Eine Untersuchung zur Endozytose in Pflanzenzellen [Elektronische Ressource] / vorgelegt von Nadia Ruthardt

Nadia

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Eine Untersuchung zur Endozytose in Pflanzenzellen



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen
Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation


vorgelegt von

Diplom-Biologin
Nadia Ruthardt
aus Hanau



Berichter: Universitätsprofessor Dr. rer. nat. R. Fischer
Priv. Doz. Dr. rer. nat. Christoph Peterhänsel


Tag der mündlichen Prüfung: 31. Mai 2005



Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.






An Investigation of Endocytosis in Plant Cells




Dissertation submitted by

Dipl. Biol. Nadia Ruthardt
RWTH Aachen


Parts of this work have been published:

Ruthardt N, Gulde N, Spiegel H, Fischer R, Emans N (in press) 4D imaging of trans-
vacuolar strand dynamics in tobacco BY-2 cells. Protoplasma
Paciorek T, Zažímalová E, Ruthardt N, Petrášek J, Stierhof YD, Kleine-Vehn J, Morris
DA, Emans N, Jürgens G, Geldner N, and Friml J (in press) Auxin inhibits endocytosis
and promotes its own efflux from cells. Nature


1 Introduction 1
1.1 Endocytosis in Animals 1
1.2 Endocytosis in Plants 5
1.3 The Plant Vacuole 8
1.4 Study Objectives 10
2 Material and Methods 14
2.1 Material 14
2.1.1 Chemicals and Consumables 14
2.1.2 Enzymes and Reactionkits 14
2.1.3 Antibodies and Enzyme-conjugated Antibodies for Detection 14
2.1.4 Synthetic Oligonucleotides 15
2.1.5 Plasmids 17
2.1.6 Bacterial Strains and Plants 18
2.1.7 Media, Buffers and Solutions 19
2.1.8 Devices and Equipment 19
2.2 Molecularbiological Methods 20
2.2.1 Isolation of Plasmid DNA from E. coli 20
2.2.2 DNA Restriction 20
2.2.3 Dephosphorylation of linear DNA 21
2.2.4 Ligation of DNA 21
2.2.5 Agarose Gel-electrophoresis 21
2.2.6 Polymerase Chain Reaction (PCR) 21
2.2.6.1 Standard PCR 21
2.2.6.2 PCR for Ve2 amplification 22
2.2.7 DNA Purification 23
2.2.8 Sequencing of Plasmid DNA 23
2.2.9 Growth and Transformation of E. coli and A. tumefaciens 23
2.2.9.1 Cultivation of Escherichia coli 23
2.2.9.2 Preparation of electro-competent E.coli 23
2.2.9.3 Electrotransformation of E.coli 24
2.2.9.4 Cultivation of Agrobacterium tumefaciens 24
2.2.9.5 Preparation of electro-competent A. tumefaciens 25
2.2.9.6 Electro-transformation of A. tumefaciens 25
2.2.9.7 Identification of transformed bacteria by PCR 25
2.3 Protein Analysis 25
2.3.1 Total Soluble Protein Extraction from BY-2 cells 25
2.3.2 SDS-Page 26
2.3.3 Immunoblotting 26

2.4 Cell Biological Methods 27
2.4.1 Cultivation of BY2 Suspension Cultures and Tobacco plants 27
2.4.2 Stable Transformation of BY-2 cells 27
2.4.3 ation of tobacco plants 28
2.4.3.1 Cultivation of sterile tobacco plants 28
2.4.3.2 Stable transforma28
2.4.4 Vacuolar Membrane Staining 29
2.4.5 Confocal Laser Scanning Microscopy 29
2.4.6 Preparation of Protoplasts 30
2.4.7 pIgR Dimerization Assay 30
2.4.8 Immunocytochemistry 31
2.4.9 Uptake Assays 32
3 Results 34
3.1 Structure and Dynamics of BY-2 cell vacuoles 35
3.1.1 Vacuolar Structure 35
3.1.2 Vacuolar Dynamics 36
3.1.3 Endosome Tracking 40
3.2 Analysis of endocytic trafficking pathways with selected GFP fusion
proteins 43
3.2.1 GFP-KDEL control construct 43
3.2.1.1 Cloning of GFP-KDEL 43
3.2.1.2 Microscopy 44
3.2.2 AtGPIP1-GFP 45
3.2.2.1 Analysis of transgenic BY-2 suspension cells 45
3.2.2.2 Endosome labeling with FM4-64 48
3.2.2.3 Immunofluorescence 48
3.2.2.4 BFA treatment 49
3.2.2.5 ent and endosome labeling with FM4-64 50
3.2.3 pIgR-GFP 53
3.2.3.1 Cloning of pIgR-GFP 53
3.2.3.2 Analysis of transgenic BY-2 cells 55
3.2.3.3 FM4-64 labeling 57
3.2.3.4 JIM84 staining 61
3.2.3.5 Dimerization Assay 62
3.2.4 Ve-GFP 67
3.2.4.1 Cloning of Ve2-GFP 67
3.2.4.2 Cloning of Ve2mut-GFP 69
3.2.4.3 Analysis of transgenic N. tabacum cv. BY-2 cells 71
3.2.4.4 Confocal microscopy analysis of transgenic N. tabacum cv. Petite
Havana SR1 plants 72
3.2.5 Ve2 ∆KKF-GFP 73
3.2.5.1 Cloning of Ve2 ∆KKF-GFP and Ve2mut ∆KKF-GFP 73

