Periodic Reporting for period 1 - MingleIFT (Multi-color and single-molecule fluorescence imaging of intraflagellar transport in the phasmid chemosensory cilia of C. Elegans)
Reporting period: 2020-03-01 to 2022-02-28
The overarching objective of this project was to develop a multi-color and single-molecule imaging/image analysis toolkit to understand (i) how different ciliary components assemble into anterograde IFT trains to enter the sensory cilia of C. elegans and (ii) how this process is perturbed during chemotaxis.
To achieve the overall objective, the research methodology was structured into 3 aims, which are as follows:
Aim 1: Develop a multi-color and single-molecule imaging toolbox in chemosensory cilia of C. elegans.
Aim 2: Unravel how different ciliary proteins reach the base of the cilia, assemble into anterograde IFT trains and enter the cilia.
Aim 3: Determine how IFT dynamics and cilia structure is perturbed by mutations of ciliary proteins as well as in response to external stimuli.
The progress made during this project is briefly highlighted below and elaborated in greater detail in the next section.
Aim1:
A simple imaging strategy, termed small-window illumination microscopy (SWIM), was developed in order to perform long duration, fast, dual-color, single molecule imaging in C. elegans. Further, a tracking and image analysis pipeline was developed to study protein dynamics in sensory neurons as well as map the ultrastructure of sensory cilia.
Aim2 and Aim3:
There are a several studies that have emerged from performing SWIM microscopy in the sensory cilia of C. elegans.
To achieve the overall objective, the research methodology was structured into 3 aims, which are as follows:
Aim 1: Develop a multi-color and single-molecule imaging toolbox in chemosensory cilia of C. elegans.
Aim 2: Unravel how different ciliary proteins reach the base of the cilia, assemble into anterograde IFT trains and enter the cilia.
Aim 3: Determine how IFT dynamics and cilia structure is perturbed by mutations of ciliary proteins as well as in response to external stimuli.
The progress made during this project is briefly highlighted below and elaborated in greater detail in the next section.
Aim1:
A simple imaging strategy, termed small-window illumination microscopy (SWIM), was developed in order to perform long duration, fast, dual-color, single molecule imaging in C. elegans. Further, a tracking and image analysis pipeline was developed to study protein dynamics in sensory neurons as well as map the ultrastructure of sensory cilia.
Aim2 and Aim3:
There are a several studies that have emerged from performing SWIM microscopy in the sensory cilia of C. elegans.
The implementation of the project has resulted in development of a new imaging technique and a new image analysis pipeline. The exploitation of the developed methods has resulted in several interesting research findings.
• Small-window illumination microscopy (SWIM) advances single molecule imaging in chemosensory neurons of C. elegans.
A new microscopy technique, SWIM was implemented to obtain detailed single molecule information in C. elegans chemosensory cilia (Figure). The essential idea is to control the epi-fluorescence illumination beam size to only illuminate the region of interest. Using a high laser power, one can bleach all molecules that are within the illuminated region, but any new molecule that enters the region is not bleached and can be imaged until it bleaches.
Dissemination: The methodology will be described in a book chapter in Springers Protocols, Single Molecule Analysis – Third Edition (accepted) and a methods paper will be submitted in a special issue of Optics Communications on Advances in Localization Microscopy
• Ultrastructure and IFT motor-entry dynamics at the ciliary base uncovered using single-molecule tracking microscopy.
Intraflagellar transport (IFT) along a microtubule-based axoneme orchestrates the growth and maintenance of sensory cilia, elongated signalling hubs protruding out of eukaryotic cells. Kinesin-2 motors drive anterograde IFT trains, assembled at the cilia base, into the cilium, importing cargo proteins hitchhiking on the trains. SWIM allows us to visualize and quantify the entry of individual anterograde kinesin-2 motors, kinesin-II and OSM-3, and cargo proteins associated with anterograde IFT trains, IFT dynein and tubulin, into cilia located at the tip of dendrites of chemosensory neurons in C. elegans.
Dissemination: Final analysis is ongoing and the manuscript is under preparation.
• Ciliary proteins utilize contrasting mechanisms to reach the ciliary base, assemble into trains and enter the cilia in C. elegans
The IFT proteins associated with cilia, have to be transported from the cell body to the cilia base, moving across the dendrites (several 10s of microns), where they assemble into IFT trains and cross the transition barrier to enter the cilia. By performing SWIM in the chemosensory neurons of C. elegans, we elucidate the different mechanistic strategies employed to orchestrate entry of proteins inside cilia of C. elegans. Further, we image changes in the dendritic transport of ciliary proteins in response to chemical repellants, to unravel the mechanism of regulation of IFT transport during chemotaxis.
Dissemination: Final experiments and analysis is ongoing.
• We have published a review article with focus on the regulation of intraflagellar transport motors in cilia and its relevance to ciliary structure, function and disease. Dissemination: Review titled ‘Mechanisms of Regulation in Intraflagellar Transport’ was published in the special issue of the Journal Cells: Primary Cilia in the Nervous System: Structure, Function and Disease Mechanisms. (https://doi.org/10.3390/cells11172737(opens in new window)).
• We utilize SWIM to understand tubulin dynamics in chemosensory cilia of C. elegans. IFT trains travel bidirectionally along the microtubule-based backbone – the axoneme. Project is driven by a PhD student, E. Loseva, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Final experiments and analysis is ongoing.
• We utilize quantitative fluorescence microscopy to study the regulation of kinesin motors in the chemosensory cilia of C. elegans by kinases DYF-5 and DYF-18. Project is driven by a PhD student, W. Mul, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Experiments and analysis is ongoing.
>> The felloship will be (has been) acknowledged for funding all the above discussed research works.
>> Invited talks, presentations and posters in conferences and online platforms
The results of this work have been disseminated in 3 invited talks, 1 virtual talk in a webinar series and 6 conferences (4 talks and 3 posters).
>> Public Outreach
A video including clay animation titled Worm Lab Tour (https://youtu.be/T_ZjuqJLTr0(opens in new window)) was made to explain the research performed in my host laboratory, which is suitable for general audience. It has been and will be shared at numerous platforms.
• Small-window illumination microscopy (SWIM) advances single molecule imaging in chemosensory neurons of C. elegans.
A new microscopy technique, SWIM was implemented to obtain detailed single molecule information in C. elegans chemosensory cilia (Figure). The essential idea is to control the epi-fluorescence illumination beam size to only illuminate the region of interest. Using a high laser power, one can bleach all molecules that are within the illuminated region, but any new molecule that enters the region is not bleached and can be imaged until it bleaches.
Dissemination: The methodology will be described in a book chapter in Springers Protocols, Single Molecule Analysis – Third Edition (accepted) and a methods paper will be submitted in a special issue of Optics Communications on Advances in Localization Microscopy
• Ultrastructure and IFT motor-entry dynamics at the ciliary base uncovered using single-molecule tracking microscopy.
Intraflagellar transport (IFT) along a microtubule-based axoneme orchestrates the growth and maintenance of sensory cilia, elongated signalling hubs protruding out of eukaryotic cells. Kinesin-2 motors drive anterograde IFT trains, assembled at the cilia base, into the cilium, importing cargo proteins hitchhiking on the trains. SWIM allows us to visualize and quantify the entry of individual anterograde kinesin-2 motors, kinesin-II and OSM-3, and cargo proteins associated with anterograde IFT trains, IFT dynein and tubulin, into cilia located at the tip of dendrites of chemosensory neurons in C. elegans.
Dissemination: Final analysis is ongoing and the manuscript is under preparation.
