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WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

Patel-King RS, Gilberti RM, Hom EF, King SM - Mol. Biol. Cell (2013)

Bottom Line: We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement.The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz.Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

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Smed-wd60(RNAi) planaria exhibit reduced gliding velocity and an altered mode of locomotion. (A) RT-PCR analysis of mRNA levels in untreated control planaria and in animals fed either with the empty L4440 vector or dsRNA from the Smed-wd60(RNAi) construct. The first-strand cDNA was prepared using a random hexamer as primer, and the reaction was continued for 25 cycles. Planaria fed with the WD60 dsRNA have unchanged levels of mRNAs encoding actin, IFT88, and outer-arm dynein IC2. Two RT-PCR products (325 and 502 base pairs), however, were detected for WD60. Sequence analysis revealed that both encoded the same region of WD60, but that the larger one also contained two unspliced introns. Levels of the 325–base pair product were greatly reduced in planaria fed with WD60 dsRNA, whereas levels of the 502–base pair product were unchanged. The 502–base pair product was not obtained following treatment of the RNA sample with DNaseI before PCR (not shown), indicating that it derives from genomic DNA. (B) Map of the two RT-PCR products for WD60 shown in A, indicating the location of the two introns. (C) Products obtained by RT-PCR after 40 cycles following DNaseI treatment of the mRNA sample and first-strand cDNA synthesis using an oligo-dT primer. The two additional products derive from partially spliced polyadenylated mRNA for WD60. (D) Bar chart illustrating the velocity of movement of control and Smed-wd60(RNAi) planaria. The controls moved by cilia-driven gliding at a rate of 1.48 ± 0.21 mm/s (mean ± SD; n = 7), whereas the knockdown animals used waves of muscle contraction and traveled at 0.55 ± 0.19 mm/s (n = 13). (E) The tracks taken by individual planaria over a 30-s period. These images were prepared from the decompiled videos by overlaying every 100th frame using Photoshop (and see Supplemental Movie S1). (F) Mode of planarian movement. Controls move with a smooth, cilia-driven gliding motion, whereas Smed-wd60(RNAi) animals use peristaltic waves of muscle contraction. These images were taken from the decompiled combined video (see Supplemental Movie S2).
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Figure 5: Smed-wd60(RNAi) planaria exhibit reduced gliding velocity and an altered mode of locomotion. (A) RT-PCR analysis of mRNA levels in untreated control planaria and in animals fed either with the empty L4440 vector or dsRNA from the Smed-wd60(RNAi) construct. The first-strand cDNA was prepared using a random hexamer as primer, and the reaction was continued for 25 cycles. Planaria fed with the WD60 dsRNA have unchanged levels of mRNAs encoding actin, IFT88, and outer-arm dynein IC2. Two RT-PCR products (325 and 502 base pairs), however, were detected for WD60. Sequence analysis revealed that both encoded the same region of WD60, but that the larger one also contained two unspliced introns. Levels of the 325–base pair product were greatly reduced in planaria fed with WD60 dsRNA, whereas levels of the 502–base pair product were unchanged. The 502–base pair product was not obtained following treatment of the RNA sample with DNaseI before PCR (not shown), indicating that it derives from genomic DNA. (B) Map of the two RT-PCR products for WD60 shown in A, indicating the location of the two introns. (C) Products obtained by RT-PCR after 40 cycles following DNaseI treatment of the mRNA sample and first-strand cDNA synthesis using an oligo-dT primer. The two additional products derive from partially spliced polyadenylated mRNA for WD60. (D) Bar chart illustrating the velocity of movement of control and Smed-wd60(RNAi) planaria. The controls moved by cilia-driven gliding at a rate of 1.48 ± 0.21 mm/s (mean ± SD; n = 7), whereas the knockdown animals used waves of muscle contraction and traveled at 0.55 ± 0.19 mm/s (n = 13). (E) The tracks taken by individual planaria over a 30-s period. These images were prepared from the decompiled videos by overlaying every 100th frame using Photoshop (and see Supplemental Movie S1). (F) Mode of planarian movement. Controls move with a smooth, cilia-driven gliding motion, whereas Smed-wd60(RNAi) animals use peristaltic waves of muscle contraction. These images were taken from the decompiled combined video (see Supplemental Movie S2).

Mentions: Analysis of mRNA by RT (reverse transcription)-PCR from first-strand cDNA prepared using a random hexamer as the primer revealed that two bands for WD60 were present. One product obtained was the originally targeted 325–base pair fragment that encodes the central portion of the WD60 protein, whereas the second product of 502 base pairs consisted of this same exonic segment but included two unspliced introns (Figure 5, A and B). The 502–base pair product was not observed when first-strand cDNA was prepared using oligo-dT as the primer for reverse transcriptase, nor was it obtained when the original RNA sample was treated with DNaseI. Consequently, this second product derives from genomic DNA. Of interest, when first-strand cDNA was prepared using an oligo-dT primer, we obtained the 325–base pair product and two minor additional products that included one or the other of the two introns (Figure 5C). Thus planaria maintain a detectable pool of partially spliced polyadenylated mRNA for WD60. RT-PCR using total RNA derived from Smed-wd60(RNAi) planaria further revealed that ingestion of WD60 dsRNA resulted in greatly decreased amounts of the 325–base pair product compared with empty vector or no vector controls but had essentially no effect on transcript levels for actin, IFT88, or IC2 (Figure 5A).


WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

Patel-King RS, Gilberti RM, Hom EF, King SM - Mol. Biol. Cell (2013)

Smed-wd60(RNAi) planaria exhibit reduced gliding velocity and an altered mode of locomotion. (A) RT-PCR analysis of mRNA levels in untreated control planaria and in animals fed either with the empty L4440 vector or dsRNA from the Smed-wd60(RNAi) construct. The first-strand cDNA was prepared using a random hexamer as primer, and the reaction was continued for 25 cycles. Planaria fed with the WD60 dsRNA have unchanged levels of mRNAs encoding actin, IFT88, and outer-arm dynein IC2. Two RT-PCR products (325 and 502 base pairs), however, were detected for WD60. Sequence analysis revealed that both encoded the same region of WD60, but that the larger one also contained two unspliced introns. Levels of the 325–base pair product were greatly reduced in planaria fed with WD60 dsRNA, whereas levels of the 502–base pair product were unchanged. The 502–base pair product was not obtained following treatment of the RNA sample with DNaseI before PCR (not shown), indicating that it derives from genomic DNA. (B) Map of the two RT-PCR products for WD60 shown in A, indicating the location of the two introns. (C) Products obtained by RT-PCR after 40 cycles following DNaseI treatment of the mRNA sample and first-strand cDNA synthesis using an oligo-dT primer. The two additional products derive from partially spliced polyadenylated mRNA for WD60. (D) Bar chart illustrating the velocity of movement of control and Smed-wd60(RNAi) planaria. The controls moved by cilia-driven gliding at a rate of 1.48 ± 0.21 mm/s (mean ± SD; n = 7), whereas the knockdown animals used waves of muscle contraction and traveled at 0.55 ± 0.19 mm/s (n = 13). (E) The tracks taken by individual planaria over a 30-s period. These images were prepared from the decompiled videos by overlaying every 100th frame using Photoshop (and see Supplemental Movie S1). (F) Mode of planarian movement. Controls move with a smooth, cilia-driven gliding motion, whereas Smed-wd60(RNAi) animals use peristaltic waves of muscle contraction. These images were taken from the decompiled combined video (see Supplemental Movie S2).
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Figure 5: Smed-wd60(RNAi) planaria exhibit reduced gliding velocity and an altered mode of locomotion. (A) RT-PCR analysis of mRNA levels in untreated control planaria and in animals fed either with the empty L4440 vector or dsRNA from the Smed-wd60(RNAi) construct. The first-strand cDNA was prepared using a random hexamer as primer, and the reaction was continued for 25 cycles. Planaria fed with the WD60 dsRNA have unchanged levels of mRNAs encoding actin, IFT88, and outer-arm dynein IC2. Two RT-PCR products (325 and 502 base pairs), however, were detected for WD60. Sequence analysis revealed that both encoded the same region of WD60, but that the larger one also contained two unspliced introns. Levels of the 325–base pair product were greatly reduced in planaria fed with WD60 dsRNA, whereas levels of the 502–base pair product were unchanged. The 502–base pair product was not obtained following treatment of the RNA sample with DNaseI before PCR (not shown), indicating that it derives from genomic DNA. (B) Map of the two RT-PCR products for WD60 shown in A, indicating the location of the two introns. (C) Products obtained by RT-PCR after 40 cycles following DNaseI treatment of the mRNA sample and first-strand cDNA synthesis using an oligo-dT primer. The two additional products derive from partially spliced polyadenylated mRNA for WD60. (D) Bar chart illustrating the velocity of movement of control and Smed-wd60(RNAi) planaria. The controls moved by cilia-driven gliding at a rate of 1.48 ± 0.21 mm/s (mean ± SD; n = 7), whereas the knockdown animals used waves of muscle contraction and traveled at 0.55 ± 0.19 mm/s (n = 13). (E) The tracks taken by individual planaria over a 30-s period. These images were prepared from the decompiled videos by overlaying every 100th frame using Photoshop (and see Supplemental Movie S1). (F) Mode of planarian movement. Controls move with a smooth, cilia-driven gliding motion, whereas Smed-wd60(RNAi) animals use peristaltic waves of muscle contraction. These images were taken from the decompiled combined video (see Supplemental Movie S2).
Mentions: Analysis of mRNA by RT (reverse transcription)-PCR from first-strand cDNA prepared using a random hexamer as the primer revealed that two bands for WD60 were present. One product obtained was the originally targeted 325–base pair fragment that encodes the central portion of the WD60 protein, whereas the second product of 502 base pairs consisted of this same exonic segment but included two unspliced introns (Figure 5, A and B). The 502–base pair product was not observed when first-strand cDNA was prepared using oligo-dT as the primer for reverse transcriptase, nor was it obtained when the original RNA sample was treated with DNaseI. Consequently, this second product derives from genomic DNA. Of interest, when first-strand cDNA was prepared using an oligo-dT primer, we obtained the 325–base pair product and two minor additional products that included one or the other of the two introns (Figure 5C). Thus planaria maintain a detectable pool of partially spliced polyadenylated mRNA for WD60. RT-PCR using total RNA derived from Smed-wd60(RNAi) planaria further revealed that ingestion of WD60 dsRNA resulted in greatly decreased amounts of the 325–base pair product compared with empty vector or no vector controls but had essentially no effect on transcript levels for actin, IFT88, or IC2 (Figure 5A).

Bottom Line: We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement.The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz.Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

Show MeSH
Related in: MedlinePlus