3.2.5.2 Analysis of transgenic N. tabacum cv. BY-2 cells 74
3.3 Development and application of an assay for quantitative measurement of
endocytosis in plant cells 77
3.3.1 Uptake Assay 78
3.3.2 Compound Testing 80
3.3.3 Auxins and Polar Auxin Transport Inhibitors 89
4 Discussion 99
4.1 Structure and Dynamics of BY-2 Vacuoles 100
4.2 Analysis of endocytic trafficking pathways of selected GFP fusion proteins
107
4.2.1 AtGPIP1-GFP 107
4.2.2 pIgR-GFP 111
4.2.3 Ve2-GFP 115
4.3 Uptake Assay 118
5 Summary 126
6 Zusammenfassung 129
7 References 132


Introduction 1
1 Introduction
Endocytosis is the uptake of extracellular substances into cells through the
internalization of the plasma membrane derived vesicles. It is a fundamental process
found in all eukaryotes required for diverse cellular functions such as turnover and
degradation of plasma membrane proteins and receptors, nutrient uptake, elimination of
pathogenic microorganisms, spread of morphogens and cell-to-cell communication at
synapses (Olson and Grose, 1997; Pfeiffer and Vincent, 1999; Gitan and Eide, 2000;
Lin and Scheller, 2000; Di Fiore and De Camilli, 2001; Loder and Melikian, 2003;
Naslavsky et al., 2004). In the following, a short overview on endocytosis in animals
and plants is given including differences and similarities in both systems based on
current knowledge.
1.1 Endocytosis in Animals
According to the type of cargo and molecular machinery driving its internalization,
several forms of endocytosis have been defined in mammalian cells. These endocytic
pathways include clathrin-mediated, caveolae/lipid raft-mediated, clathrin- and
caveolae-independent endocytosis, fluid-phase endocytosis, and phagocytosis
(Steinman and Swanson, 1995; Mellman, 1996; Nichols and Lippincott-Schwartz, 2001;
Nabi and Le, 2003). Most knowledge on endocytosis derives from studies on receptor-
mediated clathrin-dependent endocytosis. This is highly regulated and serves to down-
regulate receptors, permits transmission of signals or the selective uptake of extra-
cellular ligands. A central feature of clathrin-dependent endocytosis is the
macromolecular lattice (clathrin coat) required for the budding of endocytic vesicles
(Mellman, 1996). This special coat consists of the structural protein clathrin and several
adaptor proteins and interacts with the large GTPase dynamin (van der Bliek et al.,
1993; Damke et al., 1994; De Camilli et al., 1995) as well as with cytoskeletal and
signaling protein complexes (Engqvist-Goldstein and Drubin, 2003). The internalization
of specific surface receptors and their ligands by receptor-mediated endocytosis starts
with selective sequestration of the receptors into specialized structures on the cell-
surface termed clathrin-coated pits (CCP). These sites of rapid endocytosis are
responsible for the internalization of a large variety of surface receptors and proteins.
Introduction 2
The selective accumulation in coated pits and internalization of receptors usually
involves at least two types of signals: the tyrosine-based motif and the dileucine-based
motif (LL) where one leucine can be replaced by an isoleucine (Trowbridge et al., 1993;
Marks et al., 1997). Both motifs can be found in the cytoplasmic tail of the poly-Ig
receptor (pIgR), a well studied, fast internalizing receptor responsible for the serosal-to-
mucosal transepithelial transport of dimeric IgA and pentameric IgM via the
transcytotic pathway in secretory epithelial cells (Okamoto et al., 1992). The most
thoroughly examined of these motifs is probably the tyrosine-motif that consists of a
sequence of 4-6 amino acids, specifically containing a tyrosine that is crucial for proper
endocytic targeting such as the YXX Φ motif (where Y refers to tyrosine, X refers to any
amino acid residue, and Φ refers to hydrophobic residues with a bulky side chain)
(Warren et al., 1998). Although receptors bearing tyrosine-based internalization signals
all initially depend on clustering into clathrin-coated pits for efficient endocytosis, they
are not necessarily destined for the same location within the cell.
The recruitment of clathrin is initiated by adaptor proteins such as AP2 (Chang
et al., 1993; Peeler et al., 1993; Sorkin and Carpenter, 1993; Carpenter et al., 1995;
Sorkin et al., 1995), AP180 (Prasad and Lippoldt, 1988; Hao et al., 1999; Ford et al.,
2001) and epsins (Chen et al., 1998; Kalthoff et al., 2002) that interact with plasma
membrane phospholipids, cytoplasmic domains of receptors, and synaptotagmin (Wang
et al., 1993; Zhang et al., 1994). Clathrin is a multimeric protein with a characteristical
triskelion structure build of three 180-kDa heavy chains complexed by a 30-35 kDa