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Sonstige beteiligte Institutionen
Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology.
Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family – not only in classical mechanosensory structures.
This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function.
The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons – a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.
Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.
Functional and genetic dissection of mechanosensory organs of \(Drosophila\) \(melanogaster\)
(2016)
In Drosophila larvae and adults, chordotonal organs (chos) are highly versatile mechanosensors
that are essential for proprioception, touch sensation and hearing. Chos share molecular,
anatomical and functional properties with the inner ear hair cells of mammals. These multiple
similarities make chos powerful models for the molecular study of mechanosensation.
In the present study, I have developed a preparation to directly record from the sensory neurons
of larval chos (from the lateral chos or lch5) and managed to correlate defined mechanical inputs
with the corresponding electrical outputs. The findings of this setup are described in several case
studies.
(1) The basal functional lch5 parameters, including the time course of response during continuous
mechanical stimulation and the recovery time between successive bouts of stimulation, was
characterized.
(2) The calcium-independent receptor of α-latrotoxin (dCIRL/Latrophilin), an Adhesion class G
protein-coupled receptor (aGPCR), is identified as a modulator of the mechanical signals
perceived by lch5 neurons. The results indicate that dCIRL/Latrophilin is required for the
perception of external and internal mechanical stimuli and shapes the sensitivity of neuronal
mechanosensation.
(3) By combining this setup with optogenetics, I have confirmed that dCIRL modulates lch5
neuronal activity at the level of their receptor current (sensory encoding) rather than their ability
to generate action potentials.
(4) dCIRL´s structural properties (e.g. ectodomain length) are essential for the mechanosensitive
properties of chordotonal neurons.
(5) The versatility of chos also provides an opportunity to study multimodalities at multiple levels.
In this context, I performed an experiment to directly record neuronal activities at different
temperatures. The results show that both spontaneous and mechanically evoked activity increase
in proportion to temperature, suggesting that dCIRL is not required for thermosensation in chos.
These findings, from the development of an assay of sound/vibration sensation, to neuronal
signal processing, to molecular aspects of mechanosensory transduction, have provided the first
insights into the mechanosensitivity of dCIRL.
In addition to the functional screening of peripheral sensory neurons, another
electrophysiological approach was applied in the central nervous system: dCIRL may impact the
excitability of the motor neurons in the ventral nerve cord (VNC). In the second part of my work,
whole-cell patch clamp recordings of motor neuron somata demonstrated that action potential
firing in the dCirl\(^K\)\(^O\) did not differ from control samples, indicating comparable membrane
excitability.
During my PhD I studied two principal biological aspects employing Drosophila melanogaster. Therefore, this study is divided into Part I and II.
Part I: Bruchpilot and Complexin interact to regulate synaptic vesicle tethering to the
active zone cytomatrix
At the presynaptic active zone (AZ) synaptic vesicles (SVs) are often physically linked to an electron-dense cytomatrix – a process referred to as “SV tethering”. This process serves to concentrate SVs in close proximity to their release sites before contacting the SNARE complex for subsequent fusion (Hallermann and Silver, 2013). In Drosophila, the AZ protein Bruchpilot (BRP) is part of the proteinous cytomatrix at which SVs accumulate (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Intriguingly, truncation of only 1% of the C-terminal region of BRP results in a severe defect in SV tethering to this AZ scaffold (hence named brpnude; Hallermann et al., 2010b).
