Browsing by Subject "Drosophila"

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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.
Effects of N-terminal mutations of human androgen receptor on polyglutamine toxicity
(2008) Funderburk, Sarah F.; Cato, Andrew C. B.
Nine neurodegenerative diseases are caused by polyglutamine (polyQ) tract amplification in different proteins. The cytotoxicity of each of these proteins is associated with a misfolding of the mutant protein, resulting in the subsequent alteration of cellular processes and interactions as well as the interrelated formation of insoluble aggregates and other conformationally toxic species. However, the diseases differ in their pathology and tissue specificity of action, which may be due to protein context/regions neighboring the polyQ stretch. For the purpose of the studies presented in ths work, the polyQ containing human androgen receptor (AR) that causes the disorder spinal and bulbar muscular atrophy (SBMA) was used to model polyQ toxicity. In previous investigations, two putative phosphorylation sites of the AR were identified, and it was demonstrated that mutation of these sites appeared to cause conformational change in the protein. Therefore, these N-terminal serine residues were exchanged to alanine in the wild type AR (ARQ22/ARQ22dm) or a receptor with an amplified polyQ stretch (ARQ77/ARQ77dm). These mutants were then used to characterize variance in types of aggregates and the associated toxic profiles due to the different protein conformations that arose from the serine mutations. Evaluating changes in aggregation and toxicity in cultured cells and in a Drosophila model of SBMA, it was found that the effects of the conformational changes differed depending on the length of the polyQ stretch. Mutations in the ARQ22 resulted in a marked increase in aggregation as well as decreased survival rates and altered locomotion behavior in Drosophila. These results were similar but not as severe as the ARQ77/SBMA model. In quite the opposite manner, mutations in the ARQ77 caused a decrease in aggregation and a lessened toxic effect in Drosophila. Moreover, it was found that inhibitor compounds used to ameliorate polyQ toxicity were not as efficient in inhibiting the varied toxicities exhibited by both the ARQ22dm and ARQ77dm. Therefore, two distinct amino acid sites that profoundly modulate polyQ toxicity in the AR have been identified. These results can be further utilized to understand the conformational changes in the AR that lead to aggregation as well as the types of aggregates that lead to toxicity.
Funktionelle Charakterisierung der Phosphatase RDGC in Drosophila melanogaster Photorezeptorzellen
(2018) Strauch, Lisa; Huber, Armin
Phosphorylation of important components like rhodopsin and TRP plays a big role in the phototransduction cascade of Drosophila melanogaster. The analyzed phosphatase RDGC is needed for the dephosphorylation of both components. It is yet knwon thet RDGC is expressed in three isoforms which will be named RDGC-S, RDGC-M and RDGC-L. Nothing has been known about the origin of RDGC-M. The present work shows thet RDGC-M is generated by using an alternative translation start codon and an alternative splice site within the RNA of the short isoform. Analysis of the subcellular localization showed membrane assoziation of RDGC-M and -L whereas RDGC-S is found in the soluble fraction. Recominant expression in S2-cells identified acylation of RDGC-M and -L as the source of the membrane association. In addition, acylation of RDGC-L isolated from flies was directly proven by using a biochemical assay. To functionally characterize the three isoforms, mutant flies with different RDGC expression paterns were created and analyzed. As a result, it was shown that rhodopsin hyperphosphorylation that is found in the rdgc nullmutant as well as the associated retinal degeneration is prevented by the expression of any RDGC isoform. Regarding TRP channel phosphorylation none of the three isoforms is mandatory for the dephosphorylation of TRP at Ser936. However, the results revealed thet the total amount of RDGC that is available, in particular RDGC-M, affects the kinetics of the TRP-S936 dephosphorylation. An increased expression of RDGC-M in the absence of RDGC-S leads to a faster dephosphorylation of TRP-S936. Such a change in TRP-S936 dephosphorylation kinetics was not observed in flies overexpressing RDGC-S in an rdgc-nullmutangt backgroundand therefore cannot be attributed to the increased amount of the corresponding protein. Taken together this study shows thet the tgree RDGC isoforms differ in their subcellular localization due to differences in the N-termini. This may be the reason for kinetic differences in the dephosphorylation of TRP-S936 by RDGC-S or RDGC-M. Apart from these findings, all RDGC isoforms are able to dephosphorylate rhodopsin.
