Browsing by Person "Huber, Armin"

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Application of fluorescent proteins for functional dissection of the drosophila visual system
(2021) Smylla, Thomas; Wagner, Krystina; Huber, Armin
The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.
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.
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.
Quantitative Proteomanalyse von Pseudomonaden zur Aufklärung biotechnologisch relevanter Stoffwechselwege
(2013) Simon, Oliver; Huber, Armin
The main focus of this work was a quantitative proteome analysis of a variety of Pseudomonas strains with respect to the biotechnological synthesis of the base chemicals glyoxylic acid, butanol and vanillin. In addition, effects of the terpene citronellol on the proteome of P. aeruginosa were investigated. A second key aspect of this work involved the establishment of proteomics methods for the analysis of complex samples, especially for the analysis of membrane proteins. Using carbonate extraction followed by label-free MS-based quantification allowed the identification and quantification of a significant number of hydrophobic proteins which were not covered by the 2D-DIGE approach. In addition, the GeLCMSMS workflow was found to be a simple and efficient method for the analysis of total bacterial lysates. Using this method, about 30% of all proteins encoded by the P. putida KT2440 genome could be identified and quantified. In conclusion, this work demonstrated that different proteomics methods can substantially contribute to biotechnological strain development and the understanding of cellular networks.
tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel
(2024) Zeger, Matthias; Stanisławczyk, Lena Sarah; Bulić,Marija; Binder, Andrea Maria; Huber, Armin
In polarized cells, the precise regulation of protein transport to and from the plasma membrane is crucial to maintain cellular function. Dysregulation of intracellular protein transport in neurons can lead to neurodegenerative diseases such as Retinitis Pigmentosa, Alzheimer’s and Parkinson’s disease. Here we used the light-dependent transport of the TRPL (transient receptor potential-like) ion channel in Drosophila photoreceptor cells to study the role of Rab proteins in TRPL recycling. TRPL is located in the rhabdomeric membrane of dark-adapted flies, but it is transported out of the rhabdomere upon light exposure and localizes at the Endoplasmatic Reticulum within 12 h. Upon subsequent dark adaptation, TRPL is recycled back to the rhabdomeric membrane within 90 min. To screen for Rab proteins involved in TRPL recycling, we established a tissue specific (ts) CRISPR/Cas9-mediated knock- out of individual Rab genes in Drosophila photoreceptors and assessed TRPL localization using an eGFP tagged TRPL protein in the intact eyes of these mutants. We observed severe TRPL recycling defects in the knockouts of Rab3, Rab4, Rab7, Rab32, and RabX2. Using immunohistochemistry, we further showed that Rab3 and RabX2 each play a significant role in TRPL recycling and also influence TRPL transport. We localized Rab3 to the late endosome in Drosophila photoreceptors and observed disruption of TRPL transport to the ER in Rab3 knock-out mutants. TRPL transport from the ER to the rhabdomere ensues from the trans-Golgi where RabX2 is located. We observed accumulated TRPL at the trans-Golgi in RabX2 knock-out mutants. In summary, our study reveals the requirement of specific Rab proteins for different steps of TRPL transport in photoreceptor cells and provides evidence for a unique retrograde recycling pathway of TRPL from the ER via the trans-Golgi
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.