Publikationsserver der RWTH Aachen University
Eine Untersuchung zur Endozytose in Pflanzenzellen
Abstract
dc:descriptionEndocytosis is a fundamental property of all eukaryotic life. In contrast to animal cells the endosomal system of plants is largely unknown. Recent research indicates a central role of endocytosis as a regulatory mechanism in plant morphogenesis and growth (Baluska et al., 2003; Geldner et al., 2003). Motivated by the growing interest in plant endocytosis, this work was intended to examine the endocytic pathway and its regulation in plant cells by different approaches and techniques. Three-dimensional time-lapse (4D) imaging was used to investigate the structure and dynamics of the plant vacuole, the terminal compartment of the endocytic pathway, and endosome trafficking in living tobacco BY-2 cells. Staining of the vacuolar membrane and endocytic compartments was obtained by using internalization of membrane bound, fluorescent styryl dyes. 4D imaging of the labeled vacuolar membrane revealed a structurally complex and dynamic morphology. The vacuole is traversed by multiple trans-vacuolar strands (TVS), which moved along each other and were able to fuse. New strands were created by fission of large membrane sheets. These observations lead to propose a model for TVS homeostasis whereby fusion and fission of TVS drive the alterations in TVS number that are observed across the cell cycle in plant cells (Kutsuna and Hasezawa, 2002). By double labeling, endocytic vesicle traffic was followed within the dynamic TVS. Membrane traffic and internalization was studied via different GFP fusion constructs. GFP fused to the GPI-anchored cell surface protein AtGPIP1 from A. thaliana allowed to visualize the secretory pathway in connection to endocytosis of dye labeled plasma membrane and assess the effect of Brefeldin A (BFA) on both pathways in plant cells. BFA is a fungal toxin and affects Golgi structure, endocytic recycling and secretion in animal cells. Imaging of the BFA effects in the cellular context in plant cells showed the formation of ER-Golgi hybrids and so-called “BFA compartments”. Double labeling of secretory and endocytic pathway in one cell demonstrated that the “BFA compartment” is a composite structure consistent of endosomal and secretory membrane components. The regulation of endocytosis in plant cells was analyzed by an artificial receptor construct comprising of the cytosolic tail of the wild-type rabbit poly-Ig receptor and GFP on the extracellular side. The endocytic behavior of this fusion protein was compared with a second fusion protein containing the pIgR tail with a mutated endocytosis signal fused to GFP. The observed decrease in endocytosis of the mutant construct suggests the functionality of mammalian endocytosis signals in plant cells and indicates that the prerequisites for receptor-mediated endocytosis in plant cells exist. Furthermore, relocation of both fusion proteins to the cell plate during cytokinesis suggests that also the targeting signals within the pIgR tail are recognized. In addition to signal mediated endocytosis of the artificial receptor also the endocytosis signal of a native plant receptor-like protein was intended for examination. The Ve2 protein from tomato was suggested as cell-surface receptor based on the sequence (Kawchuk et al., 2001). As the sequence also contains a potential endocytosis signal, Ve2 and a mutant version of Ve2 with altered endocytosis signal were chosen for fusion to GFP. However, the fusion proteins were localized to the ER, even after removal of a potential ER localization signal. In the last part of this work, an assay was developed that quantified membrane internalization in plant cells for screening of compounds on their effects on endocytosis. Testing of BFA confirmed the results obtained on cellular context by microscopy of transgenic cells expressing AtGPIP1-GFP and showed inhibition of recycling. In addition to its role in Golgi-mediated secretion and endocytic recycling, it was found that BFA also inhibited endocytic uptake in this study. The assay was further used to screen auxins and inhibitors of polar auxin transport. The polar auxin transport inhibitor TIBA was shown to inhibit membrane cycling of the auxin efflux carrier PIN1 thereby resulting in inhibition of auxin transport (Geldner et al., 2001). The inhibition of membrane internalization by TIBA was confirmed in uptake assays. Auxins demonstrated a reversing effect on endocytosis in uptake assays. Taken together with the findings that inhibition of membrane cycling of PIN1 blocks auxin transport, this suggests an auxin signaling pathway, where auxins mediate their own transport within the plant by endocytosis-mediated modulation of the activity of cell surface proteins by their levels at the plasma membrane.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2005
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Ruthardt, Nadia
- Contributors dc:contributor
-
- Fischer, Rainer
Subjects
dc:subject × 9Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng