Dynein-Driven Retrograde Intraflagellar Transport Is Triphasic in C. elegans Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

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The WD Repeat-containing Protein IFTA-1 Is Required for Retrograde Intraflagellar Transport

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0571 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5053-5062 ◽

Cited By ~ 65

Author(s):

Oliver E. Blacque ◽

Chunmei Li ◽

Peter N. Inglis ◽

Muneer A. Esmail ◽

Guangshuo Ou ◽

...

Keyword(s):

Protein Complexes ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Bardet Biedl Syndrome ◽

C Elegans ◽

Gene Mutant ◽

Strong Homology ◽

Sensory Cilia ◽

Wd Repeat ◽

Mammalian Protein

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet–Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.

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Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3

The Journal of Cell Biology ◽

10.1083/jcb.200605179 ◽

2006 ◽

Vol 174 (7) ◽

pp. 931-937 ◽

Cited By ~ 76

Author(s):

Miki Imanishi ◽

Nicholas F. Endres ◽

Arne Gennerich ◽

Ronald D. Vale

Keyword(s):

Atpase Activity ◽

Single Molecule ◽

Intramolecular Interaction ◽

Optical Trap ◽

Coiled Coil ◽

Intraflagellar Transport ◽

Wild Type ◽

C Elegans ◽

Sensory Cilia

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3–null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

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Faculty Opinions recommendation of Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022137.251725 ◽

2004 ◽

Author(s):

Vladimir I Gelfand

Keyword(s):

Intraflagellar Transport ◽

C Elegans ◽

Sensory Cilia

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Intraflagellar transport motors in Caenorhabditis elegans neurons

Biochemical Society Transactions ◽

10.1042/bst0320682 ◽

2004 ◽

Vol 32 (5) ◽

pp. 682-684 ◽

Cited By ~ 13

Author(s):

J.M. Scholey ◽

G. Ou ◽

J. Snow ◽

A. Gunnarson

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Protein Complexes ◽

Intraflagellar Transport ◽

Time Lapse ◽

Protein Fusion ◽

C Elegans ◽

Sensory Cilia ◽

Macromolecular Protein ◽

Green Fluorescent

IFT (intraflagellar transport) assembles and maintains sensory cilia on the dendritic endings of chemosensory neurons within the nematode Caenorhabditis elegans. During IFT, macromolecular protein complexes called IFT particles (which carry ciliary precursors) are moved from the base of the sensory cilium to its distal tip by anterograde IFT motors (kinesin-II and Osm-3 kinesin) and back to the base by retrograde IFT-dynein [Rosenbaum and Witman (2002) Nat. Rev. Mol. Cell Biol. 3, 813–825; Scholey (2003) Annu. Rev. Cell Dev. Biol. 19, 423–443; and Snell, Pan and Wang (2004) Cell 117, 693–697]. In the present study, we describe the protein machinery of IFT in C. elegans, which we have analysed using time-lapse fluorescence microscopy of green fluorescent protein-fusion proteins in concert with ciliary mutants.

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Ectosome uptake by glia sculpts Caenorhabditis elegans sensory cilia

10.1101/2021.02.14.430969 ◽

2021 ◽

Author(s):

Adrià Razzauti ◽

Patrick Laurent

Keyword(s):

Caenorhabditis Elegans ◽

Cell Surface ◽

Glial Cells ◽

Intraflagellar Transport ◽

Membrane Receptors ◽

Sensory Function ◽

C Elegans ◽

Sensory Cilia

AbstractCilia are sensory organelles protruding from cell surface. A tight regulation of membrane receptors traffic in and out of cilia is achieved by the action of Intraflagellar Transport (IFT). Here, we show that ectosomes bud from a subset of C. elegans sensory cilia. Packing and disposal of ciliary receptors in ectosomes complement their retrieval by IFT. Mutations in ciliary retrieval genes increase export of the salt sensor GCY-22 from ASER neurons by ectosomes, preventing its accumulation in ASER cilia. Ectosomes are produced from two ciliary locations: cilia tip and/or cilia base. Ectosomes budding from cilia tip are released in the environment. Ectosomes produced from the cilia base are concomitantly phagocytosed by the associated glial cells. Although ectocytosis does not require glia to occur, ectosome phagocytosis by the contacting glia contributes to maintain cilia shape and sensory function. We suggest this coordinated neuron-glia interaction is required for proper cilia function.

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Analysis of Intraflagellar Transport in C. elegans Sensory Cilia

Methods in Cell Biology ◽

10.1016/s0091-679x(08)93013-2 ◽

2009 ◽

pp. 235-266 ◽

Cited By ~ 10

Author(s):

Limin Hao ◽

Seyda Acar ◽

James Evans ◽

Guangshuo Ou ◽

Jonathan M. Scholey

Keyword(s):

Intraflagellar Transport ◽

C Elegans ◽

Sensory Cilia

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Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

10.1101/2021.12.19.473369 ◽

2021 ◽

Author(s):

Michael Clupper ◽

Rachael Gill ◽

Malek Elsayyid ◽

Denis Touroutine ◽

Jeffrey L. Caplan ◽

...

Keyword(s):

Super Resolution ◽

Intraflagellar Transport ◽

Extracellular Vesicle ◽

Specific Protein ◽

Secondary Site ◽

Selective Enrichment ◽

C Elegans ◽

Sensory Cilia ◽

Membrane Compartment ◽

Super Resolution Microscopy

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

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Sensory cilia act as a specialized venue for regulated EV biogenesis and signaling

10.1101/2021.02.04.429799 ◽

2021 ◽

Cited By ~ 1

Author(s):

Juan Wang ◽

Inna A. Nikonorova ◽

Malan Silva ◽

Jonathon D. Walsh ◽

Peter Tilton ◽

...

Keyword(s):

Fluorescent Protein ◽

Coiled Coil ◽

Super Resolution ◽

Intraflagellar Transport ◽

Sensory Stimulation ◽

Intercellular Signaling ◽

C Elegans ◽

Sensory Cilia ◽

Ciliated Neurons

AbstractExtracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study signaling EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

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