Untersuchungen zur Endozytose in Pflanzenzellen mittels artifizieller Rezeptoren

The aim of this thesis was the creation of artificial receptors at the plant cell membrane that were capable of binding extra cellular molecules and transporting them into the plant cell through the endocytic pathway. This would then create the basis for a novel mechanism for the phyto-remediation of environmental contaminants using transgenic plants. The artificial receptor was created by exchanging the native binding domain of the polymeric Ig receptor for a single chain antibody fragment (scFv29) specific for a small peptide epitope. In this configuration, the scFv29-pIgR fusion protein contained a endocytosis signal in its cytoplasmic domain. This endocytosis signal is highly potent in mammalian cells and to investigate its role in plant cells, a second fusion protein was created where the endocytosis signal was made defective by amno acid replacement (scFv29-pIgRmut). as a further control, a fusion protein was created between the scFv29 and the constant, trans membrane and cytoplasmic domain of the beta-chain of the human T-cell receptor, which possesses no signalling information in the cytoplasmic domain. These receptors were stably expressed in both tobacco plants and tobacco cell suspension cultures (BY-2 cultures). A functional ELISA measured the accumulation of the fusion proteins through quantitation of the concentration of the scFv29 domain. Accumulation levels were high in the leaves and roots of transgenic plants, indicating that the fusion proteins found in the roots could potentially be used to bind extra cellular molecules. Further analysis in-vitro demonstrated that the receptor fusion protein was intact and capable of binding to be specific peptide recognised by the single chain antibody. Immuno-fluorescence confocal microscopy was used to determine the sub-cellular localization of the artificial receptors. The scFv29-pIgR fusion protein was found to be at the plasma membrane of suspension cell protoplasts and was found in intra-cellular vesicles. The intra-cellular location of the artificial receptor was explained by the presence of the functional endocytosis signal in the cytoplasmic tail, for the version of the receptor lacking the endocytosis signal was found exclusively at the plasma membrane. The control T-cell receptor based fusion protein was found at the plasma membrane as well as in the endoplasmic reticulum. Immuno-electron microscopy was used to identify the intra-cellular vesicles as endocytic multi-vesicular bodies. Furthermore, electron microscopy demonstrated that the receptor was correctly orientated in the membrane with the single chain antibody domain facing outward. In order to assess whether the artificial receptors were capable of binding extra-cellular molecules in vivo, a green fluorescent protein was produced fused to the target peptide epitope recognized by the single chain antibody incorporated into the artificial receptors. It was shown that this protein bound to the cell surface of tobacco cell lines or plant root hairs expressing the artificial receptors when it was added to the extra-cellular medium. The Labeled fluorescent protein was only endocytosed significantly in cells expressing the scFv29-pIgR construct where there was an endocytosis signal in the cytoplasmic tail. Therefore, this thesis work demonstrated the feasibility of assembling and expressing artificial receptors in plant cells. Secondly, it showed that these receptors were expressed at the cell surface where they could bind extra-cellular molecules. The uptake of these molecules was enhanced by the presence of an endocytosis signal in the cytoplasmic domain of the artificial receptor showing that the plant cell endocytic apparatus recognizes this signal and that it can be co-opted for uptake of artificial receptors carrying molecules from the extra-cellular environment. The thesis demonstrates that this approach may be of interest for creating plants that are capable of removing pollutants from the environment.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2003

Author dc:creator

• Hoppmann, Verena

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Endozytose
• poly Ig Rezeptor
• single chain Antikörperfragment
• Pflanzenzelle
• Phytoremediation

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-120680%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:58851