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

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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Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

The Journal of Cell Biology ◽

10.1083/jcb.201411041 ◽

2015 ◽

Vol 208 (6) ◽

pp. 683-692 ◽

Cited By ~ 41

Author(s):

Wenjing Li ◽

Peishan Yi ◽

Guangshuo Ou

Keyword(s):

Caenorhabditis Elegans ◽

Intraflagellar Transport ◽

Cytoplasmic Dynein ◽

Regulatory Mechanisms ◽

Light Chains ◽

Induced Mutations ◽

Functional Studies ◽

Cilium Formation ◽

Dynein Intermediate Chain ◽

Intermediate Chain

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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XBX-1 Encodes a Dynein Light Intermediate Chain Required for Retrograde Intraflagellar Transport and Cilia Assembly in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0677 ◽

2003 ◽

Vol 14 (5) ◽

pp. 2057-2070 ◽

Cited By ~ 84

Author(s):

Jenny C. Schafer ◽

Courtney J. Haycraft ◽

James H. Thomas ◽

Bradley K. Yoder ◽

Peter Swoboda

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Sensory Neurons ◽

Intraflagellar Transport ◽

Anterograde Transport ◽

Behavioral Abnormalities ◽

Genes Encoding ◽

Intermediate Chain

Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.

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Faculty Opinions recommendation of The molecular identities of the Caenorhabditis elegans intraflagellar transport genes dyf-6, daf-10 and osm-1.

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

10.3410/f.1046282.496218 ◽

2006 ◽

Author(s):

Shai Shaham

Keyword(s):

Caenorhabditis Elegans ◽

Intraflagellar Transport

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Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

10.1101/2021.09.14.460298 ◽

2021 ◽

Author(s):

Jeffrey C Medley ◽

Shilpa Hebbar ◽

Joel T Sydzyik ◽

Anna Y. Zinovyeva

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Paternal Genome ◽

Single Nucleotide ◽

C Elegans ◽

Homology Directed Repair ◽

Maternal Genome ◽

Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure

The Journal of Cell Biology ◽

10.1083/jcb.200707090 ◽

2008 ◽

Vol 180 (5) ◽

pp. 973-988 ◽

Cited By ~ 80

Author(s):

Andrew R. Jauregui ◽

Ken C.Q. Nguyen ◽

David H. Hall ◽

Maureen M. Barr

Keyword(s):

Caenorhabditis Elegans ◽

Intraflagellar Transport ◽

Basal Bodies ◽

Structural Components ◽

Transition Zones ◽

C Elegans ◽

Cell Type Specific ◽

Children And Young Adults ◽

Stage Renal Disease ◽

End Stage

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type–specific phenotypes.

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Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Nucleic Acids Research ◽

10.1093/nar/gkt805 ◽

2013 ◽

Vol 41 (20) ◽

pp. e193-e193 ◽

Cited By ~ 93

Author(s):

Changchun Chen ◽

Lorenz A. Fenk ◽

Mario de Bono

Keyword(s):

Caenorhabditis Elegans ◽

Homologous Recombination ◽

Genome Editing

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Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.118.301532 ◽

2018 ◽

Vol 210 (3) ◽

pp. 781-787 ◽

Cited By ~ 79

Author(s):

Gregoriy A. Dokshin ◽

Krishna S. Ghanta ◽

Katherine M. Piscopo ◽

Craig C. Mello

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Single Stranded Dna

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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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