Structure and Function of the
Contractile Vacuoles of
Chlamydomonas
Inaugural
reinhardtii
Dissertation
-
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultat
der Universitat
zu
Koln
vorgelegt von
Karin Komsic-Buchmann
aus
Koln
2013
Table of Content
1
Prolog
I
Introduction
1
1
The Contractile Vacuole
2
1.1
Occurrence
2
1.2
Structural Characteristics
2
1.3
Cellular Processes and Proteins
3
1.3.1
Composition of the CV
Content and Electrochemical Gradient
1.3.2
Water Flow is Mediated by an Aquaporin
1.3.3
Membrane
Trafficking and Related
3
4
Processes
2
Chlamydomonas
3
CVs in Chlamydomonas reinhardtii
11
4
Aim of This
13
II
Material and Methods
5
Chlamydomonas Strains, Culture
6
10
Study
15
and Growth
16
5.1
Chlamydomonas Strains
16
5.2
Chlamydomonas Culture Conditions
16
5.3
Growth Determination
17
Microscopically Analysis of Chlamydomonas Strains
6.1
Light Microscopy
6.2
Transmission Electron
6.2.1
7
reinhardtii
6
18
18
19
Microscopy
Epon, according to Luft (1961)
20
Molecular Biological Techniques
20
7.1
Buffers and Solutions
20
7.2
RNA Work
21
7.3
7.2.1
Isolation of RNA with TriReagent
21
7.2.2
Isolation of RNA with peqGOLD Plant RNA Kit
22
7.2.3
DNasel-Treatment
22
7.2.4
cDNA-Synthesis
22
DNA Work
23
7.3.1
Software used in DNA work
23
7.3.2
DNA Isolation
23
7.3.3
Standard PCR and RT-PCR
7.3.4
RESDA—Restriction Enzyme Site Directed
7.3.5
Primer walk
23
Amplification
24
25
xi
7.4
PCR-
25
7.3.6
Quantitative real-time
7.3.7
Purification of PCR Products
26
7.3.8
Cloning of PCR-Products
26
7.3.9
Generation of GFP Fusion Constructs
26
qPCR
27
PCR
7.3.10
Colony
7.3.11
Sequencing of
DNA
27
Transformation of Chlamydomanas reinhardtii
27
28
7.4.1
Insertional Mutagenesis
7.4.2
Screening for Osmoregulatory Mutants
7.4.3
Transformation of GFP Fusion Constructs into
28
Chlamydomonas
28
reinhardtii
8
7.4.4
Mutant Rescue
29
7.4.5
Suppressor Mutation
29
29
Biochemical Methods
8.1
Buffers and Solutions
29
8.2
Sample Preparation for SDS-Page
30
8.3
Protein Assay modified after Neuhoff
30
8.4
SDS-Page
31
8.5
Western Blot
31
33
III
Results
9
Characterization of the CVs of
9.1
9.2
Chlamydomonas reinhardtii CC3395
Basic Characterization of the CVs of Chlamydomonas reinhardtii
of
Light Microscope Observations
9.1.1
Analysis
9.1.2
Analysis of Electron Microscopy
Effect of the Culture Conditions
on
9.4
11
34
38
9.2.1
Light Microscopy
9.2.2
The Alteration of the Cytosolic Osmotic Potential under Different
38
39
Effect of Different Osmotic Environments
on
the CVs
41
9.3.1
Growth
9.3.2
Analysis
9.3.3
Analysis of Electron Microscopy
46
9.3.4
Expression Levels of Genes possibly Involved in CV Function
49
Water
41
Analysis
of
41
Light Microscopy
Permeability
of the CV and the Plasma Membranes in
50
Chlamydomonas reinhardtii CC3395 Membrane
10
34
37
the CVs
Culture Conditions
9.3
34
Aquaporins in Chlamydomonas reinhardtii
52
10.1
Localization of GFP-tagged Aquaporins
52
10.2
Does the CreMIPl-GFP Fusion Construct affect the CV Physiology?
59
Identification of Novel Genes involved in the CVs in
11.1
Insertional Mutagenesis
Chlamydomonas
62
62
xii
11.2
Identification of the Insertion Loci in the putative Osmoregulatory
Mutants
11.3
64
Characterization of the
11.3.1
The Growth of
Osmoregulatory
Osmo28,
Osmo32 and Osmo75
CC3395 in Media with Different Osmotic
11.3.2
Mutants
70
compared
to
71
Strength
Microscopically Examination of Osmo28,
Osmo32 and Osmo75
-
Light and Electron Microscopy
11.3.3
72
Does the CreMIPl-GFP Fusion Construct also Localize at the CVs in
the Osmoregulatory Mutants Osmo28, Osmo32 and Osmo75?
11.4
Rescue of the
Osmoregulatory
77
Mutant Osmo75
11.4.1
Growth
11.4.2
Examination of the Expression Levels of SEC6 in the Rescued Cell
79
Experiment of the Rescued Cell Lines Osmo75-SEC6GFP
79
Lines Osmo75-SEC6GFP
11.4.3
80
Characterization of the Rescued Cell Lines Osmo75-SEC6GFP using
Light and Electron Microscopy
11.4.1
83
Does the Rescue of Osmo75 with the SEC6-GFP Fusion Construct
also Rescue the
84
Long-Flagella Phenotype?
IV
Discussion
87
12
The Contractile Vacuoles in
Chlamydomonas reinhardtii
12.1
Basic Characterization of the CVs of Chlamydomonas reinhardtii CC3395
12.2
Effect of the Culture Conditions on the CVs
12.3
Effect of Different Osmotic Environments
12.4
Water
Permeability of the
on
88
..
88
90
the CVs
91
CV and the Plasma Membranes in
Cnfomydomonas reinhardtii CC3395
94
13
Aquaporins in Chlamydomonas reinhardtii
14
Identification of Novel Genes involved in the CVs in Chlamydomonas
reinhardtii
95
100
14.1
Insertional Mutagenesis
14.2
Identification of the Insertion Loci in the putative Osmoregulatory
100
Mutants
100
14.3
Characterization of the Osmoregulatory Mutants
102
14.4
Rescue of the
105
Osmoregulatory
Mutant Osmo75
15
Conclusion
Ill
16
References
115
V
Appendix
I