION
OF
THE
Y-TURCS
WITH
GEL
FILTRATION
CHROMATOGRAPHY.
44
2.2.6
CROSS-LINKING
OF
PURIFIED
Y-TURCS
.
45
2.2.7
FURTHER
PURIFICATION
OF
THE
Y-TURCS
WITH
SUCROSE
GRADIENT
.
46
2.3
CRYO-EM
ANALYSES
OF
THE
Y-TURC
.
47
2.3.1
STARTING
MODELS
OF
THE
Y-TURC
.
47
2.3.2
THE
Y-TURC
CRYO-EM
MODEL
WITH
AN
ANISOTROPIC
RESOLUTION
.
48
2.3.3
GRIDS
OPTIMIZATION
FOR
CRYO-EM
ANALYSIS
.
49
2.3.4
CRYO-EM
STRUCTURE
OF
THE
Y-TURC
.
51
2.4
FUNCTIONAL
ANALYSES
OF
PURIFIED
Y-TURCS
.
53
2.4.1
BIOCHEMICAL
ANALYSIS
OF
THE
ACTIN
IN
THE
Y-TURC
.
53
2.4.2
FUNCTIONAL
ANALYSIS
OF
GCP6-ID0
.
53
2.4.2.1
DOMAIN
ORGANIZATION
OF
GCP6-ID0
.
54
2
4.2.2
GCP6-ID0
BINDS
TO
GCP2
AND
GCP5
.
56
2.5
ASSEMBLY
OF
THE
Y-TURC
.
57
2.6
THE
MAKING
OF
*
ACTIVE
*
Y-TURCS
.
59
2.6.1
MISMATCHES
BETWEEN
THE
Y-TURC
AND
MICROTUBULES
.
59
2.6.2
CEP215
DOES
NOT
AFFECT
THE
CONFORMATION
OF
PURIFIED
Y-TURCS
.
59
2.6.3
CEP215
REQUIRES
CO-FACTORS
TO
PROMOTE
MICROTUBULE
NUCLEATION
.
60
2.7
MOZART
1
IS
INDISPENSABLE
FOR
THE
ACTIVITY
Y-TURC
.
62
2.8
NEDD1
DEPLETION
DOES
NOT
AFFECT
THE
INTEGRITY
AND
ACTIVITY
OF
Y-TURC
.
63
3.
DISCUSSION
.
65
3.1
THE
Y-TURC
CRYO-EM
STRUCTURE
.
65
3.1.1
LOCATION
OF
GCP4-5-4-6
WITHIN
THE
Y-TURC
.
65
3.1.2
POTENTIAL
FUNCTIONS
OF
GCP4
AND
GCP5
.
65
3.1.3
GCP6-ID0:
*
SNOW
WHITE
AND
THE
SEVEN
DWARFS
*
.
66
3.1.4
TO
*
LOCK
*
THE
Y-TURC
.
67
3.2
ASSEMBLY
PROCESS
OF
THE
Y-TURC
.
67
3.3
ACTIN
IN
THE
Y-TURC.
68
3.4
Y-TUBULIN:
A
*
GTPASE
*
?
.
69
3.5
Y-TUBULIN
AS
THE
CENTRAL
PLAYER
IN
MICROTUBULE
NUCLEATION
.
69
3.6
Y-TURC-ASSOCIATED
FACTORS
.
70
3.6.1
MISSING
FACTORS
IN
THE
CRYO-EM
RECONSTRUCTION.
70
3.6.2
MISSING
FACTORS
IN
MASS
SPECTROMETRY:
*
TO
BIND
OR
NOT
TO
BIND,
THIS
IS
A
QUESTION
*
.
70
II
3.6.3
CDK5RAP2
IN
X.
LAEVIS
EGG
EXTRACTS
.
71
3.6.4
TWO
CLASSES
OF
NEDD1
.
71
3.7
ADDITIONAL
FUNCTIONS
OF
Y-TURC
.
72
3.8
HYPOTHESIS
FOR
MICROTUBULE
NUCLEATION
.
72
3.9
MICROTUBULE
NUCLEATION:
S.
CEREVISIAE
VS.
VERTEBRATES
.
72
3.10
CLOSING
REMARKS
AND
PERSPECTIVES
.
73
4.
MATERIALS
AND
METHODS
.
74
4.1
PLASMIDS
.
74
4.2
ANTIBODIES
.
74
4.3
Y-TURC
PURIFICATION
.
76
4.4
Y-TURC
SALT
TREATMENT
.
77
4.5
IMMUNOPRECIPITATION
.
78
4.6
SILVER
STAINING
.
78
4.7
IMMUNOBLOTTING
.
79
4.8
IN
VITRO
MICROTUBULE
NUCLEATION
.
79
4.9
DNASEL
INHIBITION
ASSAY
.
80
4.10
SUCROSE
GRADIENT
.
80
4.11
PROTEIN
PURIFICATION.
81
4.12
PULL-DOWN
ASSAY
.
81
4.13
CEP215N
ACTIVITY
ASSAY
IN
X.
LAEVIS
EGG
EXTRACTS
.
82
4.14
NEGATIVE
STAIN
EM.
82
4.15
PLUNGE
FREEZING
.
82
4.16
MICROTUBULE
REGROWTH
ASSAY
.
82
5.
PUBLICATIONS
AND
POSTER
PRESENTATIONS.
84
6.
REFERENCES
.
85
7.
ACKNOWLEDGMENT
.
104
ERKLARUNG
.
108
III |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Liu, Peng 1988- |
| author_GND | (DE-588)1196704945 |
| author_facet | Liu, Peng 1988- |
| author_role | aut |
| author_sort | Liu, Peng 1988- |
| author_variant | p l pl |
| building | Verbundindex |
| bvnumber | BV046996991 |
| ctrlnum | (OCoLC)1196228763 (DE-599)DNB1217738215 |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV046996991 |
| illustrated | Illustrated |
| index_date | 2024-07-03T15:55:26Z |
| indexdate | 2024-07-10T08:59:41Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032404730 |
| oclc_num | 1196228763 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | IX, 108 Seiten Illustrationen 30 cm |
| publishDate | 2020 |
| publishDateSearch | 2020 |
| publishDateSort | 2020 |
| record_format | marc |
| spelling | Liu, Peng 1988- Verfasser (DE-588)1196704945 aut Structural and functional analyses of microtubule nucleation Peng Liu Heidelberg 2020 IX, 108 Seiten Illustrationen 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Heidelberg 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1217738215/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032404730&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Liu, Peng 1988- Structural and functional analyses of microtubule nucleation |
| subject_GND | (DE-588)4113937-9 |
| title | Structural and functional analyses of microtubule nucleation |
| title_auth | Structural and functional analyses of microtubule nucleation |
| title_exact_search | Structural and functional analyses of microtubule nucleation |
| title_exact_search_txtP | Structural and functional analyses of microtubule nucleation |
| title_full | Structural and functional analyses of microtubule nucleation Peng Liu |
| title_fullStr | Structural and functional analyses of microtubule nucleation Peng Liu |
| title_full_unstemmed | Structural and functional analyses of microtubule nucleation Peng Liu |
| title_short | Structural and functional analyses of microtubule nucleation |
| title_sort | structural and functional analyses of microtubule nucleation |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1217738215/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032404730&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT liupeng structuralandfunctionalanalysesofmicrotubulenucleation |
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