8 research outputs found
Einzelmoleküluntersuchung der Ligandenbindung eines hyperpolarisationsaktivierten und zyklisch Nukleotid-gesteuerten Ionenkanals
Hyperpolarisationsaktivierte und zyklisch Nukleotid-gesteuerte (HCN-) Ionenkanäle bilden eine Familie von Ionenkanälen mit ungewöhnlichen Eigenschaften. HCN-Kanäle werden durch Hyperpolarisation des Membranpotenzials aktiviert und die Kanalaktivierung wird durch die Bindung von zyklischem Adenosinmonophosphat (cAMP) moduliert. Das Wechselspiel zwischen der spannunginduzierten Aktivierung und der Ligandenbindung ist äußerst komplex: So verändert einerseits die Ligandenbindung die Spannungsabhängigkeit des Kanals und andererseits die spannungsabhängige Kanalaktivierung die Ligandenaffinität. Dieses Wechselspiel wollte ich in meiner Arbeit genauer untersuchen. Mein Ziel war es, Bindungsereignisse einzelner Moleküle mit fluoreszenzoptischen Methoden zu verfolgen. Ich habe hierzu ein fluoreszenzierendes Analog zyklischer Nukleotide (Atto488cAMP) verwendet und dessen Bindungseigenschaften charakterisiert. An Plasmamembransheets habe ich mit TIRF-Mikroskopie die Atto488cAMP-Bindung an heterolog exprimierte HCN2- Kanäle und an isolierte Bindestellen sowohl makroskopisch als auch auf Einzelmolekülebene untersucht. Die Auswertung der Einzelmoleküldaten war kompliziert, da es auch unspezifische Bindungsereignisse gab und das Signal-zu-Rauschverhältnis nur moderat war. Anschließend erweiterte ich den Messaufbau, um an lebenden Zellen parallel Elektrophysiologie und TIRF-Mikroskopie durchführen zu können. Mit dem Aufbau habe ich makroskopische Fluoreszenzmessungen etabliert. Aufbauend auf diesen Ergebnissen können nun die Bindungszeiten einzelner Ligandenmoleküle an HCN2-Kanälen bei vorgegebenen Membranpotenzialen studiert werden.Hyperpolarization-activated and cyclic nucleotide-gated (HCN) ion channels belong to a family of ion channels with unusual properties. HCN channels are activated upon hyperpolarization of the membrane potential and the channel activation is modulated upon binding of cyclic adenosine monophosphate. The interplay between the voltage-induced activation and ligand binding is extremely complex: on one hand ligand binding changes the voltage dependence of the channel and on the other hand, the voltage-dependent channel activation changes the binding affinity. I wanted to investigate this interplay in more detail. My aim was to track binding of individual molecules by a fluorescence optical approach. For this I used a fluorescent analog of cyclic adenosine monophosphate (Atto488cAMP) and characterized its binding properties. With plasma membrane sheets and TIRF-microscopy I examined Atto488cAMP binding of heterologously expressed HCN2 channels as well as of a isolated binding sites both macroscopically and at a single-molecule level. The analysis of the single-molecule data was complicated, as there were non-specific binding events, and as the signal-to-noise ratio was only moderate. I expanded the experimental set-up, in order to perform electrophysiology and TIRF microscopy at living cells in parallel. With this set-up I established macroscopic binding studies. Based on these results, the binding times of individual ligands at HCN2 channels can now be studied at diffenerent membrane potentials
Loss of CaMKI function disrupts salt aversive learning in C. elegans
The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior is likely advantageous to avoid areas without food. While some genes have been implicated in this salt aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt aversive learning behavior in C. elegans. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 leads to an altered pattern of sensory- evoked calcium responses that may underlie salt aversive learning. Our study implicates the conserved CaMKI/CMK-1 as an essential cell-autonomous regulator for behavioral plasticity to environmental salt in C. elegans
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Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.
New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worms cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons
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Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.
New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm's cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons
Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.
New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm's cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons
Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap
New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm\u27s cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons