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Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility

Lechtreck KF, Witman GB - J. Cell Biol. (2007)

Bottom Line: An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes.Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1).In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity.

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. karl.lechtreck@umassmed.edu

ABSTRACT

Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an approximately 540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP.

HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3, which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3, and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 (Cole et al., 1998) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼106 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes (Hou et al., 2004). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.
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fig1: HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3, which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3, and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 (Cole et al., 1998) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼106 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes (Hou et al., 2004). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.

Mentions: C. reinhardtii hydin is a polypeptide of ∼540 kD encoded by a single copy gene, HY3, spanning ∼17,700 bp on linkage group I. To generate an antibody to C. reinhardtii hydin, a HY3 fragment (Fig. 1 A, fragment A), representing exon 3, was amplified from genomic DNA by PCR and cloned into the bacterial expression vector pMAL-cRI v.2 encoding maltose-binding protein (MBP). The MBP-hydin peptide was expressed in Escherichia coli, purified, and used for antibody production. A BamHI–SalI piece of fragment A was cloned into pGEX-6P-1, and the purified GST-hydin peptide was used for affinity purification of the antibody. The purified anti- hydin antibody stained a single band of ∼540 kD in Western blots of axonemes of control cells (Fig. 1 B, control lane).

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Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility

Lechtreck KF, Witman GB - J. Cell Biol. (2007)

HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3, which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3, and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 (Cole et al., 1998) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼106 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes (Hou et al., 2004). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.
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fig1: HY3 gene-silencing vector and anti-hydin antibody. (A) C. reinhardtii HY3, which encodes hydin, is a gene of 17.7 kb. Fragment A, corresponding to exon 3 of HY3, and a BamHI–SalI piece of fragment A were cloned into bacterial expression vectors, and the fusion proteins were used for antibody production and purification. A gene-silencing vector was constructed from fragment A, fragment S (another PCR product of HY3), a triple HA tag, and the promoter and terminator region of the LC8 gene. (B) Coomassie-stained gel (a; 4–20% SDS-PAGE) and Western blot (b; 7.5% SDS-PAGE) of isolated axonemes of CC3395 (control) and the HY3 RNAi strains hyN3 and hyN4. Anti-hydin specifically stained a band of ∼540 kD that was strongly reduced in the HY3 RNAi strains. (C) Western blots probed with anti-hydin and anti-IFT172 (Cole et al., 1998) comparing the amount of hydin present in deflagellated cells (CB) and isolated flagella (Fla) or axonemes (Ax). (a) Equivalent numbers of cell bodies and flagellar pairs from ∼106 cells were loaded. (b) Equal amounts (∼25 μg) of cell body and axonemal protein were loaded. IFT172, an intraflagellar transport protein used as a control, is present in the cell body and flagella; a considerable amount remains with the axonemes (Hou et al., 2004). (D) Immunofluorescence images of methanol-fixed cells of strains CC3395 (control), hyN4, and hyS2 labeled with anti-acetylated tubulin (a, d, and g) and anti-hydin (b, e, and h). Merged images (c, f, and i) reveal the localization of hydin to the flagella of wild-type cells and the reduction of hydin in the hydin RNAi cells. Note the shorter flagella in the latter. At least part of the fluorescence in the cell bodies stained with anti-hydin is background caused by chlorophyll autofluorescence. Bar, 5 μm.
Mentions: C. reinhardtii hydin is a polypeptide of ∼540 kD encoded by a single copy gene, HY3, spanning ∼17,700 bp on linkage group I. To generate an antibody to C. reinhardtii hydin, a HY3 fragment (Fig. 1 A, fragment A), representing exon 3, was amplified from genomic DNA by PCR and cloned into the bacterial expression vector pMAL-cRI v.2 encoding maltose-binding protein (MBP). The MBP-hydin peptide was expressed in Escherichia coli, purified, and used for antibody production. A BamHI–SalI piece of fragment A was cloned into pGEX-6P-1, and the purified GST-hydin peptide was used for affinity purification of the antibody. The purified anti- hydin antibody stained a single band of ∼540 kD in Western blots of axonemes of control cells (Fig. 1 B, control lane).

Bottom Line: An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes.Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1).In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity.

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA. karl.lechtreck@umassmed.edu

ABSTRACT

Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an approximately 540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP-radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP.

View Similar Images In: Results  - Collection
View Article: MedlinePlus - PubMed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2063982&rFormat=json&query=null&req=5