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

Abstract

Femtosecond-laser ablation is used in combination with fluorescence microscopy to study how intraflagellar transport in Caenorhabditis elegans is affected by cutting off supply via the dendrite. It is found that IFT reacts in a remarkable, multistep response, allowing rapid adaptation to the environment.

Mechanisms of Regulation in Intraflagellar Transport

“…In C. elegans chemosensory cilia, fluorescence recovery after photobleaching experiments and modelling have shown that new tubulin, transported by IFT is incorporated in the axoneme at the ciliary tip and at the end of the proximal segment, where the B-tubules end [ 48 ]. A study employing dendritic laser ablation to block the supply of new material into cilia showed that IFT components redistribute after ablation to the base, followed by shortening of the axoneme [ 149 ]. More recently, similar behavior was observed in response to the chemical repellents the chemosensory cilia sense [ 32 ].…”

TRPV channel OCR-2 is distributed alongC. eleganschemosensory cilia by diffusion in a local interplay with intraflagellar transport

“…Rapid redistribution of IFT dynein and tubulin away from the DS is reminiscent of the laser ablation and azide dosing experiments we have performed before (Mijalkovic et al, 2020). Here it was shown that ablation of the phasmid chemosensory neurons at the dendrite (a couple of micrometers before the start of the cilium), resulted in the activation of retrograde IFT, leading to a redistribution of IFT components out of the DS towards the base on a time scale similar to the SDS-addition experiments presented here.…”

“…Remarkably, the shortening appears to stop at the end of the PS, where the axoneme consists of microtubule doublets. In a previous study, femtosecondlaser ablation of the phasmid-neuron dendrites was shown to also cause the collapse of the DS, but not the PS, suggesting that the PS, consisting of microtubule doublets, is more stable than the DS, which consists of microtubule singlets (Mijalkovic et al, 2020). In contrast to IFT dynein, part of the tubulin appears to leave the cilium, as is evident from the increased TBB-4 fluorescence in the dendrite (Figure 1C).…”

“…Whereas the SDS-evoked avoidance response occurs almost instantaneously after SDS addition, the redistribution of ciliary components starts much later, about a minute after addition, suggesting that the redistribution is not an intrinsic part of the avoidance response. Laser-ablation experiments of the C. elegans phasmid dendrite and live imaging of IFT and calcium in Chlamydomonas have shown that the distribution of IFT components and IFT dynamics can be altered almost instantaneously (Collingridge et al, 2013; Mijalkovic et al, 2020). The cause of the delay in our SDS-addition experiments is hence not the inability of the cilia or IFT to react rapidly.…”

Differentiated dynamic response in C. elegans chemosensory cilia

“…The kymograph indicates that the proximal segments remain intact and no further shortening occurs, presumably because the doublet microtubules in the proximal segment are more stable than the singlet microtubules in the distal segment. 33 The transmembrane protein OCR-2 partially redistributes from the tip into the distal segment (Figure 4D,E). A fraction of OCR-2 remains at the tip, suggesting that the ciliary membrane is not remodeled, e.g.…”