Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.

Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.

The presynaptic active zone (AZ) of chemical synapses is a highly dynamic compartment where synaptic vesicle fusion and neurotransmitter release take place. During evolution the AZ was optimized for speed, accuracy, and reliability of chemical synaptic transmission in combination with miniaturization and plasticity. Single-molecule localization microscopy (SMLM) offers nanometer spatial resolution as well as information about copy number, localization, and orientation of proteins of interest in AZs. This type of imaging allows quantifications of activity dependent AZ reorganizations, e.g., in the context of presynaptic homeostatic potentiation. In combination with high-pressure freezing and optogenetic or electrical stimulation AZs can be imaged with millisecond temporal resolution during synaptic activity. Therefore SMLM allows the determination of key parameters in the complex spatial environment of AZs, necessary for next generation simulations of chemical synapses with realistic protein arrangements.

In Nervensystemen bedürfen Informationsweitergabe und Gedächtnisformation eines präzisen Zusammenspiels von Synapsen in Zeit und Raum. Synaptische Transmission basiert strukturell auf mesoskopischen cytosolischen Kompartimenten an der präsynaptischen Membran, sogenannten Aktiven Zonen (AZ). Ihre Cytomatrix, bestehend aus zentralen Gerüstproteinen wie Rab3 interacting molecule (RIM), ermöglicht eine schnelle Freisetzung synaptischer Vesikel. Die Defizienz der lokal häufigsten Isoform RIM1α resultiert an einer komplexen zentralen Säugersynapse, die des hippocampalen Moosfaserboutons (MFB) zu im Cornu ammonis (CA)3 befindlichen Pyramidalzellen, in einer dezimierten Langzeitplastizität. Auf Verhaltensebene zeigen diese Mäuse eine reduzierte Lernfähigkeit. Die vorliegende Dissertation widmet sich grundlegend der bisher unbekannten dreidimensionalen (3D) AZ-Ultrastruktur des MFB in akuten Hippocampusschnitten der adulten Wildtyp- und RIM1α-Knock-Out-Maus (RIM1α\(^{-/-}\)). In einer methodischen Entwicklungsphase wurde ein neuartiges, anspruchsvolles Protokoll der nahezu artefaktfreien (near to native) Synapsenpräparation am MFB mittels Hochdruckgefrierung und Gefriersubstitution sowie der 3D-Modellierung mittels Elektronentomographie etabliert. In einer zweiten Experimentier- und Analysephase ermöglichte die hochwertige synaptische Gewebeerhaltung in beiden Genotypen eine standardisierte, auf Programmierskripten basierte Quantifizierung der AZ-Ultrastruktur bis auf die Ebene eines individuell gedockten synaptischen Vesikels. Dieser Dissertation gelingt der Nachweis, dass eine Defizienz von RIM1α zu einer multidimensionalen ultrastrukturellen Veränderung der AZ und ihres Vesikelpools am MFB führt. Neben einer Reduktion, Dezentralisierung und strukturellen Veränderung (eng) gedockter Vesikel – der ultrastrukturellen Messgrößen von unmittelbar freisetzungsfähigen Vesikeln – verdichtet sich der distaler lokalisierte Vesikelpool auf zugleich größeren, heterogenen AZ-Flächen mit erweitertem synaptischem Spalt. Vorliegende Untersuchungen tragen zum Verständnisgewinn über eine zentrale Rolle von RIM1α für das Docking und die Organisation von Vesikeln der AZ im MFB bei. Darüber hinaus stellen die präzisen ultrastrukturellen Analysen eine morphologische Grundlage für weiterführende Studien mit Hilfe modernster Techniken dar, beispielsweise über die Auswirkungen der geänderten RIM1α\(^{-/-}\) AZ-Ultrastruktur auf die präsynaptische Plastizität sowie in Korrelation zum Gedächtnis und Lernen der Tiere.

Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.