Funktionen N- und C-terminaler Proteindomänen für Assemblierung, subzelluläre Lokalisation und Physiologie der TRP-Ionenkanäle in Drosophila Photorezeptoren
(2011) Oberacker, Tina; Huber, Armin
In the photoreceptor cells of Drosophila melanogaster, the cation channels TRP and TRPL are responsible for generating the receptor potential. Previous publications have shown that the TRPL ion channel changes its subcellular localization depending on light conditions, while TRP is located in the rhabdomeres irrespective of light conditions. TRP and TRPL form tetramers. However, it is under debate in the scientific literature if TRP and TRPL form ho-momultimers only or also heteromultimers. In the present study, co-immunoprecipitations (Co-IPs) of untagged TRP or TRPL channels demonstrated that the tetrameric channels in the photoreceptors of Drosophila are composed of homomultimers exclusively. Co-IPs of eGFP-tagged TRP or TRPL channels showed that the tetramers consist of eGFP-tagged and the corresponding untagged channel subunits. To study the biochemical and physiological properties of the cytosolic N- and C-termini of TRP and TRPL, eGFP-tagged chimeric TRP/TRPL ion channels were generated and ex-pressed in the photoreceptor cells R1-R6 of Drosophila. The effect of these termini on trans-location of channels between the rhabdomere and the cell body and on ion channel assembly was studied. Studies of the subcellular localization of eGFP-tagged chimeras showed that a chimera com-posed of the transmembrane regions of TRP and both the N- and the C-terminus of TRPL displayed a light-dependent translocation behavior like TRPL. Interestingly, the translocation of this chimera was much faster than the TRPL-eGFP translocation. The exchange of either the N-terminus or the C-terminus of TRPL with the respective termini of TRP caused a locali-zation of these chimeras mainly in the cell body. This localization neither corresponded to the translocation of TRPL nor to the rhabdomeric localization of TRP. Therefore, motifs inducing light-dependent translocation of TRPL must be located in both termini and are only effective in concert. Co-IPs of the eGFP-tagged chimeras demonstrated that the C-terminus of TRPL appears to be more important than the N-terminus of TRPL. For interaction with the TRP channel the C-terminus of TRP also seems to be more important than the N-terminus. TRPL-TRPL or TRP-TRP interaction via the N-terminus could only be observed by Co-IPs in certain chimeras. In contrast to the termini, the transmembrane regions of both ion channels are not necessary for interaction. Assuming that an interaction between the eGFP-tagged chimera and endogenous channel subunits might cause a mislocalization of the endogenous subunit, immunocytochemical stu-dies were carried out. On cross sections through the eyes of dark and light adapted flies ex-pressing eGFP-tagged chimeras the localization of these eGFP-tagged channels was visua-lized by the eGFP fluorescence, while the localization of the endogenous subunits was de-termined by labeling with specific antibodies. It was found that those chimeras which show a strong interaction with the endogenous channels in Co-IPs cause a mislocalization of the corresponding endogenous channels. This thesis clarified that TRP and TRPL exclusively form homomutlimers in the photorecep-tors of Drosophila. Furthermore, it could be shown, which termini are responsible for the TRP and TRPL homomultimerization and which regions of the TRPL ion channel are necessary for TRPL translocation.
Overwintering and reproduction biology of Drosophila suzukii Matsumura (Diptera: Drosophilidae)
(2018) Zerulla, Florian Niklas; Zebitz, Claus P. W.
Drosophila suzukii (Matsumura) was introduced to southern Europe and the United States of America in 2008 through fruit imports from Southeast Asia and spread in the following years all over Europe, as well as South and North America. D. suzukii is a polyphagous pest which infests fruits of soft-skinned wild and cultivated plants. In contrast to the well-known D. melanogaster, healthy and ripe fruits are preferred. The infestation is caused by female D. suzukii who damage the fruit skin to deposit eggs underneath with the help of their serrated ovipositor. The feeding of hatching larvae and secondary infections, which can easily penetrate through the damaged fruit, can lead to complete yield losses. A short reproductive period, a large range of host plants and infestation of the fruits, shortly before harvesting makes it extremely difficult to control the pest. Due to comparatively late infestation, the possible period of application during ripening and harvesting is limited. The same applies to the frequency of application of insecticides at this time. Possible residues on the harvested products also carry the risk of rejection of the fruit on the market. Furthermore, an incomplete knowledge of the biology, especially overwintering biology under European environmental conditions makes an effective control of this invasive pest extremely difficult. Therefore, the main research topics are the induction and refraction of the postulated diapause, the detection of any possible hibernation sites and the influence of temperature on the oviposition behaviour of D. suzukii. Based on field experiments it could be shown that successful wintering could probably only take place in forest areas. After freezing, the forest was the only place where D. suzukii could resume its flight activity at warmer temperatures. It has also been confirmed that female flies are more resistant to temperatures below freezing than male flies. Accordingly, after a frost period, hardly any male D. suzukii were caught in bait traps. Similar results have also been obtained in laboratory tests, showing that D. suzukii adapted to low temperatures and shortday conditions had lower temperature preferences and a decreased mortality after changing environmental conditions. It can therefore be assumed that overwintering D. suzukii can spread better in spring due to a lower temperature preference