Scientific summary
from my PhD thesis 'Sensing Cilia'
In order to investigate the dynamics of ciliary proteins involved in signal transduction, sensitive (single-molecule) fluorescence microscopy was applied. Chapter 2 describes the labelling and imaging strategy that was used for most of this work. Proteins of interest were endogenously labeled with a fluorescent protein to be able to observe their dynamics, unperturbed by the unwanted side effects of over-expression. C. elegans strains containing fluorescent-protein insertions used in this thesis made by colleagues before me were generated using MoSCI. Strains made by me were made using the CRISPR/Cas9 system. To image the labeled proteins inside living C. elegans, a custom-built, laser-illuminated, widefield epifluorescence microscope with sensitive detection was employed. Initial data analysis was usually performed using the ImageJ plugin KymographClear and the stand-alone program KymographDirect. Single-molecule tracking and analysis was performed using custom-written MATLAB routines.
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Light-sheet microscopy
The downside of the setup described in chapter 2, is that the worms have to be mounted on a coverslip. This limits the degrees of freedom to position the sample to three: x, y and z. Although the setup is excellent for sensitive single-molecule imaging of for example intraflagellar transport (IFT) components, the epi-illumination can cause out-of-focus background fluorescence. If one wants to make z-stacks, this background fluorescence decreases the signal-to-noise ratio drastically. To only illuminate the detection objective focal plane, and to be able to position the worm with four degrees of freedom, a light-sheet microscope was constructed (Chapter 3). In this instrument, an illumination objective was placed perpendicular to a detection objective. In this configuration, the illumination objective can be used to only illuminate the plane that is in focus of the detection objective, eliminating out-of-focus background fluorescence. Since both objectives are fixed in an objective holder, the stage is used to focus and position the area of interest in the field of view. The stage consists of three accurate closed-loop linear positioning piezomotor stages and a fourth rotational stage, allowing four degrees of freedom. For detection, initially a 100×, N.A. 1.1 Plan achromat objective was used. This objective, however, induced too many optical aberrations, severely affecting image sharpness. The 60×, N.A. 1.0 NIR Apo objective that was implemented in the final design, offered better image sharpness albeit at slightly lower magnification, resolution and collection efficiency. This turned out to be a limiting factor for imaging IFT-components in cilia at high resolution. We found, however, that the instrument is well suited for volumetric imaging, for example of the C. elegans nervous system, allowing recording of three-dimensional movies of a neuronal network in action.
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Cutting of the dendrite
In C. elegans, a sensory cilium protrudes from the dendrite of a sensory neuron. As a consequence, the distance between cilium and sites of protein synthesis in the soma is substantial. For ciliary development and function as chemosensor, IFT components continuously cycle from the ciliary base to the tip and back again. Chapter 4 investigates how dendritic input of ciliary components affects IFT and ciliary function. The real-time response of IFT components to femtosecond laser ablation of the dendrite was visualized using fluorescence microscopy. We found that the response occurs in three stages. First, IFT dynein is activated within seconds, redistributing IFT components towards the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Additional experiments show that activation of retrograde transport is not triggered by ATP depletion. Taken together, these results indicate that laser ablation triggers a specific mechanism allowing the IFT system to rapidly adapt to environmental cues.
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Live imaging of Single-Molecule tip turnarounds
At the forefront of sensing, the tip of the cilia is an intriguing site. At the tip, anterograde trains arrive, and retrograde trains leave to return to the base. To investigate what happens during this turnaround, we performed single-molecule imaging of four key IFT components: OSM-3, IFT-A component CHE-11, IFT-B component OSM-6 and IFT-dynein (XBX-1) (chapter 5). Our data show that OSM-3, IFT-A and IFT-dynein turn around almost instantly after arrival at the tip. IFT-B component OSM-6 however, is retained at the tip for a short while, possibly undergoing modification before joining a retrograde train. This indicates that the anterograde IFT-trains disassemble into at least the IFT-A, IFT-B, IFT-dynein and OSM-3 complexes before returning to the base. Taken together, the SM-approach as employed in this chapter is a valuable tool to dissect directional switches in bidirectional intracellular transport driven by opposite-polarity, microtubule-based motors.
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Chemosensor dynamics
C. elegans chemosensory cilia are at the forefront of the signal-transduction cascade that can lead to behavioral change. To assess the real-time response of involved ciliary components, we exposed C. elegans expressing fluorescently labeled tubulin, the molecular motor IFT-dynein, and the transmembrane calcium-channel OCR-2 to aversive chemicals (chapter 6). The experiments demonstrate a remarkable, robust and reversible redistribution of ciliary components out and into the ciliary distal segment. Genetically severing the link between OCR-2 and IFT shows that OCR-2 relies on both active transport by IFT and diffusion for its steady-state ciliary distribution. Single-molecule fluorescence imaging of OCR-2 was performed to elucidate the mechanisms underlying the steady-state distribution and reversible redistribution. From advanced analysis of the single-molecule trajectories, the picture arises that OCR-2 distribution and dynamics are governed by location-specific motility of OCR-2: OCR-2 is mostly transported by IFT in dendrite and transition zone, while diffusion in the ciliary membrane is much more prominent further along the cilium, with mostly normal diffusion in the proximal segment and sub-diffusion in the distal segment. At the ciliary tip, OCR-2 is mostly static, possibly held in place by interactions with other proteins. Taken together, the data demonstrates an intricate interplay between OCR-2’s transportation modes that enable it to function as ciliary signal transducer. On a broader scale, these insights into the dynamics of ciliary signal transduction components contribute to a wider understanding of IFT dynamics and to cilia as chemosensory organelles.
