Browsing by Subject "Endocytose"

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Charakterisierung der lichtinduzierten Internalisierung des Ionenkanals TRPL aus Drosophila melanogaster
(2012) Oberegelsbacher, Claudia; Huber, Armin
The light-dependent isomerization of rhodopsin (Rh1), which takes place in the compound eyes of Drosophila, leads to the activation of the visual signaling cascade. The result is a depolarizing receptor potential caused by the Ca2+-influx through the two cation channels TRP and TRPL. This Ca2+ influx subsequently mediates a change in the subcellular localization of the TRPL channel by inducing its translocation. TRPL of dark-adapted flies is located inside the rhabdomeres, whereas upon illumination, TRPL translocates to a yet unidentified storage compartment in the cell body of the photoreceptor cell. The translocation is reversible; however, the underlying mechanism remains largely unclear. Based on the observation of TRPL-containing vesicles on immunocytochemical sections of illuminated flies a vesicular transport mechanism has been proposed for TRPL translocation. In the present work, the mechanism underlying light-dependent TRPL internalization was studied. Using immunocytochemical techniques, a co-localization of rhodopsin and TRPL was observed in endocytic vesicles. Like many other G-protein coupled receptors, Rhodopsin undergoes endocytosis following activation. The rate of Rh1 internalization depends on the amount of metarhodopsin and, therefore, on the light quality used for illumination. The internalization rate was determined by counting Rh1- and TRPL-positive vesicles observed upon illumination with different light qualities. Surprisingly, the light quality that induced the highest number of Rh1-positive vesicles (i.e. blue light) caused the lowest number of TRPL-positive vesicles, while illumination with orange light induced strong TRPL internalization, but poor Rh1 endocytosis. Likewise, time courses of TRPL internalization were significantly faster in orange light compared to blue light. These findings may indicate a competition between TRPL and Rh1 for a common internalization factor. Analysis of endocytosis in different mutants showed that the internalization of TRPL required Ca2+ influx mediated by the activation of the phototransduction cascade, whereas internalization of Rhodopsin was Ca2+-independent. Therefore, the trigger for activating TRPL and Rh1 endocytosis seems to be different, although both types of internalization were mechanistically similar and depended on dynamin function. The internalization of Rhodopsin is mediated by Rab5. A screen of dominant negative Rab mutants revealed that the light-induced internalization of TRPL is mediated by Rab5 and RabX4. Accordingly, the involvement of Rab5 constitutes another common feature in the endocytosis of TRPL and Rh1. Arrestins play an important role in regulating the endocytosis of rhodopsin. Whereas arrestin2 mediates the inactivation of metarhodopsin, arrestin1 is responsible for subsequent rhodopsin endocytosis. The endocytosis of TRPL is independent of arrestins, but arrestin2 fulfills an important function regarding the stability of the TRPL protein in the rhabdomere. In the present work, the analysis of different arr2 alleles revealed a complete degradation of the TRPL protein after ten days in darkness, but not in light. This finding suggests that arrestin2 has a possible function as a scaffolding protein in the rhabdomer of dark-adapted flies, but not of light-adapted flies, when TRPL is located in a storage compartment in the cell body. There is another fundamental difference between the two transport mechanisms regarding the fate of the protein after it has been internalized. Rhodopsin undergoes rapid lysosomal degradation whereas the trafficking of TRPL is described as a recycling mechanism. In this work, it was possible to show colocalization of TRPL with recycling endosomes indicating an involvement of these compartments in TRPL trafficking. Furthermore, rhodopsin but not TRPL showed colocalization with a lysosomal marker in light-adapted flies, providing additional evidence for the recycling of the TRPL channel.
Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.