Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function, cilia are dependent on intraflagellar transport (IFT). Here, we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We find that chemical stimulation results in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents results in different responses, not only in IFT, ciliary structure, and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.

To survive, Caenorhabditis elegans depends on sensing soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Cilia rely on intraflagellar transport (IFT) to facilitate the distribution of cargo, such as TPs, along the ciliary axoneme. Here, we use fluorescence imaging of living worms and perform single-molecule tracking experiments to elucidate the dynamics underlying the ciliary distribution of the sensory TP OCR-2. Quantitative analysis reveals that the ciliary distribution of OCR-2 depends on an intricate interplay between transport modes that depends on the specific location in the cilium: in dendrite and transition zone, directed transport is predominant. Along the cilium motion is mostly due to normal diffusion together with a small fraction of directed transport, while at the ciliary tip subdiffusion dominates. These insights in the role of IFT and diffusion in ciliary dynamics contribute to a deeper understanding of ciliary signal transduction and chemosensing.

Cilia are vital for the cell's ability to sense its environment and rely on a process called intra flagellar transport (IFT) for their development, maintenance and function in signal-transduction. In IFT, motor proteins transport ciliary components along the polarized microtubule axoneme of the cilium. Among the cargoes of IFT are transmembrane proteins involved in signal transduction. As a model system, we study C. elegans chemosensory cilia, which we study using fluorescence microscopy with single-molecule sensitivity. First, we demonstrate that IFT machinery and ciliary components, including TRPV transmembrane channel protein OCR-2 are redistributed away from the ciliary tip upon external chemical stimulation, in a robust, extensive and reversible way. To elucidate the dynamics underlying this dramatic protein redistribution, we performed single-molecule imaging of OCR-2 in live C. elegans. Advanced analysis of the single-molecule trajectories shows that, in dendrite and transition zone, active transport is the prevailing motility mode of OCR-2. In the proximal and distal segments, however, motility is a much more complex, location-specific interplay between active transport, normal diffusion and sub diffusion. At the tip, confinement of the membrane proteins plays an important role. Together, our data and analysis demonstrate an intricate interplay between modes of transportation that ensure the proper ciliary distribution of OCR-2. These insights in the dynamics of cellular signal-transduction contributes to a wider understanding of IFT dynamics and to cilia as chemosensory organelles.

Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal velocity and changes therein, including pauses. We use SM imaging to study the dynamics of motor proteins and their cargo in the cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, wide-field fluorescence microscope and mostly open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in C. elegans and data analysis.

Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes.

Bidirectional transport driven by motor proteins is essential for the proper distribution of cargo, and therefore vital for many cellular processes. Cilia are polar, microtubule-based cellular sensing hubs that rely on a process called intraflagellar transport (IFT) for their development, maintenance and function in signal-transduction. IFT trains, consisting of cargo and the IFT-A and IFT-B protein complexes, are assembled at the ciliary base and driven co-operatively by kinesin-II and OSM-3 motors to the ciliary tip. The trains reverse direction at the tip and are transported back to the base by IFT dynein. The mechanism of IFT turnaround at the ciliary tip remains unknown. Here, we employ single-molecule fluorescence microscopy and single-particle tracking in the phasmid cilia of living C. elegans to probe IFT tip turnaround. Single-molecule trajectories reveal direct, pausing and diffusive turns in a sub-micrometer long turnaround region at the ciliary tip. Strikingly, while most IFT dyneins, OSM-3s and IFT-A particles turn almost instantaneously (within 600ms), IFT-B particles pause on average for 3 s before returning, with pauses lasting as long as 15 s. Further analysis reveals that IFT dynein and OSM-3 also exhibit diffusive behavior at the ciliary tip, whereas IFT-A and IFT-B are more spatially constrained. Our findings suggest that IFT trains dissociate at the tip and re-associate in retrograde moving trains. IFT-B, different than the other components, requires substantial remodeling while remaining spatially constrained at the tip, before it docks to an IFT-dynein driven retrograde train. Stochastic simulations of several tip turnaround scenarios support this model. Our data provides the first in vivo single-molecule quantification of IFT tip turnarounds, providing new insights into how bidirectional intracellular transport is organized and regulated.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these noncentrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to noncentrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes noncentrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that noncentrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of noncentrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.