Consistent with these findings, cell-specific overexpression of a C-terminal BRP fragment, named mBRPC-tip (corresponds to 1% absent in brpnude; m = mobile) phenocopied the brpnude mutant in behavioral and functional experiments. These data indicate that mBRPC-tip suffices to saturate putative SV binding sites, which induced a functional tethering deficit at motoneuronal AZs. However, the molecular identity of the BRP complement to tether SVs to the presynaptic AZ scaffold remains unknown. Moreover, within larval motoneurons membrane-attached C-terminal portions of BRP were sufficient to tether SVs to sites outside of the AZ. Based on this finding a genetic screen was designed to identify BRP interactors in vivo. This screen identified Complexin (CPX), which is known to inhibit spontaneous SV fusion and to enhance stimulus evoked SV release (Huntwork and Littleton, 2007; Cho et al., 2010; Martin et al., 2011). However, so far CPX has not been associated with a function upstream of priming/docking and release of SVs. This work provides morphological and functional evidence, which suggests that CPX promotes recruitment of SVs to the AZ and thereby curtails synaptic short-term depression. Together, the presented findings indicate a functional interaction between BRP and CPX at Drosophila AZs.
Part II: The Adhesion-GPCR Latrophilin/CIRL shapes mechanosensation
The calcium independent receptor of α-latrotoxin (CIRL), also named Latrophilin, represents a prototypic Adhesion class G-protein coupled-receptor (aGPCR). Initially, Latrophilin was identified based on its capacity to bind the α-component of latrotoxin (α-LTX; Davletov et al., 1996; Krasnoperov et al., 1996), which triggers massive exocytotic activity from neurons of the peripheral nervous system (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). As a result Latrophilin is considered to play a role in synaptic transmission. Later on, Latrophilins have been associated with other biological processes including tissue polarity (Langenhan et al., 2009), fertility (Prömel et al., 2012) and synaptogenesis (Silva et al., 2011). However, thus far its subcellular localization and the identity of endogenous ligands, two aspects crucial for the comprehension of Latrophilin’s in vivo function, remain enigmatic.
Drosophila contains only one latrophilin homolog, named dCirl, whose function has not been investigated thus far.
This study demonstrates abundant dCirl expression throughout the nervous system of Drosophila larvae. dCirlKO animals are viable and display no defects in development and neuronal differentiation. However, dCirl appears to influence the dimension of the postsynaptic sub-synaptic reticulum (SSR), which was accompanied by an increase in the postsynaptic Discs-large abundance (DLG). In contrast, morphological and functional properties of presynaptic motoneurons were not compromised by the removal of dCirl. Instead, dCirl is required for the perception of mechanical challenges (acoustic-, tactile- and proprioceptive stimuli) through specialized mechanosensory devices, chordotonal organs (Eberl, 1999). The data indicate that dCirl modulates the sensitivity of chordotonal neurons towards mechanical stimulation and thereby adjusts their input-output relation. Genetic interaction analyses suggest that adaption of the molecular mechanotransduction machinery by dCirl may underlie this process. Together, these results uncover an unexpected function of Latrophilin/dCIRL in mechanosensation and imply general modulatory roles of aGPCR in mechanoception.
To unravel the role of single genes underlying certain biological processes, scientists often use amorphic or hypomorphic alleles. In the past, such mutants were often created by chance. Enormous approaches with many animals and massive screening effort for striking phenotypes were necessary to find a needle in the haystack. Therefore at the beginning chemical mutagens or radiation were used to induce mutations in the genome. Later P-element insertions and inaccurate jump-outs enabled the advantage of potential larger deletions or inversions. The mutations were characterized and subsequently kept in smaller populations in the laboratories. Thus additional mutations with unknown background effects could accumulate.
The precision of the knockout through homologous recombination and the additional advantage of being able to generate many useful rescue constructs that can be easily reintegrated into the target locus made us trying an ends-out targeting procedure of the two core clock genes period and timeless in Drosophila melanogaster. Instead of the endogenous region, a small fragment of approximately 100 base pairs remains including an attP-site that can be used as integration site for in vitro created rescue constructs. After a successful ends-out targeting procedure, the locus will be restored with e.g. flies expressing the endogenous gene under the native promoter at the original locus coupled to a fluorescence tag or expressing luciferase.
