Periodic Reporting for period 3 - HITSCIL (How intraflagellar transport shapes the cilium: a single-molecule systems study)
Período documentado: 2021-09-01 hasta 2023-02-28
Sensory cilia are organelles extending like antennas from many eukaryotic cells, with crucial functions in sensing and signalling. Cilia consist of an axoneme built of microtubules, enveloped by a specialized membrane. Ciliary development and maintenance depend critically on a specific, microtubule-based intracellular transport mechanism, intraflagellar transport (IFT). In my laboratory, we study the chemosensory cilia of C. elegans, which sense water-soluble molecules in the animal’s environment for chemotaxis. Over the past years, we have developed a unique set of quantitative, single-molecule fluorescence microscopy tools that allow us to visualize and quantify IFT dynamics with unprecedented detail in living animals. So far, our focus has been on the cooperation of the motor proteins driving IFT. The overall objective of the HITSCIL project is to zoom out and shed light on the connection between ciliary structure, chemosensory function and IFT, from a systems perspective. The four major aims of the project are to:
• determine how directional changes in IFT are regulated and are affected by external disturbances,
• understand the dynamics of the axonemal microtubules and how IFT affects these dynamics and vice versa,
• study how sensory ciliary function affects IFT and ciliary structure,
• further develop our (single-molecule) fluorescence microscopy toolbox by improving instrumentation and using better fluorescent probes and sensors.
These experiments will place my lab in a unique position to push forward our understanding of the relationship between structure, function and dynamics of transport of this fascinating and fundamental organelle.
• determine how directional changes in IFT are regulated and are affected by external disturbances,
• understand the dynamics of the axonemal microtubules and how IFT affects these dynamics and vice versa,
• study how sensory ciliary function affects IFT and ciliary structure,
• further develop our (single-molecule) fluorescence microscopy toolbox by improving instrumentation and using better fluorescent probes and sensors.
These experiments will place my lab in a unique position to push forward our understanding of the relationship between structure, function and dynamics of transport of this fascinating and fundamental organelle.
Within the project, we have made important progress on the following topics.
Technical: we have build a new microscope, a light-sheet microscope, optimized for live, adult C. elegans imaging (published). In addition we have achieved some important progress in single-molecule imaging in live, adult worms, namely that we can now image substantially faster processes and that we can do 2-color imaging. Furthermore we can now routinely determine neuronal activity using imaging of fluorescent Ca2+ sensors. This latter approach (as well as our routine in vivo worm imaging) can be performed in a microfluidics chip, which allows us to expose the worms in a well-controlled way to different chemical stimuli (e.g. badly tasting compounds). Lastly, we have greatly improved our single-molecule trajectory analysis tools, allowing now for automated identification of directional turns, bouts of diffusion, active transport or pauses.
We are now applying the novel tools and are close to publishing new results in the coming months on:
- single-molecule imaging of the Ca2+ channel OCR-2 that is involved in chemosensing. We show that this membrane protein is distributed over the cilium (with substantial accumulation in the ciliary tip) via a complex, location-dependent combination of active transport via intraflagellar transport and free diffusion in the membrane.
- single-molecule imaging of the motor proteins in intraflagellar transport. Here we focus on the so-called turnaround: motors that fall off transport trains and dock on another one moving in the opposite direction, switching from a 'driver' to a 'passenger' role. We had shown before that this aspect is mechanistically very important for IFT, now, with better time resolution we can see in detail what happens during a directional switch.
- we have now directly observed that there is a direct link between the function of the cilia (i.e. sensing of repellent chemicals), intraflagellar transport (IFT) and ciliary shape / structure. We found that two different classes of chemicals result in completely different neuronal responses, but also completely differently effect on IFT and ciliary shape.
Technical: we have build a new microscope, a light-sheet microscope, optimized for live, adult C. elegans imaging (published). In addition we have achieved some important progress in single-molecule imaging in live, adult worms, namely that we can now image substantially faster processes and that we can do 2-color imaging. Furthermore we can now routinely determine neuronal activity using imaging of fluorescent Ca2+ sensors. This latter approach (as well as our routine in vivo worm imaging) can be performed in a microfluidics chip, which allows us to expose the worms in a well-controlled way to different chemical stimuli (e.g. badly tasting compounds). Lastly, we have greatly improved our single-molecule trajectory analysis tools, allowing now for automated identification of directional turns, bouts of diffusion, active transport or pauses.
We are now applying the novel tools and are close to publishing new results in the coming months on:
- single-molecule imaging of the Ca2+ channel OCR-2 that is involved in chemosensing. We show that this membrane protein is distributed over the cilium (with substantial accumulation in the ciliary tip) via a complex, location-dependent combination of active transport via intraflagellar transport and free diffusion in the membrane.
- single-molecule imaging of the motor proteins in intraflagellar transport. Here we focus on the so-called turnaround: motors that fall off transport trains and dock on another one moving in the opposite direction, switching from a 'driver' to a 'passenger' role. We had shown before that this aspect is mechanistically very important for IFT, now, with better time resolution we can see in detail what happens during a directional switch.
- we have now directly observed that there is a direct link between the function of the cilia (i.e. sensing of repellent chemicals), intraflagellar transport (IFT) and ciliary shape / structure. We found that two different classes of chemicals result in completely different neuronal responses, but also completely differently effect on IFT and ciliary shape.
We are further sharpening our research tools and applying them (with important progress) to the following topics:
- understand how motor activity in C. elegans chemosensory neurons is regulated
- understand how Tubulin transport takes place and axoneme / microtubule (the backbone of the cilium) length is regulated
- understand what signalling pathways and secondary messengers are involved in the two different responses we observed in chemosensing
- understand how components of the IFT machinery (train particles, motors) arrive and leave the cilium: what is the contribution (and mechanism) of dendritic transport, how much exchange between outside / inside cilium takes place.
- understand how motor activity in C. elegans chemosensory neurons is regulated
- understand how Tubulin transport takes place and axoneme / microtubule (the backbone of the cilium) length is regulated
- understand what signalling pathways and secondary messengers are involved in the two different responses we observed in chemosensing
- understand how components of the IFT machinery (train particles, motors) arrive and leave the cilium: what is the contribution (and mechanism) of dendritic transport, how much exchange between outside / inside cilium takes place.