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

2021 ◽  
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.

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

2006 ◽  
Vol 174 (7) ◽  
pp. 931-937 ◽  
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.

scholarly journals MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

2017 ◽  
Author(s):  
Peishan Yi ◽  
Chao Xie ◽  
Guangshuo Ou
Keyword(s):  
Cell Cycle ◽  
Sensory Neurons ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Genetic Screen ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Neuronal Cilia ◽  
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.

Intraflagellar transport motors in Caenorhabditis elegans neurons

2004 ◽  
Vol 32 (5) ◽  
pp. 682-684 ◽  
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.

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

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.

scholarly journals Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

1999 ◽  
Vol 10 (2) ◽  
pp. 345-360 ◽  
Author(s):  
Dawn Signor ◽  
Karen P. Wedaman ◽  
Lesilee S. Rose ◽  
Jonathan M. Scholey
Keyword(s):  
Signal Transduction ◽  
Fluorescent Protein ◽  
Partial Purification ◽  
Localization Pattern ◽  
Chemosensory Neurons ◽  
C Elegans ◽  
Microtubule Motors ◽  
Sensory Cilia ◽  
Green Fluorescent ◽  
Kinesin Family

Chemosensation in the nervous system of the nematodeCaenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

A cilia-mediated environmental input induces solitary behaviour in Caenorhabditis elegans and Pristionchus pacificus nematodes

Nematology ◽  
2018 ◽  
Vol 20 (3) ◽  
pp. 201-209 ◽  
Author(s):  
Eduardo Moreno ◽  
Ralf J. Sommer
Keyword(s):  
Caenorhabditis Elegans ◽  
Social Behaviour ◽  
Intraflagellar Transport ◽  
Large Protein ◽  
Pristionchus Pacificus ◽  
C Elegans ◽  
The Social ◽  
Genes Encoding ◽  
Social Behaviours ◽  
Ciliated Neurons

Nematodes respond to a multitude of environmental cues. For example, the social behaviours clumping and bordering were described as a mechanism of hyperoxia avoidance in Caenorhabditis elegans and Pristionchus pacificus. A recent study in P. pacificus revealed a novel regulatory pathway that inhibits social behaviour in a response to an as yet unknown environmental cue. This environmental signal is recognised by ciliated neurons, as mutants defective in intraflagellar transport (IFT) proteins display social behaviours. The IFT machinery represents a large protein complex and many mutants in genes encoding IFT proteins are available in C. elegans. However, social phenotypes in C. elegans IFT mutants have never been reported. Here, we examined 15 previously isolated C. elegans IFT mutants and found that most of them showed strong social behaviour. These findings indicate conservation in the inhibitory mechanism of social behaviour between P. pacificus and C. elegans.

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

2017 ◽  
Vol 27 (10) ◽  
pp. 1448-1461.e7 ◽  
Author(s):  
Peishan Yi ◽  
Wen-Jun Li ◽  
Meng-Qiu Dong ◽  
Guangshuo Ou
Keyword(s):  
Intraflagellar Transport ◽  
C Elegans ◽  
Sensory Cilia

scholarly journals Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons

2004 ◽  
Vol 6 (11) ◽  
pp. 1109-1113 ◽  
Author(s):  
Joshua J. Snow ◽  
Guangshuo Ou ◽  
Amy L. Gunnarson ◽  
M. Regina S. Walker ◽  
H. Mimi Zhou ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
C Elegans ◽  
Sensory Cilia

scholarly journals Quantitative assessment of near-infrared fluorescent proteins

2021 ◽  
Author(s):  
Kiryl Piatkevich ◽  
Hanbin Zhang ◽  
Stavrini Papadaki ◽  
Xiaoting Sun ◽  
Luxia Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Quantitative Assessment ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Oligomeric State ◽  
C Elegans ◽  
The Right ◽  
Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

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