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

Li W, Yi P, Ou G - J. Cell Biol. (2015)

Bottom Line: 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 mutants.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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Affiliation: Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

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IFT of LIS-1, IFT velocities, and the proposed model. (A) Colocalization of GFP::LIS-1 with OSM-6::mCherry (red) in cilia (left), and a kymograph of GFP::LIS-1 motility (right). Micrograph bar, 5 µm; kymograph horizontal bar, 2 µm; vertical bar, 5 s. (B) Velocities of IFT particle and dynein subunits (mean ± SE). (C) A proposed model of subunits in the IFT–dynein complex and multiple turnaround sites on the cilium.
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fig5: IFT of LIS-1, IFT velocities, and the proposed model. (A) Colocalization of GFP::LIS-1 with OSM-6::mCherry (red) in cilia (left), and a kymograph of GFP::LIS-1 motility (right). Micrograph bar, 5 µm; kymograph horizontal bar, 2 µm; vertical bar, 5 s. (B) Velocities of IFT particle and dynein subunits (mean ± SE). (C) A proposed model of subunits in the IFT–dynein complex and multiple turnaround sites on the cilium.

Mentions: Next, we used these conditional mutants to study the contribution of the dynein components to cilium formation. We first showed that 29% (dyci-1), 19% (dlc-1), and 18% (dli-1) of the conditional mutants failed to take up DiI, whereas <4% of dylt-3 or none of dyrb-1 conditional mutants developed the Dyf phenotype (Fig. 3 A). Consistently, we found that the cilium length was significantly reduced from 8.3 µm in WT animals to 4.4 µm (dyci-1), 4.9 µm (dlc-1), and 4.8 µm (dli-1) in the conditional mutants but was not altered in dylt-3-sg or dyrb-1-sg animals (Fig. 3, A and B). IFT cargo includes IFT particles and ciliary receptors such as a transient receptor potential vanilloid channel OSM-9 (Qin et al., 2005). We did not find that OSM-6::GFP or OSM-9::GFP moved in the remaining cilia of the dyci-1-sg and dlc-1-sg conditional mutants (Figs. 3 B and S3 A). These ciliary defects are similar to those detected in che-3– and xbx-1– mutants. To further test whether DYCI-1 and DLC-1 function with IFT–dynein, we determined the cellular localization of GFP-tagged DYCI-1 and DLC-1. We showed that they were distributed along the cilia and underwent the same biphasic IFT as XBX-1 (Fig. 3 C and see Fig. 5 B). Furthermore, we genetically introduced XBX-1::YFP into the dyci-1-sg conditional mutant and GFP::DYCI-1 into the xbx-1(ok279) mutant. We found that XBX-1::YFP and GFP::DYCI-1 formed aggregates around the transitional zone and were not motile in cilia (Fig. 3, D and E). The interdependence of XBX-1 and DYCI-1 in IFT further indicated that they may function together with IFT–dynein.


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

Li W, Yi P, Ou G - J. Cell Biol. (2015)

IFT of LIS-1, IFT velocities, and the proposed model. (A) Colocalization of GFP::LIS-1 with OSM-6::mCherry (red) in cilia (left), and a kymograph of GFP::LIS-1 motility (right). Micrograph bar, 5 µm; kymograph horizontal bar, 2 µm; vertical bar, 5 s. (B) Velocities of IFT particle and dynein subunits (mean ± SE). (C) A proposed model of subunits in the IFT–dynein complex and multiple turnaround sites on the cilium.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362450&req=5

fig5: IFT of LIS-1, IFT velocities, and the proposed model. (A) Colocalization of GFP::LIS-1 with OSM-6::mCherry (red) in cilia (left), and a kymograph of GFP::LIS-1 motility (right). Micrograph bar, 5 µm; kymograph horizontal bar, 2 µm; vertical bar, 5 s. (B) Velocities of IFT particle and dynein subunits (mean ± SE). (C) A proposed model of subunits in the IFT–dynein complex and multiple turnaround sites on the cilium.
Mentions: Next, we used these conditional mutants to study the contribution of the dynein components to cilium formation. We first showed that 29% (dyci-1), 19% (dlc-1), and 18% (dli-1) of the conditional mutants failed to take up DiI, whereas <4% of dylt-3 or none of dyrb-1 conditional mutants developed the Dyf phenotype (Fig. 3 A). Consistently, we found that the cilium length was significantly reduced from 8.3 µm in WT animals to 4.4 µm (dyci-1), 4.9 µm (dlc-1), and 4.8 µm (dli-1) in the conditional mutants but was not altered in dylt-3-sg or dyrb-1-sg animals (Fig. 3, A and B). IFT cargo includes IFT particles and ciliary receptors such as a transient receptor potential vanilloid channel OSM-9 (Qin et al., 2005). We did not find that OSM-6::GFP or OSM-9::GFP moved in the remaining cilia of the dyci-1-sg and dlc-1-sg conditional mutants (Figs. 3 B and S3 A). These ciliary defects are similar to those detected in che-3– and xbx-1– mutants. To further test whether DYCI-1 and DLC-1 function with IFT–dynein, we determined the cellular localization of GFP-tagged DYCI-1 and DLC-1. We showed that they were distributed along the cilia and underwent the same biphasic IFT as XBX-1 (Fig. 3 C and see Fig. 5 B). Furthermore, we genetically introduced XBX-1::YFP into the dyci-1-sg conditional mutant and GFP::DYCI-1 into the xbx-1(ok279) mutant. We found that XBX-1::YFP and GFP::DYCI-1 formed aggregates around the transitional zone and were not motile in cilia (Fig. 3, D and E). The interdependence of XBX-1 and DYCI-1 in IFT further indicated that they may function together with IFT–dynein.

Bottom Line: 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 mutants.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Show MeSH
Related in: MedlinePlus