Periodic Reporting for period 4 - HITSCIL (How intraflagellar transport shapes the cilium: a single-molecule systems study)
Reporting period: 2023-03-01 to 2024-05-31
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 and serve as a model system for human cilia, which are part of almost all human cells and can be affected by cilium-specific diseases, ciliopathies. 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 were to:
Aim 1. Further develop our (single-molecule) fluorescence microscopy toolbox by improving instrumentation and using better fluorescent probes and sensors.
Aim 2. Determine how directional changes in IFT are regulated and are affected by external disturbances.
Aim 3. Understand the dynamics of the axonemal microtubules and how IFT affects these dynamics and vice versa.
Aim 4. Study how sensory ciliary function affects IFT and ciliary structure.
In the project we have made major progress on all the four aims, see in detail below.
Taken together these results have provided new insights in how cilia and intraflagellar transport work. In particular, our most remarkable findings were that chemosensing and IFT within cilia affect each other in major ways, that IFT trains are dynamic assemblies formed at the ciliary base in a sequential process, with the motor proteins binding last, and that while IFT trains are stable asemblies, the (kinesin) motors dock on and off in a dynamic way, governing the transport.
Aim 1. Further develop our (single-molecule) fluorescence microscopy toolbox by improving instrumentation and using better fluorescent probes and sensors.
Aim 2. Determine how directional changes in IFT are regulated and are affected by external disturbances.
Aim 3. Understand the dynamics of the axonemal microtubules and how IFT affects these dynamics and vice versa.
Aim 4. Study how sensory ciliary function affects IFT and ciliary structure.
In the project we have made major progress on all the four aims, see in detail below.
Taken together these results have provided new insights in how cilia and intraflagellar transport work. In particular, our most remarkable findings were that chemosensing and IFT within cilia affect each other in major ways, that IFT trains are dynamic assemblies formed at the ciliary base in a sequential process, with the motor proteins binding last, and that while IFT trains are stable asemblies, the (kinesin) motors dock on and off in a dynamic way, governing the transport.
The final results of HITSCIL are:
Aim 1.
a. We have developed and applied a novel light-sheet microscope. [van Krugten et al. J. of Microscopy 2021].
b. We have developed a new illumination method allowing higher sensitivity and longer-term single-molecule fluorescence imaging in living animals, SWIM (Small Window Illumination Micrscopy) [Mitra et al. Optics Communications 2023].
c. We have improved our data analysis pipeline. We can now discriminate within single-molecule trajectories, periods of diffusive motion and of directed motion and extract the relevant motility parameters [van Krugten et al. Communications Biology 2022].
d. We have published a protocol article, describing our single-molecule imaging approach in detail [Loseva et al. Methods in Molecular Biology 2024]. discrimination We have furthermore developed a 2-color imaging approach and substantially faster imaging.
Aim 2.
a. Using faster imaging and 2-color imaging we have directly visualized how single-kinesin motors hop from one IFT train to another train (often moving in the opposite direction), via detachment from one train, free diffusion in solution and attachment to another train [Zhang et al. PNAS USA 2021].
b. Using single-molecule imaging we have directly visualized IFT trains assembling at the ciliary base. We have shown that motors and cargo assemble in a sequential process, in time and place [Mitra et al. Nature Comm 2024].
c. We used a similar approach to also focus on the earlier steps of IFT-A, IFT-B and BBSome subcomplexes assembling into trains [Mitra et al. Science Advances, in press].
d. We investigated the role of kinase DYF-5 in regulating IFT and found that it blocks the assembly of retrograde trains at the ciliary tip [Mul et al. Mol Biol Cell in press].
e. We wrote a review article on the regulation of IFT [Mul et al. Cells 2022].
Aim 3.
a. A detailed single-molecule study showing how tubulin is transported within the cilium (via free diffusion and, to a lesser extent via active transport with IFT) and incorporated in the axonemes is finalized, a manuscript is in preparation [Loseva et al. In preparation]
Aim 4.
a. We cut the dendrite connecting the chemosensory soma with cilia using laser abblation and observed major changes in IFT and ciliary structure, suggesting a well-defined mechanism to alter IFT and ciliary structure [Mijalkovic et al. Mol Biol Cell 2020].
b. We applied chemical cues to living C. elegans using microfluidics and observed major changes in IFT: hyperosmotic stimuli resulted in accumulation of the IFT machinery towards the ciliary tip, while chemical stimuli resulted in the opposite: accumulation at the base resulting in shortening and sensory inactivation of the cilia (like with laser abblation) [Bruggeman et al. Cell Reports 2022].
c. Detailed methods of this approach have been described in a protocol article [Bruggeman et al. 2024].
All publication have been open acces, including detailed methods, software and sharing of C. elegans strains. We have also presented these results at international conferences (invited and selected presentations and posters) and seminars. Furthermore, HITSCIL has resulted in two PhD theses (van Krugten & Mul) and a third one is in preparation (Loseva).
Aim 1.
a. We have developed and applied a novel light-sheet microscope. [van Krugten et al. J. of Microscopy 2021].
b. We have developed a new illumination method allowing higher sensitivity and longer-term single-molecule fluorescence imaging in living animals, SWIM (Small Window Illumination Micrscopy) [Mitra et al. Optics Communications 2023].
c. We have improved our data analysis pipeline. We can now discriminate within single-molecule trajectories, periods of diffusive motion and of directed motion and extract the relevant motility parameters [van Krugten et al. Communications Biology 2022].
d. We have published a protocol article, describing our single-molecule imaging approach in detail [Loseva et al. Methods in Molecular Biology 2024]. discrimination We have furthermore developed a 2-color imaging approach and substantially faster imaging.