We also linked this project to other research interests of our work group, like the epigenetic related ADAR-editing project of the Timeless protein, a promising newly discovered feature of time point specific timeless mRNA modification after transcription with yet unexplored consequences. The editing position within the Timeless protein is likewise interesting and not only noticed for the first time. This will render new insights into the otherwise not-satisfying investigation and quest for functional important sequences of the Timeless protein, which anyway shows less homology to other yet characterized proteins.
Last but not least, we bothered with the question of the role of Shaggy on the circadian clock. The impact of an overexpression or downregulation of Shaggy on the pace of the clock is obvious and often described. The influence of Shaggy on Period and Timeless was also shown, but for the latter it is still controversially discussed. Some are talking of a Cryptochrome stabilization effect and rhythmic animals in constant light due to Shaggy overexpression, others show a decrease of Cryptochrome levels under these conditions. Also the constant light rhythmicity of the flies, as it was published, could not be repeated so far. We were able to expose the conditions behind the Cryptochrome stabilization and discuss possibilities for the phenomenon of rhythmicity under constant light due to Shaggy overexpression.
Neuropathische Schmerzen treten in der klinischen Praxis häufig auf und
beeinträchtigen in hohem Maße die Lebensqualität der Patienten. Bedingt durch eine
Schädigung somatosensorischer Nervenstrukturen im peripheren oder zentralen
Nervensystem ist der Schmerz meist durch eine Allodynie, Hyperalgesie oder
einschießende Schmerzen charakterisiert. Diese Symptome lassen sich durch
gängige Schmerzmittel kaum lindern. In jüngerer Zeit rückte die Blut-Rückenmarkschranke immer mehr in den Fokus verschiedener Untersuchungen, da auch
einige nicht-schmerzhafte Erkrankungen (z.B. Multiple Sklerose und Amyotrophe
Lateralsklerose) zur Veränderung dieser Barriere mit folgender Permeabilitätserhöhung
durch reduzierte Tight Junction-Protein-Expression führen. Die Blut-
Rückenmarkschranke dichtet Gefäße des Rückenmarks ab und verhindert das
Eindringen toxischer oder proanalgetischer Mediatoren in das zentrale Nervensystem.
Dafür ist die Funktion der Tight Junction-Proteine zur Aufrechterhaltung dieser Barriere
essentiell. Dennoch konnte die Rolle der Blut-Rückenmarkschranke bei Neuropathie
noch nicht vollständig erörtert werden. Die Untersuchungen meiner Arbeitsgruppe
konnten bereits zeigen, dass eine lockere Ligatur des N. ischiadicus bei Ratten (CCI;
chronic constriction injury) zur Entwicklung einer thermischen und mechanischen
Hyperalgesie sowie eingeschränkten motorischen Funktionen führt. Zudem konnte
eine Störung der Blut-Rückenmarkschranke nach einer CCI nachgewiesen werden, da
es Tracern unterschiedlicher molekularer Größe möglich war das Rückenmark zu
penetrieren. Daher sollte im Rahmen dieser Dissertation untersucht werden, inwieweit
es zu einer Veränderung unterschiedlicher Tight Junction-Proteine nach peripherer
Nervenverletzung (CCI) kommt. Hierbei konnte gezeigt werden, dass eine CCI zu einer
Herabregulation der mRNA-Expression von Claudin-1, Claudin-19, Tricellulin und
Occludin im Rückenmark führt, wobei diese Veränderungen insbesondere 7 und 14 d
nach der CCI auftraten. Die membranäre Expression dieser Proteine im Rückenmark
blieb bis auf Occludin unverändert, das 7 d nach der CCI signifikant reduziert war. Am
deutlichsten waren jedoch Claudin-5 und ZO-1 verändert. Folglich vermindert eine CCI
signifikant die Claudin-5-mRNA sowie die Immunreaktivität in isolierten Kapillaren des
Rückenmarks. Das Ankerprotein ZO-1 war sogar auf allen Ebenen, also in der Genals
auch Proteinexpression, und darüber hinaus in den Rückenmarkskapillaren
signifikant reduziert.
Die Interpretation dieser Ergebnisse legt nahe, dass ZO-1, und zum Teil auch
Claudin-5, für die gestörte Blut-Rückenmarkschranke verantwortlich sind und die
anderen untersuchten Proteine vermutlich nur eine untergeordnete Rolle spielen. Die
Bedeutung der Tight Junction-Proteine in der Blut-Rückenmarkschranke konnte somit
weiter untermauert werden. In zukünftigen Untersuchungen wäre es wichtig den
Signalweg, der zur Veränderung der Tight Junction-Proteine führt sowie die
subzelluläre Lokalisation zu untersuchen, um Möglichkeiten zum Wiederverschluss
der Barriere zu finden. Somit könnten Therapien zur Aufrechterhaltung der Blut-
Rückenmarkhomöostase den neuropathischen Schmerz unter Umständen kausal, und
nicht nur symptomatisch, behandelbar machen.
Optogenetics became successful in neuroscience with Channelrhodopsin-2 (ChR2), a light-gated cation channel from the green alga Chlamydomonas reinhardtii, as an easy applicable tool. The success of ChR2 inspired the development of various photosensory proteins as powerful actuators for optogenetic manipulation of biological activity. However, the current optogenetic toolbox is still not perfect and further improvements are desirable. In my thesis, I engineered and characterized several different optogenetic tools with new features.
(i) Although ChR2 is the most often used optogenetic actuator, its single-channel conductance and its Ca2+ permeability are relatively low. ChR2 variants with increased Ca2+ conductance were described recently but a further increase seemed possible. In addition, the H+ conductance of ChR2 may lead to cellular acidification and unintended pH-related side effects upon prolonged illumination. Through rational design, I developed several improved ChR2 variants with larger photocurrent, higher cation selectivity, and lower H+ conductance.
(ii) The light-activated inward chloride pump NpHR is a widely used optogenetic tool for neural silencing. However, pronounced inactivation upon long time illumination constrains its application for long-lasting neural inhibition. I found that the deprotonation of the Schiff base underlies the inactivation of NpHR. Through systematically exploring optimized illumination schemes, I found illumination with blue light alone could profoundly increase the temporal stability of the NpHR-mediated photocurrent. A combination of green and violet light eliminates the inactivation effect, similar to blue light, but leading to a higher photocurrent and therefore better light-induced inhibition.
(iii) Photoactivated adenylyl cyclases (PACs) were shown to be useful for light-manipulation of cellular cAMP levels. I developed a convenient in-vitro assay for soluble PACs that allows their reliable characterization. Comparison of different PACs revealed that bPAC from Beggiatoa is the best optogenetic tool for cAMP manipulation, due to its high efficiency and small size. However, a residual activity of bPAC in the dark is unwanted and the cytosolic localization prevents subcellular precise cAMP manipulation. I therefore introduced point mutations into bPAC to reduce its dark activity. Interestingly, I found that membrane targeting of bPAC with different linkers can remarkably alter its activity, in addition to its localization. Taken together, a set of PACs with different activity and subcellular localization were engineered for selection based on the intended usage. The membrane-bound PM-bPAC 2.0 with reduced dark activity is well-tolerated by hippocampal neurons and reliably evokes a transient photocurrent, when co-expression with a CNG channel.
(iv) Bidirectional manipulation of cell activity with light of different wavelengths is of great importance in dissecting neural networks in the brain. Selection of optimal tool pairs is the first and most important step for dual-color optogenetics. Through N- and C-terminal modifications, an improved ChR variant (i.e. vf-Chrimson 2.0) was engineered and selected as the red light-controlled actuator for excitation. Detailed comparison of three two-component potassium channels, composed of bPAC and the cAMP-activated potassium channel SthK, revealed the superior properties of SthK-bP. Combining vf-Chrimson 2.0 and improved SthK-bP “SthK(TV418)-bP” could reliably induce depolarization by red light and hyperpolarization by blue light. A residual tiny crosstalk between vf-Chrimson 2.0 and SthK(TV418)-bP, when applying blue light, can be minimized to a negligible level by applying light pulses or simply lowering the blue
light intensity.
Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor.
G protein-coupled receptors of the Adhesion family (aGPCRs) comprise the second largest group within the GPCR realm with over 30 mammalian homologs. They contain a unique structure with unusually large extracellular domains (ECDs) holding many structural folds known to mediate cell-cell and cell-matrix interactions. Furthermore, aGPCRs undergo autoproteolytic cleavage at the GPCR proteolysis site (GPS), an integral portion of the GPCR autoproteolysis inducing (GAIN) domain. Thus far, it is largely unknown if and how self-cleavage affects aGPCR activation and signaling and how these signals may shape the physiological function of cells.
Latrophilin, alternatively termed the calcium-independent receptor of α-latrotoxin (CIRL) constitutes a highly conserved, prototypic aGPCR and has been assigned roles in various biological processes such as synaptic development and maturation or the regulation of neurotransmitter release. The Drosophila melanogaster homolog dCIRL is found in numerous sensory neurons including the mechanosensory larval pentascolopidial chordotonal organs (CHOs), which rely on dCIRL function in order to sense mechanical cues and to modulate the mechanogating properties of present ionotropic receptors.
This study reveals further insight into the broad distribution of dCirl expression throughout the larval central nervous system, at the neuromuscular junction (NMJ), as well as subcellular localization of dCIRL in distal dendrites and cilia of chordotonal neurons. Furthermore, targeted mutagenesis which disabled GPS cleavage of dCIRL left intracellular trafficking in larval CHOs unaffected and proved autoproteolysis is not required for dCIRL function in vivo. However, substitution of a threonine residue, intrinsic to a putative tethered agonist called Stachel that has previously been documented for several other aGPCRs, abrogated receptor function. Conclusively, while this uncovered the presence of Stachel in dCIRL, it leaves the question about the biological relevance of the predetermined breaking point at the GPS unanswered. In an independent approach, the structure of the “Inter-RBL-HRM” (IRH) region, the region linking the N-terminal Rhamnose-binding lectin-like (RBL) and the hormone receptor motif (HRM) domains of dCIRL, was analyzed. Results suggest random protein folding, excessive glycosylation, and a drastic expansion of the size of IRH. Therefore, the IRH might represent a molecular spacer ensuring a certain ECD dimension, which in turn may be a prerequisite for proper receptor function.
Taken together, the results of this study are consistent with dCIRL’s mechanoceptive faculty and its role as a molecular sensor that translates mechanical cues into metabotropic signals through a yet undefined Stachel-dependent mechanism.