• Ciliary proteins utilize contrasting mechanisms to reach the ciliary base, assemble into trains and enter the cilia in C. elegans
The IFT proteins associated with cilia, have to be transported from the cell body to the cilia base, moving across the dendrites (several 10s of microns), where they assemble into IFT trains and cross the transition barrier to enter the cilia. By performing SWIM in the chemosensory neurons of C. elegans, we elucidate the different mechanistic strategies employed to orchestrate entry of proteins inside cilia of C. elegans. Further, we image changes in the dendritic transport of ciliary proteins in response to chemical repellants, to unravel the mechanism of regulation of IFT transport during chemotaxis.
Dissemination: Final experiments and analysis is ongoing.
• We have published a review article with focus on the regulation of intraflagellar transport motors in cilia and its relevance to ciliary structure, function and disease. Dissemination: Review titled ‘Mechanisms of Regulation in Intraflagellar Transport’ was published in the special issue of the Journal Cells: Primary Cilia in the Nervous System: Structure, Function and Disease Mechanisms. (https://doi.org/10.3390/cells11172737(opens in new window)).
• We utilize SWIM to understand tubulin dynamics in chemosensory cilia of C. elegans. IFT trains travel bidirectionally along the microtubule-based backbone – the axoneme. Project is driven by a PhD student, E. Loseva, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Final experiments and analysis is ongoing.
• We utilize quantitative fluorescence microscopy to study the regulation of kinesin motors in the chemosensory cilia of C. elegans by kinases DYF-5 and DYF-18. Project is driven by a PhD student, W. Mul, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Experiments and analysis is ongoing.
>> The felloship will be (has been) acknowledged for funding all the above discussed research works.
>> Invited talks, presentations and posters in conferences and online platforms
The results of this work have been disseminated in 3 invited talks, 1 virtual talk in a webinar series and 6 conferences (4 talks and 3 posters).
>> Public Outreach
A video including clay animation titled Worm Lab Tour (https://youtu.be/T_ZjuqJLTr0(opens in new window)) was made to explain the research performed in my host laboratory, which is suitable for general audience. It has been and will be shared at numerous platforms.
Several parts of the project are at the later stage of development and I expect at least 5 peer-reviewed manuscripts and 1 book chapter to be published as a result of this work, within the next 1-2 years. Further, I have already published an important review in the field, which provides thorough mechanistic insights in the regulation of intraflagellar transport in primary cilia.
A simple imaging/image analysis toolkit was developed in order to study the dynamics and localization of single protein molecules in adult living C. elegans. This technique could be easily extended to study protein dynamics in any cell type in adult C. elegans or even in other multicellular model organisms. Hopefully, this work will inspire other laboratories around the globe to incorporate aspects of this technology to perform research in diverse topics.
A fundamental understanding of cilia structure, function, ciliary-length regulation, IFT dynamics and IFT regulation is essential to have a conceptual understanding of ciliopathies, providing the basis for future research to find cures for ciliopathies. The imaging/image analysis methodology established during this project was employed to address several key questions regarding mechanisms involved during IFT in sensory neurons of C. elegans.
The scientific and personal growth during the course of the Mingle IFT grant has shaped my future research ideas and the choice of my career direction. I am currently actively applying for group leader positions all around Europe, to get an opportunity to perform my own independent scientific research.
A simple imaging/image analysis toolkit was developed in order to study the dynamics and localization of single protein molecules in adult living C. elegans. This technique could be easily extended to study protein dynamics in any cell type in adult C. elegans or even in other multicellular model organisms. Hopefully, this work will inspire other laboratories around the globe to incorporate aspects of this technology to perform research in diverse topics.
A fundamental understanding of cilia structure, function, ciliary-length regulation, IFT dynamics and IFT regulation is essential to have a conceptual understanding of ciliopathies, providing the basis for future research to find cures for ciliopathies. The imaging/image analysis methodology established during this project was employed to address several key questions regarding mechanisms involved during IFT in sensory neurons of C. elegans.
The scientific and personal growth during the course of the Mingle IFT grant has shaped my future research ideas and the choice of my career direction. I am currently actively applying for group leader positions all around Europe, to get an opportunity to perform my own independent scientific research.