Biology subjects

Haasnoot, G. H.

Publications and source records attributed to Haasnoot, G. H..

4 recordsLinked to original sources

bioRxiv2025

Removal of the Ciliary Gate Allows Axoneme Extension in the Absence of Retrograde IFT

The transition zone (TZ) is a selective barrier that maintains ciliary compartmentalization by controlling protein entry and exit. Cilia assembly requires the crossing of this barrier by intraflagellar transport (IFT) trains, scaffolded by IFT-A and IFT-B complexes, which move cargo bidirectionally using kinesin-2 and dynein-2 motors. In Caenorhabditis elegans, IFT-A loss abolishes retrograde transport, resulting in truncated cilia packed with IFT material. Here, we show that blocking TZ assembly prevents dynein-2 and IFT-B accumulation inside IFT-A-deficient cilia and partially rescues axoneme length. Single-particle imaging reveals that this rescue occurs without recovery of retrograde IFT. Instead, IFT particles exit cilia by passively diffusing through the disrupted TZ. Moreover, IFT-A/TZ double mutants shed ciliary extracellular vesicles (EVs) abnormally enriched in IFT components, providing a second clearance route. We conclude that TZ removal alters ciliary responses to retrograde transport defects, promoting diffusion and EV release to clear IFT machinery and facilitate axoneme extension. Highlights- TZ loss provides alternative routes for clearing IFT machinery stalled in IFT-A mutant cilia - Axoneme extension is possible without retrograde IFT when the TZ barrier is removed - Disrupted TZ enables exit of IFT particles by passive diffusion in retrograde IFT-deficient cilia - Excess IFT machinery is discarded in ciliary EVs when retrograde IFT and gating are compromised

cell biology↗

bioRxiv2025

Sensory stimuli and cilium trafficking defects trigger the release of ciliary extracellular vesicles from multiple ciliary locations

The primary cilium is a signaling organelle that extends from many cell types to detect and relay extracellular signals. Beyond its signaling role, the cilium also produces cilia-derived extracellular vesicles (cEVs), although the mechanisms underlying their biogenesis and functions remain poorly understood. We characterized the cEV biogenesis in vivo using ciliated sensory neurons of C. elegans. In response to sensory cues, interruption of the intraflagellar transport (IFT) -a ciliary trafficking machinery carrying cargoes along the cilium-occurs together with ciliary membrane fission, resulting in the release of cEVs. Similarly, mutants disrupting IFT and ciliary receptor trafficking also enhance cEV production. To investigate how IFT influences the rate and location of cEV biogenesis, we selected a membrane marker that spans the entire length of the ciliary membrane independently of IFT. Single-molecule tracking demonstrates that the tetraspanin TSP-6 enters and diffuses within the cilia and does it independently of IFT. Lack of receptor retrieval or receptor entry in the cilium induces membrane budding from ciliary or periciliary membranes, respectively. Prior to fission, these membrane buds get enriched in TSP-6 as well as signaling receptors. Coupling receptor buildup with their export by cEVs provides a mechanism to preserve ciliary function and to modulate ciliary signaling. HIGHLIGHTS- The cone-shaped tetraspanin TSP-6 crosses the transition zone and moves by passive diffusion within the ciliary membrane, independently of IFT. - The production of ciliary EVs loaded with TSP-6 increases upon acute sensory stimulation or when IFT of ciliary membrane proteins is disrupted. - Depending on the nature of the perturbations, ciliary EVs remove excess material from the distal, proximal or periciliary segments of the cilia.

cell biology↗

bioRxiv2022

A small excitation window allows long-duration imaging of OCR-2-associated vesicles in C. elegans sensory neurons

Single-particle imaging using laser-illuminated widefield epi-fluorescence microscopy is a powerful tool to investigate molecular processes in vivo. Performing high-quality single-molecule imaging in such biological systems, however, remains a challenge due to difficulties in controlling the number of fluorescing molecules, photobleaching, and the autofluorescence background. Here, we show that by exciting only a small, 5-15 {micro}m wide region in chemosensory neurons in live C. elegans, we can significantly improve the duration and quality of single-molecule imaging. Small-window illumination microscopy (SWIM) allows long-duration single-particle imaging since fluorescently labelled proteins are only excited upon entering the small excited area, limiting their photobleaching. Remarkably, we also find that using a small excitation window significantly improves the signal-to-background ratio of individual particles. With the help of theoretical calculations, we explain that the improved signal-to-background ratio is due to reduced background, mostly caused by out-of-focus autofluorescence. We demonstrate the potential of this approach by studying the dendritic transport of a ciliary calcium channel protein, OCR-2, in the chemosensory neurons of C. elegans. We reveal that OCR-2-associated vesicles are continuously transported back and forth along the length of the dendrite and can switch between directed and diffusive states. Furthermore, we perform single-particle tracking of OCR-2-associated vesicles to quantitatively characterize the transport dynamics. SWIM can be readily applied to other in vivo systems where intracellular transport or cytoskeletal dynamics occur in elongated protrusions, such as axons, dendrites, cilia, microvilli and extensions of fibroblasts.

biophysics↗

bioRxiv2022

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 found that chemical stimulation resulted in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents resulted 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.

biophysics↗