The superfamily of G protein-coupled receptors (GPCRs) comprises more than 800 members, which are divided into five families based on phylogenetic analyses (GRAFS classification): Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin. The adhesion G protein-coupled receptor (aGPCR) family forms with 33 homologs in Mammalia the second largest and least investigated family of GPCRs. The general architecture of an aGPCR comprises the GPCR characteristics of an extracellular region (ECR), a seven transmembrane (7TM) domain and an intracellular region (ICR). A special feature of aGPCRs is the extraordinary size of the ECR through which they interact with cellular and matricellular ligands via adhesion motif folds. In addition, the ECR contains a so-called GPCR autoproteolysis-inducing (GAIN) domain, which catalyzes autoproteolytic cleavage of the protein during maturation. This cleavage leads to the formation of an N-terminal (NTF) and a C-terminal fragment (CTF), which build a unit by means of hydrophobic interactions and therefore appear as a heterodimeric receptor at the cell surface. In the past, it has been shown that the first few amino acids of the CTF act as a tethered agonist (TA) that mediates the activation of the receptor through the interaction with the 7TM domain. However, the molecular mechanism promoting the TA-7TM domain interaction remains elusive. This work reveals a novel molecular mechanism that does not require the dissociation of the NTF-CTF complex to promote release of the TA and thus activation of the aGPCR. The introduction of bioorthogonal labels into receptorsignaling- relevant regions of the TA of various aGPCRs demonstrated that the TA is freely accessible within the intact GAIN domain. This suggests a structural flexibility of the GAIN domain, which allows a receptor activation independent of the NTF-CTF dissociation, as found in cleavage-deficient aGPCR variants. Furthermore, the present study shows that the cellular localization and the conformation of the 7TM domain depends on the activity state of the aGPCR, which in turn indicates that the TA mediates conformational changes through the interaction with the 7TM domain, which ultimately regulates the receptor activity. In addition, biochemical analyses showed that the GAIN domain-mediated autoproteolysis of the human aGPCR CD97 (ADGRE5/E5) promotes further cleavage events within the receptor. This suggests that aGPCRs undergo cleavage cascades, which are initialized by the autoproteolytic reaction of the GAIN domain. Thus, it can be assumed that aGPCRs are subject to additional proteolytic events. Finally, the constitutive internalization of the NTF and the CTF of E5 was demonstrated by various labeling methods. It was possible to label both fragments independently and to follow their subcellular location in vitro. In summary, these obtained results contribute to a better understanding about the molecular mechanisms of activity and signaling of aGPCRs.
Ziel der vorliegenden Arbeit war die nanoskopische Analyse struktureller Differenzierung und Plastizität präsynaptischer aktiver Zonen (AZs) an der NMJ von Drosophila melanogaster mittels hochauflösender, lichtmikroskopischer Bildgebung von Bruchpilot (Brp). In erster Linie wurde das lokalisationsmikroskopische Verfahren dSTORM angewendet. Es wurden neue Analyse-Algorithmen auf der Basis von HDBSCAN entwickelt, um eine objektive, in weiten Teilen automatisierte Quantifizierung bis auf Ebene der Substruktur der AZ zu ermöglichen. Die Differenzierung wurde am Beispiel phasischer und tonischer Synapsen, die an dieser NMJ durch Is- und Ib-Neurone gebildet werden, untersucht. Phasische Is-Synapsen mit hoher Freisetzungswahrscheinlichkeit zeigten kleinere, kompaktere AZs mit weniger Molekülen und höherer molekularer Dichte mit ebenfalls kleineren, kompakteren Brp-Subclustern. Akute strukturelle Plastizität wurde am Beispiel präsynaptischer Homöostase, bei der es zu einer kompensatorisch erhöhten Neurotransmitterfreisetzung kommt, analysiert. Interessanterweise zeigte sich hier ebenfalls eine kompaktere Konfiguration der AZ, die sich auch auf Ebene der Subcluster widerspiegelte, ohne Rekrutierung von Molekülen. Es konnte demonstriert werden, dass sich eine höhere Moleküldichte in der Lokalisationsmikroskopie in eine höhere Intensität und größere Fläche in der konfokalen Mikroskopie übersetzt, und damit der Zusammenhang zu scheinbar gegensätzlichen Vorbefunden hergestellt werden. Die Verdichtung bzw. Kompaktierung erscheint im Zusammenhang mit der Kopplungsdistanz zwischen VGCCs und präsynaptischen Vesikeln als plausibles Muster der effizienten Anordnung molekularer Komponenten der AZ. Die hier eingeführten Analysewerkzeuge und molekularbiologischen Strategien, basierend auf dem CRISPR/Cas9-System, zur Markierung von AZ-Komponenten können zukünftig zur weiteren Klärung der Bedeutung der molekularen Verdichtung als allgemeines Konzept der AZ-Differenzierung beitragen.