and a higher physical activity under cold environmental conditions than flies without adaptation to winter conditions. Bait traps were also used to determine the developmental status of the ovaries by dissecting the abdomens of weekly captured D. suzukii. This enabled a correlation between microclimatic conditions of individual habitats and the reproductive status of females to be established. In winter, the majority of female D. suzukii had “immature ovaries”, whereas in the summer most females had “mature eggs” in their abdomen. For this reason, it can be assumed that D. suzukii entered a reproductive diapause, which is apparently influenced by winter climatic conditions, nutritional status and the availability of food. In addition, it was found that the developmental status of ovaries correlates positively with oviposition. Laboratory tests were carried out to determine the highest number of egg depositions at 20 °C. Most females with “mature eggs” were also documented under these simulated conditions. We detected that the preferred surface temperature for egg depositing was very similar to the preferred ambient temperature of D. suzukii. Most of the eggs were deposited on fruits with a surface temperature of 22 °C. At this temperature, the highest net reproductive rate and intrinsic rate of population increase was found, too. Temperatures below 15 °C and above 35 °C were not preferred, which represented the thresholds for a successful development of D. suzukii. Temperatures between 10 and 15 °C and shortday conditions were the most important key stimuli for entering the reproductive diapause. Therefore, temperature had a stronger influence on oviposition behaviour than daylength. A complete disruption of diapause occurred at higher temperatures (20 °C) and longday conditions after 72 hours. The data presented in this work on the possibility of adapting D. suzukii to environmental conditions and key temperatures, which influence the development of ovaries and egg deposition, can provide an important contribution to the development of prediction and population dynamics models and can be used for long-term control strategies against D. suzukii. According to current knowledge and observations, overwintering is obviously a critical period for the survival of the populations. Therefore, the characterisation and identification of additional hibernation sites is of great importance. There, a targeted and environmentally friendly control of D. suzukii populations could be particularly efficient.
Untersuchung der lichtabhängigen Phosphorylierung des TRP-Kanals von Drosophila melanogaster
(2015) Bartels, Jonas-Peter; Huber, Armin
The phototransduction cascade in the eye of Drosophila melanogaster culminates in the opening of the ion channels TRP and TRPL. The hereby increased intracellular concentration of sodium and calcium ions underlies the formation of the photoreceptor potential. The TRP channel is subject to light-dependent phosphorylation. 15 of its phosphorylation sites exhibit increased phosphorylation upon illumination and one phosphorylation site exhibits increased phosphorylation in the dark. When this work was started, neither the function of light-dependent TRP phosphorylation nor the involved kinases and phosphatases were known. Therefore, in the present work, kinases and phosphatases that affect the phosphorylation pattern of the TRP channel were identified. Towards this end a candidate screen of 79 kinase and phosphatase mutants was performed using three different phosphospecific antibodies. In this screen, eight kinases and one phosphatase were identified that affect the phosphorylation of one or more of the three tested phosphorylation sites. Eye-specific protein kinase C (ePKC) and protein kinase C 53E (PKC53E) mutants exhibited reduced phosphorylation at T849 of the TRP channel, while mutants of the Rolled kinase and the alpha-subunit of AMP-dependent kinase showed an increased phosphorylation at this site. The mutants of Casein kinase Ialpha, Licorne, Tao, and Metallophosphoesterase (MPPE) exhibited reduced phosphorylation of the site T864. The retinal degeneration C (RDGC) phosphatase null mutant demonstrated a dramatically increased phosphorylation at the phosphorylation site S936. From these identified kinase and phosphatase mutants, only the mutants of the kinase ePKC and of the phosphatase RDGC displayed physiological abnormalities in terms of an already described slow deactivation of the light response in ERG measurements. To answer the question whether the altered phosphorylation of the TRP ion channel in the ePKC and RDGC mutants underlies the prolonged deactivation of the light response, transgenic flies were generated that express modified TRP channels. To this end, the ePKC-dependent TRP phosphorylation site T849 or the RDGC-dependent phosphorylation site S936 of the TRP channel were replaced by the amino acid alanine or aspartic acid. The biochemical analysis of these four transgenic flies revealed wild type characteristics of the modified channels with respect to subcellular localization, the interaction with the scaffold protein INAD, multimerization, and the expression rate. However, electrophysiological studies of these transgenic flies revealed a prolonged deactivation time when T849 was exchanged to alanine and in addition S936 was exchanged to aspartic acid. Whereas the exchange of a serine or threonine to alanine prevents phosphorylation, the exchange to aspartic acid typically mimics phosphorylation of this site. In the wild type situation, T849 is phosphorylated in the light and becomes dephosphorylated in the dark whereas for S936 the exact opposite is the case. The amino acid exchanges at the two phosphorylation sites thus mimic the phosphorylation pattern of dark-adapted wild type flies. In summary, the results demonstrate the involvement of eight kinases and one phosphatase in generating of the phosphorylation pattern of the TRP ion channel. Two of the phosphorylation sites of the TRP ion channel mediate a rapid deactivation of the light response.