Aim 2.
a. Using faster imaging and 2-color imaging we have directly visualized how single-kinesin motors hop from one IFT train to another train (often moving in the opposite direction), via detachment from one train, free diffusion in solution and attachment to another train [Zhang et al. PNAS USA 2021].
b. Using single-molecule imaging we have directly visualized IFT trains assembling at the ciliary base. We have shown that motors and cargo assemble in a sequential process, in time and place [Mitra et al. Nature Comm 2024].
c. We used a similar approach to also focus on the earlier steps of IFT-A, IFT-B and BBSome subcomplexes assembling into trains [Mitra et al. Science Advances, in press].
d. We investigated the role of kinase DYF-5 in regulating IFT and found that it blocks the assembly of retrograde trains at the ciliary tip [Mul et al. Mol Biol Cell in press].
e. We wrote a review article on the regulation of IFT [Mul et al. Cells 2022].
Aim 3.
a. A detailed single-molecule study showing how tubulin is transported within the cilium (via free diffusion and, to a lesser extent via active transport with IFT) and incorporated in the axonemes is finalized, a manuscript is in preparation [Loseva et al. In preparation]
Aim 4.
a. We cut the dendrite connecting the chemosensory soma with cilia using laser abblation and observed major changes in IFT and ciliary structure, suggesting a well-defined mechanism to alter IFT and ciliary structure [Mijalkovic et al. Mol Biol Cell 2020].
b. We applied chemical cues to living C. elegans using microfluidics and observed major changes in IFT: hyperosmotic stimuli resulted in accumulation of the IFT machinery towards the ciliary tip, while chemical stimuli resulted in the opposite: accumulation at the base resulting in shortening and sensory inactivation of the cilia (like with laser abblation) [Bruggeman et al. Cell Reports 2022].
c. Detailed methods of this approach have been described in a protocol article [Bruggeman et al. 2024].
All publication have been open acces, including detailed methods, software and sharing of C. elegans strains. We have also presented these results at international conferences (invited and selected presentations and posters) and seminars. Furthermore, HITSCIL has resulted in two PhD theses (van Krugten & Mul) and a third one is in preparation (Loseva).
In our view, the HITSCIL has been a success, pushing the state of the art in several aspects:
a. We have further and substantially improved our toolset to image single molecules in a living multicellular organism: increasing data quality, implementing faster and multi-color imaging, developing novel approaches for data analysis and interpretation. We are still one of the few research groups worldwide capable of routinely imaging single molecules in multicellular organisms and using this approach to increase fundamental, biological understanding at the molecular and cellular level.
b. We have provided novel insights in the dynamic nature of IFT trains: how they assemble at the ciliary base, how motor proteins dynamically attach and detach from IFT trains governing train motion and how kinases are involved in this. The detailed, quantitative insight we get from our unique approach allows direct access to the dynamics of this process, complementing the revolutionary cryo electron microscopy and tomography work performed on cilia (e.g. by Pigino, Roberts etc.), which provides snapshots at close to atomic resolution.
c. We have found that the chemosonsory function of cilia is tightly connected to intraflagellar transport. Sensory activity results in a substantial response of the IFT machinery. Surprisingly stimulation by different chemicals results in almost opposite reaction of the IFT machinery: hyperosmotic stimuli result in accumulation of the whole IFT machinery at the ciliary tip, while noxious chemicals trigger accumulation at the base, followed by partial disassembly of the cilium and (temporary) abolishment of sensory function.
Taken together, our results have shed new light on cilia, their sensory function, and intraflagellar transport. We are convinced that our unique single-molecule experiments in the model organism C. elegans will inspire discoveries in other ciliary systems, including human cells.
a. We have further and substantially improved our toolset to image single molecules in a living multicellular organism: increasing data quality, implementing faster and multi-color imaging, developing novel approaches for data analysis and interpretation. We are still one of the few research groups worldwide capable of routinely imaging single molecules in multicellular organisms and using this approach to increase fundamental, biological understanding at the molecular and cellular level.
b. We have provided novel insights in the dynamic nature of IFT trains: how they assemble at the ciliary base, how motor proteins dynamically attach and detach from IFT trains governing train motion and how kinases are involved in this. The detailed, quantitative insight we get from our unique approach allows direct access to the dynamics of this process, complementing the revolutionary cryo electron microscopy and tomography work performed on cilia (e.g. by Pigino, Roberts etc.), which provides snapshots at close to atomic resolution.
c. We have found that the chemosonsory function of cilia is tightly connected to intraflagellar transport. Sensory activity results in a substantial response of the IFT machinery. Surprisingly stimulation by different chemicals results in almost opposite reaction of the IFT machinery: hyperosmotic stimuli result in accumulation of the whole IFT machinery at the ciliary tip, while noxious chemicals trigger accumulation at the base, followed by partial disassembly of the cilium and (temporary) abolishment of sensory function.
Taken together, our results have shed new light on cilia, their sensory function, and intraflagellar transport. We are convinced that our unique single-molecule experiments in the model organism C. elegans will inspire discoveries in other ciliary systems, including human cells.