Differentiated dynamic response in C. elegans chemosensory cilia.

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.

A local interplay between diffusion and intraflagellar transport distributes TRPV-channel OCR-2 along C. elegans chemosensory cilia.

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.

Cutting off ciliary protein import: intraflagellar transport after dendritic femtosecond-laser ablation.

Primary cilia, organelles protruding from the surface of eukaryotic cells, act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), where ciliary proteins are transported between the ciliary base and tip. These proteins originate from the cell body because cilia lack protein synthesis machinery. How input from the cell body affects IFT and ciliary function is not well understood. Here, we use femtosecond-laser ablation to perturb the dendritic input of proteins to chemosensory cilia in living Caenorhabditis elegans. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to dendritic ablation. We find that the response occurs in three distinct stages. First, IFT dynein is activated within seconds, redistributing IFT components toward the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Depletion of ATP by adding azide also results in IFT slowdown and IFT components leaving the cilium, but not in activation of retrograde IFT. These results indicate that laser ablation triggers a specific mechanism important for IFT regulation that allows the cilium to rapidly adapt to changes in the outside environment.

Single-Molecule Turnarounds of Intraflagellar Transport at the C. elegans Ciliary Tip.

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.

Single-Molecule Fluorescence Microscopy in Living Caenorhabditis elegans.

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.

Microtubule minus-end binding protein CAMSAP2 controls axon specification and dendrite development.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal 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 non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal 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 non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.