Intraflagellar Transport Complex A Genes Differentially Regulate Cilium Formation and Transition Zone Gating

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

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Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans

The Journal of Cell Biology ◽

10.1083/jcb.201610070 ◽

2017 ◽

Vol 216 (6) ◽

pp. 1659-1671 ◽

Cited By ~ 26

Author(s):

Daniel Serwas ◽

Tiffany Y. Su ◽

Max Roessler ◽

Shaohe Wang ◽

Alexander Dammermann

Keyword(s):

Fluorescence Microscopy ◽

Transition Zone ◽

Electron Tomography ◽

Intraflagellar Transport ◽

Basal Bodies ◽

C Elegans ◽

Cilia Formation ◽

Syndrome Protein

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

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Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly

The EMBO Journal ◽

10.15252/embj.2018101251 ◽

2019 ◽

Vol 38 (9) ◽

Cited By ~ 7

Author(s):

Stefanie Wachter ◽

Jamin Jung ◽

Shahaan Shafiq ◽

Jerome Basquin ◽

Cécile Fort ◽

...

Keyword(s):

Intraflagellar Transport ◽

Transport Complex

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Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

Experimental Cell Research ◽

10.1016/j.yexcr.2013.06.010 ◽

2013 ◽

Vol 319 (14) ◽

pp. 2275-2281 ◽

Cited By ~ 7

Author(s):

Paul W. Howard ◽

Shall F. Jue ◽

Richard A. Maurer

Keyword(s):

Intraflagellar Transport ◽

Transport Complex

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ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

10.1101/2021.11.19.469163 ◽

2021 ◽

Author(s):

Yan-Xia Liu ◽

Wei-Yue Sun ◽

Bin Xue ◽

Rui-Kai Zhang ◽

Wen-Juan Li ◽

...

Keyword(s):

Transition Zone ◽

Phospholipase D ◽

Physiological Condition ◽

Intraflagellar Transport ◽

Outward Diffusion ◽

Ciliary Membrane ◽

Downstream Signaling ◽

Extracellular Stimuli ◽

Signaling Components ◽

Ciliary Signaling

Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase in Chlamydomonas. Under physiological condition, ARL3GDP binds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTP dissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis of Chlamydomonas cells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.

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WDR60-mediated dynein-2 loading into cilia powers retrograde IFT and transition zone crossing

10.1101/2021.09.19.459328 ◽

2021 ◽

Author(s):

Ana R. G. De-Castro ◽

Diogo R. M. Rodrigues ◽

Maria J. G. De-Castro ◽

Neide Vieira ◽

Carmen Vieira ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Transition Zone ◽

Intraflagellar Transport ◽

Live Imaging ◽

Pivotal Role ◽

Major Contributor ◽

Motor Complex ◽

Distal Side ◽

Intermediate Chain

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homolog (WDR-60) and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing the availability of the retrograde motor at the ciliary tip. Consistently, we show that less dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT and the NPHP module as a roadblock to dynein-2 passage through the transition zone.

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

10.1101/2021.05.25.445488 ◽

2021 ◽

Author(s):

Vivek Reddy Palicharla ◽

Sun-hee Hwang ◽

Bandarigoda N. Somatilaka ◽

Hemant B. Badgandi ◽

Emilie Legue ◽

...

Keyword(s):

Protein Transport ◽

Transmembrane Proteins ◽

Intraflagellar Transport ◽

Amphipathic Helix ◽

Cargo Transport ◽

Family Protein ◽

Associated Proteins ◽

Sorting Motif ◽

Necessary And Sufficient ◽

Transport Complex

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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