Periodic Reporting for period 1 - SILIA (Solid Immersion Lens Microscopy to Study Cilia Assembly)
Période du rapport: 2019-05-04 au 2021-05-03
Each cell of a human body represents a highly complex mechanism tuned to the smallest detail. Its proper functioning relies on activity and state of a plethora of its constituents - individual biomolecules. Modern electron microscopy, in particular cryo-electron microscopy, has proven instrumental for imaging in a great detail the arrangement of molecules and structures within a cell; this facilitates searching for the origin of diseases and designing drugs. However, it only provides us with static pictures or snapshots of the highly dynamic cellular environment. This project aims at developing a technique for time resolved light and cryo-electron microscopy, enabling to reconstruct time course of cellular events from individual images. The research interest of the host lab is focused on cilia and flagella, tiny organelles found on the surface of many eukaryotic cells. The cilia are known to be indispensable for vital cellular processes, such as environment sensing, cell motility, signaling and development. Defective or missing cilia often lead to severe congenital disorders called ciliopathies. On the other hand, cilia are essential for survival of many parasites, such as Trypanosoma brucei, the causative agent of Human African trypanosomiasis, in the host. Therefore, cilia are of a great interest as a potential therapeutic target. The ciliary distal tip is an essential ciliary domain; it provides capping and mechanical stabilization of the ciliary cytoskeleton, it is a turning point of the rapid intraflagellar transport trains, a sole place of cilium growth, and a place of budding of signaling vesicles. Yet the tip is the most enigmatic of all ciliary domains, with structures constituting the ciliary tip largely unknown. This hampers our understanding of how are the tip-related processes brought about and orchestrated. Current microscopes have limited capability of resolving highly dynamic traffic in such a dense and confined compartment. Thus, this project has set off to establish a method to study living cells with the highest temporal and spatial resolution. During the course of the project, such a microscopy method has been successfully developed combining the possibility of high resolution time-lapse imaging and cryo-fixation for electron microscopy and its applicability demonstrated by studying dynamic processes at the ciliary tip of Trypanosoma brucei parasites.
The scientific part of this project comprises two research aims. The first aim was building the microscope suitable for observing living cells with a high resolution and sensitivity and capable of their rapid freezing (cryogenic fixation) for a further study with electron microscopy. Following the proposed plan, this task was accomplished by developing a microscope based on solid immersion optics . Here, the sample is positioned directly on a hemispherical lens, made of a suitable material, usually with high index of refraction. The high refraction index allows to achieve higher resolution and light collection efficiency. To study dynamics of molecular processes within a cell and in cell-free biochemical systems, the microscope has been equipped with several imaging modalities: brightfield illumination, LED-based fluorescence, LED and laser-based TIRF (total internal reflection fluorescence) and interference reflection microscopy. During benchmark tests, it was observed that the system is capable of imaging challenging cellular structures, such as single unlabelled microtubules (very thin filaments) and movement of intraflagellar transport trains (a very rapid process) with resolution and sensitivity comparable to, or exceeding available commercial setups. Subsequently, cryogenic fixation with simultaneous fluorescence observation was successfully performed using this setup.
The second aim involved studying biological processes in the ciliary tip domain. There are several outstanding questions associated with the ciliary tip, including its role in length regulation of the cilium. It has been known that cilium elongates exclusively at its distal tip. One of proposed hypotheses assumes that the protein complex formed at the tip of mature cilia is involved in its stabilization and prevents an indefinite growth by blocking addition of new tubulin dimers. To explore this hypothesis, an in vitro reconstitution system based on flagellar skeletons was established. Using wild-type and knock-down mutant cell lines it was shown that the tip of mature cilia is indeed incompetent of stimulating microtubule growth. Subsequently, a role of various tip complex constituents in this process was tested using skeletons prepared of depletion mutants.
In living cells, all building blocks are delivered to the tip by intraflagellar transport. However, it remains unclear how are the intraflagellar transport trains turned around at the tip to return back to the cell body. Using the newly developed system, the intraflagellar transport events at the tip were described with unprecedented accuracy. The scientific results have been communicated in a forthcoming methodical research paper and during an invited conference talk.
The second aim involved studying biological processes in the ciliary tip domain. There are several outstanding questions associated with the ciliary tip, including its role in length regulation of the cilium. It has been known that cilium elongates exclusively at its distal tip. One of proposed hypotheses assumes that the protein complex formed at the tip of mature cilia is involved in its stabilization and prevents an indefinite growth by blocking addition of new tubulin dimers. To explore this hypothesis, an in vitro reconstitution system based on flagellar skeletons was established. Using wild-type and knock-down mutant cell lines it was shown that the tip of mature cilia is indeed incompetent of stimulating microtubule growth. Subsequently, a role of various tip complex constituents in this process was tested using skeletons prepared of depletion mutants.
In living cells, all building blocks are delivered to the tip by intraflagellar transport. However, it remains unclear how are the intraflagellar transport trains turned around at the tip to return back to the cell body. Using the newly developed system, the intraflagellar transport events at the tip were described with unprecedented accuracy. The scientific results have been communicated in a forthcoming methodical research paper and during an invited conference talk.
Development of technology for imaging and diagnostics is critical to meet the challenges of understanding and treatment of diseases. At the same time, reliability and reproducibility of biomedical experiments is receiving increasing amount of attention. In the field of microscopy imaging, this implies a minimally invasive sample treatment by limiting the exposure to strong light and chemical fixatives. This project has pushed the boundary of minimally invasive imaging while improving the spatial and temporal resolution . The capabilities of the technology have been demonstrated by acquiring novel insight into the mechanisms of cilia assembly and maintenance. Possibilities of commercial exploitation of the technology are currently being investigated.
Besides the research goals, an important part of the project was to broaden researcher’s expertise in various fields of research and technology. Originally trained as a cell biologist, the researcher benefited tremendously from aspects of this project that included optics and engineering (design and construction of the microscope) and software development and machine learning (analysis of the microscopic data). Together with acquired experience in project planning and management, this action provided the researcher with a valuable set of skills needed for his own independent research career.
In order to present this project to broader audience and promote general interest in cell biology and microscopy, the researcher participated in the The European Researchers' Night and gave a talk aimed at high school students interested in microscopy.
Besides the research goals, an important part of the project was to broaden researcher’s expertise in various fields of research and technology. Originally trained as a cell biologist, the researcher benefited tremendously from aspects of this project that included optics and engineering (design and construction of the microscope) and software development and machine learning (analysis of the microscopic data). Together with acquired experience in project planning and management, this action provided the researcher with a valuable set of skills needed for his own independent research career.
In order to present this project to broader audience and promote general interest in cell biology and microscopy, the researcher participated in the The European Researchers' Night and gave a talk aimed at high school students interested in microscopy.