Signaling compartment at the ciliary tip is formed and maintained by intraflagellar transport and functions as sensitive salt detector

AbstractPrimary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. How signaling subcompartments are established within the microtubule-based organelle, and for example support Hedgehog or cGMP signal transduction pathways, remains a central question. Here we show that a C. elegans salt-sensing receptor type guanylate cyclase, GCY-22, accumulates at a high concentration within the distal region of the cilium. This receptor uses DAF-25 (Ankmy2 in mammals) to cross the transition zone (TZ) membrane diffusion barrier in the proximal-most region of the ciliary axoneme. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the cargo-mobilizing intraflagellar transport (IFT) system. Disruption of transit across the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation, maintenance, and function of the ciliary tip compartment required for chemotaxis to low NaCl concentrations. Together, our findings reveal how a previously undescribed cilium tip cGMP signaling compartment is established and contributes to the physiological function of a primary cilium.

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TRPV channel OCR-2 is distributed along C. elegans chemosensory cilia by diffusion in a local interplay with intraflagellar transport

10.1101/2020.11.19.390005 ◽

2020 ◽

Author(s):

Jaap van Krugten ◽

Noémie Danné ◽

Erwin J.G. Peterman

Keyword(s):

Single Molecule ◽

Transmembrane Proteins ◽

Intraflagellar Transport ◽

Single Molecule Tracking ◽

C Elegans ◽

Normal Diffusion ◽

Cellular Signal ◽

Underlying Mechanisms ◽

And Function

AbstractSensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing.

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Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

10.1101/855940 ◽

2019 ◽

Cited By ~ 1

Author(s):

Punam Sonar ◽

Wiphu Youyen ◽

Augustine Cleetus ◽

Pattipong Wisanpitayakorn ◽

Iman S. Mousavi ◽

...

Keyword(s):

Single Molecule ◽

Motor Coordination ◽

Intraflagellar Transport ◽

Kinetic Properties ◽

C Elegans ◽

Slow Velocity ◽

Single Molecule Studies ◽

Fast Transport ◽

And Function

SummaryThe construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors ‘line up’ in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other’s way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

10.1101/251694 ◽

2018 ◽

Cited By ~ 1

Author(s):

Laura Vuolo ◽

Nicola L. Stevenson ◽

Kate J. Heesom ◽

David J. Stephens

Keyword(s):

Transition Zone ◽

Cell Lines ◽

Primary Cilia ◽

Point Mutations ◽

Intraflagellar Transport ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

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Interpreting the pathogenicity of Joubert Syndrome missense variants in Caenorhabditis elegans

Disease Models & Mechanisms ◽

10.1242/dmm.046631 ◽

2020 ◽

pp. dmm.046631

Author(s):

Karen I. Lange ◽

Sofia Tsiropoulou ◽

Katarzyna Kucharska ◽

Oliver E. Blacque

Keyword(s):

Caenorhabditis Elegans ◽

Primary Cilia ◽

Model Organism ◽

Joubert Syndrome ◽

Compound Heterozygous ◽

Inherited Disorders ◽

Missense Variants ◽

C Elegans ◽

Variant Alleles ◽

And Function

Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have employed a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S, G155S) in B9D2/mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment, and regulates the cilium's molecular composition and sensory/signaling functions. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and TMEM216/MKS-2. Together, these data establish C. elegans as a paradigm for interpreting JBTS mutations, and provide further insight into MKS module organisation.

