Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Bennett, Henrietta W.; Gustavsson, Anna-Karin; Bayas, Camille; Petrov, Petar N.; Mooney, Nancie A.; Moerner, W. E.; Jackson, Peter K.

doi:10.1091/mbc.e19-09-0499

Cited by 37 publications

(41 citation statements)

References 64 publications

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“…The axonemal localization of ENKD1 depends on its ability to interact with microtubules, further supporting its functions as a ciliary MAP. Notably, the length of the ENKD1-positive axoneme relative to the cilium length substantially varied from cilium to cilium, which was previously reported for components of the Inversin compartment of primary cilium (Bennett et al, 2020). Because ENKD1 did not localize with inversin, this result suggests that ENKD1 association with the ciliary axoneme might require specific tubulin modifications or ciliary binding partners.…”

Section: Discussionsupporting

confidence: 59%

“…1F, 1G, 1H). To further define the ciliary sub-compartmentalization of ENKD1, we determined its localization relative to the proximal axoneme modified by polyglutamylation as well as the inversin compartment, which was shown to vary in length from cilium to cilium (Bennett et al, 2020;Sang et al, 2011). mNG-ENKD1 did not co-localize with polyglutamylated tubulin and mScarlet-INVS, suggesting that ciliary proteins or modifications other than polyglutamylation and inversin might regulate its ciliary incorporation (Fig.…”

Section: Enkd1 Localizes To the Centrosomes And Primary Ciliamentioning

confidence: 99%

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ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

Tiryaki

Deretic

Fırat-Karalar

2021

Preprint

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Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.

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Section: Discussionsupporting

confidence: 59%

Section: Enkd1 Localizes To the Centrosomes And Primary Ciliamentioning

confidence: 99%

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

Tiryaki

Deretic

Fırat-Karalar

2021

Preprint

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“…We previously showed that Arl13b and Inpp5e localized to collecting duct cilia and localization was reduced in both Hoxb7-Cre; Tulp3 f/f and Ksp-Cre; Tulp3 f/f collecting duct cilia by P5 (Hwang et al, 2019). Nphp3 localizes to the proximal inversin zone in cilia (Bennett et al, 2020) but localization in kidney collecting ducts is unknown. Lkb1 has been reported to localize to kidney collecting duct cilia (Viau et al, 2018).…”

Section: Tulp3 Is Required For Ciliary Localization Of Lipidated Proteins In Murine Kidneysmentioning

confidence: 99%

“…The effectiveness for complete depletion of Inpp5e in cilia in Tulp3 ko might be related to direct binding between Arl13b and Inpp5e and requirement of such binding for effective ciliary retention of Inpp5e (Humbert et al, 2012;Qiu et al, 2021). Nphp3 is concentrated in the proximal ciliary inversin compartment by binding to Nek8 and Anks6 that are required downstream of Inversin for Nphp3 localization (Bennett et al, 2020). Such binding might promote some retention of Nphp3 even in the absence of Tulp3.…”

Section: Tulp3 Enriches Arl13b-dependent Lipidated Proteins In Ciliamentioning

confidence: 99%

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

Palicharla

Hwang

Somatilaka

et al. 2021

Preprint

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The tubby family protein-TULP3 coordinates with the intraflagellar transport complex-A (IFT-A) in trafficking certain transmembrane proteins to cilia. These transmembrane cargoes have short motifs that are necessary and sufficient for TULP3-mediated trafficking. However, whether TULP3 regulates trafficking of membrane-associated proteins is not well understood. Here we show that TULP3 is required for transport of the atypical GTPase ARL13B into cilia, and for ciliary enrichment of ARL13B-dependent farnesylated and myristoylated proteins. ARL13B transport requires TULP3 binding to IFT-A core but not to phosphoinositides, unlike transmembrane cargo transport that requires binding to both by TULP3. A conserved lysine in TULP3's tubby domain mediates direct ARL13B binding and trafficking of lipidated and transmembrane cargoes. An N-terminal amphipathic helix in ARL13B flanking the palmitoylation site mediates binding to TULP3 and directs trafficking to cilia even in absence of palmitoylation and RVxP sorting motif. Therefore, TULP3 transports transmembrane proteins and ARL13B into cilia by capture of short sequences through a shared tubby domain site.

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“…Two facts support the latter model; first, most of the spontaneous Ca 2+ activity and GABA-induced activity originated in the distal parts of the cilium (see Fig. 6), and second the inversin compartment has been hypothesized to work by sequestering ciliary proteins and releasing them under appropriate stimulation 32 . To test this, we analyzed the GABA-B1 receptor distribution after a short (10 min) stimulation with GABA or the GABA-B receptor agonist baclofen.…”

Section: Resultsmentioning

confidence: 82%

The β-cell primary cilium is an autonomous Ca²⁺ compartment for paracrine GABA signalling

Sanchez

Incedal

Salcedo

et al. 2021

Preprint

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The primary cilium is an organelle present in most adult mammalian cells and is thought of as an antenna for detection of a variety of signals. Here we use intact mouse pancreatic islets of Langerhans to investigate signalling properties of the primary cilium in β-cells. Using cilia-targeted Ca2+ indicators we find that the resting Ca2+ concentration in the cilium is lower than that of the cytosol, and we uncover a Ca2+ extrusion mechanism in the cilium that effectively insulates the cilium from changes in cytosolic Ca2+. Stimuli that give rise to pronounced cytosolic Ca2+ concentration increases, such as glucose- and depolarization-induced Ca2+ influx, and mobilization of Ca2+ from the ER, was accompanied by minor increases in cilia Ca2+ concentrations that were spatially restricted to a small compartment at the base. Conversely, we observe pronounced Ca2+ concentration changes in the primary cilia of islet β-cells that do not propagate into the cytosol and show that paracrine GABA signalling via cilia-localized GABA- B1-receptors is responsible for this Ca2+ signalling. Finally, we demonstrate that the cilia response to GABA involves ligand-dependent transport of GABA-B1 receptors into the cilium.

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Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Cited by 37 publications

References 64 publications

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

The β-cell primary cilium is an autonomous Ca²⁺ compartment for paracrine GABA signalling

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Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Cited by 37 publications

References 64 publications

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signalling

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The β-cell primary cilium is an autonomous Ca²⁺ compartment for paracrine GABA signalling