Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans

Loseva, Elizaveta; van Krugten, Jaap; Mitra, Aniruddha; Peterman, Erwin J. G.

doi:10.1007/978-1-0716-3377-9_7

Cited by 3 publications

(4 citation statements)

References 29 publications

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“…Images were acquired using a custom-built laser-illuminated widefield fluorescence microscope, as described previously 62 . Briefly, optical imaging was performed using an inverted microscope body (Nikon Ti E) with a 100x oil immersion objective (Nikon, CFI Apo TIRF 100x, N.A.…”

Section: Methodsmentioning

confidence: 99%

“…For imaging live C. elegans , young adult hermaphrodite worms, having PHA/PHB dendrites ∼50 μm long, were sedated in 5 mM levamisole in M9, sandwiched between an agarose pad (2% agarose in M9) and a coverslip and mounted on a microscope 62 . To perform SWIM, a small excitation window (width ranging between 7-15 μm) was used to illuminate fluorescent molecules (IFT proteins labelled with eGFP, mCherry or wrmScarlet) in a small region of PHA/PHB neurons, to image protein dynamics in either the cilia or the dendrites (see Supplementary Videos).…”

Section: Methodsmentioning

confidence: 99%

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Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

Mitra,

Gioukakis,

Mul

et al. 2024

Preprint

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Anterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in C. elegans chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results indicate that the assembly of IFT trains is a step-wise process involving the subsequent incorporation of first IFT-B, then IFT-A and finally BBSomes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

Mitra,

Gioukakis,

Mul

et al. 2024

Preprint

Self Cite

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show abstract

“…Images were acquired using a custom-built laser-illuminated widefield fluorescence microscope, as described previously 38 . Briefly, optical imaging was performed using an inverted microscope body (Nikon Ti E) with a 100x oil immersion objective (Nikon, CFI Apo TIRF 100x, N.A.…”

Section: Methodsmentioning

confidence: 99%

“…For imaging live C. elegans , young adult hermaphrodite worms were sedated in 5 mM levamisole in M9, sandwiched between an agarose pad (2% agarose in M9) and a coverslip, and mounted on a microscope 38 .…”

Section: Methodsmentioning

confidence: 99%

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans

Mitra,

Loseva,

Peterman

2024

Nat Commun

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Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

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DYF-5 regulates intraflagellar transport by affecting train turnaround

Mul,

Mitra,

Prevo

et al. 2024

Preprint

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Intraflagellar transport (IFT) coordinates the transport of cargo in cilia and is essential for ciliary function. CILK1 has been identified as a key regulator of IFT. The mechanism by which it acts has, however, remained unclear. In this study, we use fluorescence imaging and single-molecule tracking in the phasmid cilia of liveC. elegansto study the effect of the CILK1 homolog DYF-5 on the dynamics of the IFT. We show that in the absence of DYF-5, IFT components accumulate at the ciliary tip. kinesin-II is no longer restricted to the proximal segment of the cilium but is present all throughout the cilium, while its velocity is different from that of OSM-3. The frequency of IFT trains is reduced and in particular retrograde trains were rarely observed. In the absence of DYF-5, retrograde transport is vastly reduced, resulting in the accumulation of IFT components at the tip and depletion at the base. The latter results in impeded anterograde train assembly, resulting in fewer trains with irregular composition. Our results show that DYF-5 plays a key role in regulating the turnarounds of IFT trains at the ciliary tip.

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Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans

Cited by 3 publications

References 29 publications

Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans

DYF-5 regulates intraflagellar transport by affecting train turnaround

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