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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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Transport behaviors of dynein subunits in cilia. (A) IFT frequencies of IFT particle and dynein subunits. n = 64–212; mean ± SE (error bars). ***, P ≤ 0.001. (B) Kymographs (left) of DYCI-1, DLC-1, DYLT-3, and XBX-1 in the middle (M) or distal segments (D). M″ and D″ indicate turnaround events. M and D (right) are 4× enlarged images of turnarounds in the green boxes on the left. Horizontal bar, 2 µm; vertical bar, 5 s. (C) Distribution of turnaround events along the cilia. The y axis indicates the percentage of turnaround events at the specific region of cilia among the total turnaround events. n = 58–140. The broken line shows the junction between the middle and distal segments.
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fig4: Transport behaviors of dynein subunits in cilia. (A) IFT frequencies of IFT particle and dynein subunits. n = 64–212; mean ± SE (error bars). ***, P ≤ 0.001. (B) Kymographs (left) of DYCI-1, DLC-1, DYLT-3, and XBX-1 in the middle (M) or distal segments (D). M″ and D″ indicate turnaround events. M and D (right) are 4× enlarged images of turnarounds in the green boxes on the left. Horizontal bar, 2 µm; vertical bar, 5 s. (C) Distribution of turnaround events along the cilia. The y axis indicates the percentage of turnaround events at the specific region of cilia among the total turnaround events. n = 58–140. The broken line shows the junction between the middle and distal segments.

Mentions: To understand the transport behavior of IFT–dynein subunits, we generated kymographs from time-lapse movies of IFT of GFP-tagged dynein subunits (Videos 1–4). We first quantified the transport frequency as the number of transport events per minute. OSM-6::GFP and XBX-1::YFP both showed a frequency of 16 events/min; however, DYCI-1, DLC-1, and DYLT-3 displayed frequencies of ∼3, 9, and 6 events/min, respectively (Fig. 4 A). We next examined their turnaround sites. The majority of OSM-6 and XBX-1 turned around at the distal tip, whereas DYCI-1, DLC-1, and DYLT-3 returned at the middle and distal segments (Fig. 4, B and C). 76% of the GFP::DYCI-1 puncta returned along the distal segment and very few reached the distal tip, and 24% of DYCI-1 turned around at the tip of the middle segment. 60% of the DLC-1::GFP puncta turned back at the distal segment, whereas 40% of DLC-1 returned at the middle tip. Finally, 69% of the DYLT-3::GFP puncta returned in the middle segment, not even reaching the middle tip (Fig. 4, B and C). These data suggest that multiple retrograde IFT pathways may operate within a single cilium.


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)

Transport behaviors of dynein subunits in cilia. (A) IFT frequencies of IFT particle and dynein subunits. n = 64–212; mean ± SE (error bars). ***, P ≤ 0.001. (B) Kymographs (left) of DYCI-1, DLC-1, DYLT-3, and XBX-1 in the middle (M) or distal segments (D). M″ and D″ indicate turnaround events. M and D (right) are 4× enlarged images of turnarounds in the green boxes on the left. Horizontal bar, 2 µm; vertical bar, 5 s. (C) Distribution of turnaround events along the cilia. The y axis indicates the percentage of turnaround events at the specific region of cilia among the total turnaround events. n = 58–140. The broken line shows the junction between the middle and distal segments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Transport behaviors of dynein subunits in cilia. (A) IFT frequencies of IFT particle and dynein subunits. n = 64–212; mean ± SE (error bars). ***, P ≤ 0.001. (B) Kymographs (left) of DYCI-1, DLC-1, DYLT-3, and XBX-1 in the middle (M) or distal segments (D). M″ and D″ indicate turnaround events. M and D (right) are 4× enlarged images of turnarounds in the green boxes on the left. Horizontal bar, 2 µm; vertical bar, 5 s. (C) Distribution of turnaround events along the cilia. The y axis indicates the percentage of turnaround events at the specific region of cilia among the total turnaround events. n = 58–140. The broken line shows the junction between the middle and distal segments.
Mentions: To understand the transport behavior of IFT–dynein subunits, we generated kymographs from time-lapse movies of IFT of GFP-tagged dynein subunits (Videos 1–4). We first quantified the transport frequency as the number of transport events per minute. OSM-6::GFP and XBX-1::YFP both showed a frequency of 16 events/min; however, DYCI-1, DLC-1, and DYLT-3 displayed frequencies of ∼3, 9, and 6 events/min, respectively (Fig. 4 A). We next examined their turnaround sites. The majority of OSM-6 and XBX-1 turned around at the distal tip, whereas DYCI-1, DLC-1, and DYLT-3 returned at the middle and distal segments (Fig. 4, B and C). 76% of the GFP::DYCI-1 puncta returned along the distal segment and very few reached the distal tip, and 24% of DYCI-1 turned around at the tip of the middle segment. 60% of the DLC-1::GFP puncta turned back at the distal segment, whereas 40% of DLC-1 returned at the middle tip. Finally, 69% of the DYLT-3::GFP puncta returned in the middle segment, not even reaching the middle tip (Fig. 4, B and C). These data suggest that multiple retrograde IFT pathways may operate within a single cilium.

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