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STAM and Hrs Down-Regulate Ciliary TRP Receptors

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0239 ◽

2007 ◽

Vol 18 (9) ◽

pp. 3277-3289 ◽

Cited By ~ 43

Author(s):

Jinghua Hu ◽

Samuel G. Wittekind ◽

Maureen M. Barr

Keyword(s):

Growth Factor ◽

Mammalian Cells ◽

Primary Cilia ◽

Membrane Receptors ◽

Sensory Transduction ◽

Kinase Substrate ◽

C Elegans ◽

Early Endosomes ◽

Transient Receptor ◽

And Function

Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

eLife ◽

10.7554/elife.39655 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Laura Vuolo ◽

Nicola L Stevenson ◽

Kate J Heesom ◽

David J Stephens

Keyword(s):

Transition Zone ◽

Primary Cilia ◽

Complex Function ◽

Intraflagellar Transport ◽

Loss Of Function ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

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Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

10.1101/409318 ◽

2018 ◽

Cited By ~ 1

Author(s):

Martin F. Engelke ◽

Bridget Waas ◽

Sarah E. Kearns ◽

Ayana Suber ◽

Allison Boss ◽

...

Keyword(s):

Mammalian Cells ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Genetic Approach ◽

Ciliated Cells ◽

Loss Of Function ◽

Double Knockout ◽

Chemical Genetic ◽

Species Specific ◽

And Function

ABSTRACTThe trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemical-genetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition. These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function.

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Transient accumulation and bidirectional movement of KIF13B in primary cilia

10.1101/2021.07.09.451768 ◽

2021 ◽

Author(s):

Alice Dupont Juhl ◽

Zeinab Anvarian ◽

Julia Berges ◽

Daniel Wustner ◽

Lotte B Pedersen

Keyword(s):

Mammalian Cells ◽

Live Cell Imaging ◽

Primary Cilia ◽

Cell Imaging ◽

Intraflagellar Transport ◽

Cytoplasmic Dynein ◽

Ciliary Membrane ◽

Bidirectional Movement ◽

And Function ◽

Male Specific

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

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ERICH3 in Primary Cilia Regulates Cilium Formation and the Localisations of Ciliary Transport and Sonic Hedgehog Signaling Proteins

Scientific Reports ◽

10.1038/s41598-019-52830-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Mona Alsolami ◽

Stefanie Kuhns ◽

Manal Alsulami ◽

Oliver E. Blacque

Keyword(s):

Sonic Hedgehog ◽

Primary Cilium ◽

Molecular Motors ◽

Hedgehog Signaling ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Retrograde Transport ◽

Signaling Proteins ◽

Sonic Hedgehog Signaling ◽

And Function

Abstract Intraflagellar transport (IFT) is essential for the formation and function of the microtubule-based primary cilium, which acts as a sensory and signalling device at the cell surface. Consisting of IFT-A/B and BBSome cargo adaptors that associate with molecular motors, IFT transports protein into (anterograde IFT) and out of (retrograde IFT) the cilium. In this study, we identify the mostly uncharacterised ERICH3 protein as a component of the mammalian primary cilium. Loss of ERICH3 causes abnormally short cilia and results in the accumulation of IFT-A/B proteins at the ciliary tip, together with reduced ciliary levels of retrograde transport regulators, ARL13B, INPP5E and BBS5. We also show that ERICH3 ciliary localisations require ARL13B and BBSome components. Finally, ERICH3 loss causes positive (Smoothened) and negative (GPR161) regulators of sonic hedgehog signaling (Shh) to accumulate at abnormally high levels in the cilia of pathway-stimulated cells. Together, these findings identify ERICH3 as a novel component of the primary cilium that regulates cilium length and the ciliary levels of Shh signaling molecules. We propose that ERICH3 functions within retrograde IFT-associated pathways to remove signaling proteins from cilia.

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Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors

The Journal of Cell Biology ◽

10.1083/jcb.200606003 ◽

2006 ◽

Vol 174 (7) ◽

pp. 1035-1045 ◽

Cited By ~ 141

Author(s):

Xiaoyu Pan ◽

Guangshuo Ou ◽

Gul Civelekoglu-Scholey ◽

Oliver E. Blacque ◽

Nicholas F. Endres ◽

...

Keyword(s):

Motor Coordination ◽

Intraflagellar Transport ◽

Quantitative Modeling ◽

Concerted Action ◽

Action Mechanism ◽

Bardet Biedl Syndrome ◽

C Elegans ◽

And Function

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3–kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein–dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

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