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ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery.

Ahmed NT, Gao C, Lucker BF, Cole DG, Mitchell DR - J. Cell Biol. (2008)

Bottom Line: ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT.Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16.ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

ABSTRACT
Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

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ODA16 is not needed for dynein preassembly in the cytoplasm. (A) Western blots demonstrate subunit abundance in wild-type and mutant cytoplasmic extracts. HCβ mutant oda4 lacks HCβ but retains normal levels of other dynein subunits and ODA16 protein. The oda16 extract lacks ODA16 and has abnormally high levels of dynein subunits. Expression of ODA16HA in the oda16 cells (ODA16HA) restores dynein subunits to wild-type levels. Note the increased size of ODA16 due to the HA tag. (B) Axonemal outer row dynein subunits are preassembled in oda16 cytoplasm. Immunoprecipitation of dynein with anti-HCβ coprecipitates all three heavy chains and both intermediate chains from both wild-type (WT) and oda16 cytoplasmic extracts. Mock precipitation from the wild type with anti-HA (Ig) and from oda4 with anti-HCβ failed to precipitate any subunits. (C) Axonemal dynein in oda16 cytoplasm is competent to bind to axonemes. Stained gels and blots of axonemes from the wild type (WT) or pf28 cells separated by SDS-PAGE show that pf28 axonemes completely lack outer row dynein (first two lanes). To test the ability of axonemal dynein in the cytoplasmic pool to bind to axonemes, pf28 axonemes and cytoplasmic extracts were incubated together at a 1:2 stoichiometric ratio for 1 h, and washed axonemes were analyzed (last four lanes). Outer arm dynein proteins in wild-type, oda16, and ift46 extracts, but not oda4 extracts, were competent to bind to pf28 axonemes and restore dynein to wild-type levels. The stained gel and IC1 blot in C used identical sample loads; the first two lanes were originally run on the same gel as other lanes, but intervening lanes have been spliced out (indicated by black vertical lines). Gels used to prepare blots of all three HCs and IC2 in C were prepared with fivefold less protein than is shown in the gel. In addition to the mass of size standards, the position of dynein heavy chains (HC) is indicated by an arrow next to the stained gel images in A and C. Numbers adjacent to gel blot panels indicate the estimated mass in kD of each detected band.
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fig3: ODA16 is not needed for dynein preassembly in the cytoplasm. (A) Western blots demonstrate subunit abundance in wild-type and mutant cytoplasmic extracts. HCβ mutant oda4 lacks HCβ but retains normal levels of other dynein subunits and ODA16 protein. The oda16 extract lacks ODA16 and has abnormally high levels of dynein subunits. Expression of ODA16HA in the oda16 cells (ODA16HA) restores dynein subunits to wild-type levels. Note the increased size of ODA16 due to the HA tag. (B) Axonemal outer row dynein subunits are preassembled in oda16 cytoplasm. Immunoprecipitation of dynein with anti-HCβ coprecipitates all three heavy chains and both intermediate chains from both wild-type (WT) and oda16 cytoplasmic extracts. Mock precipitation from the wild type with anti-HA (Ig) and from oda4 with anti-HCβ failed to precipitate any subunits. (C) Axonemal dynein in oda16 cytoplasm is competent to bind to axonemes. Stained gels and blots of axonemes from the wild type (WT) or pf28 cells separated by SDS-PAGE show that pf28 axonemes completely lack outer row dynein (first two lanes). To test the ability of axonemal dynein in the cytoplasmic pool to bind to axonemes, pf28 axonemes and cytoplasmic extracts were incubated together at a 1:2 stoichiometric ratio for 1 h, and washed axonemes were analyzed (last four lanes). Outer arm dynein proteins in wild-type, oda16, and ift46 extracts, but not oda4 extracts, were competent to bind to pf28 axonemes and restore dynein to wild-type levels. The stained gel and IC1 blot in C used identical sample loads; the first two lanes were originally run on the same gel as other lanes, but intervening lanes have been spliced out (indicated by black vertical lines). Gels used to prepare blots of all three HCs and IC2 in C were prepared with fivefold less protein than is shown in the gel. In addition to the mass of size standards, the position of dynein heavy chains (HC) is indicated by an arrow next to the stained gel images in A and C. Numbers adjacent to gel blot panels indicate the estimated mass in kD of each detected band.

Mentions: We next examined the role of ODA16 in cytoplasmic preassembly of outer arm dynein. Although data from dikaryon rescue experiments are consistent with the presence of intact, preassembled dynein in oda16 cytoplasm (Ahmed and Mitchell, 2005), we considered an alternative hypothesis. The ODA16 protein that is supplied to oda16 mutant cytoplasm during dikaryon formation might be rapidly converting dynein from a non-preassembled form into one that is preassembled and able to be transported into flagella. We previously demonstrated that soluble extracts of wild-type cells, made by glass bead disruption in the absence of detergent, contain outer row dynein proteins preassembled into a complex that includes all three heavy chains and both intermediate chains (Fowkes and Mitchell, 1998). In extracts from some dynein assembly mutants, such as oda7, these subunits fail to preassemble, and some individual subunits show reduced cytoplasmic abundance, whereas in other mutants, such as docking complex mutant oda1, all subunits appear at normal abundance and in a stable complex. To assess dynein subunit stability in oda16 cytoplasm, blots of cytoplasmic extracts were probed with antibodies to the five larger outer row dynein proteins. All five proteins show an increase in abundance in an oda16 extract compared with their levels in extracts of wild-type or oda4 (outer arm dynein heavy chain β [ODA-HCβ] mutant) cells (Fig. 3 A), and this increase in dynein subunit abundance is rescued back to wild-type levels by expression of an ODA16HA transgene. We previously showed that the HA-tagged ODA16 transgene phenotypically complements the oda16 assembly defect and that ODA16HA is expressed in ODA16-1R(HA) flagella at levels comparable to that of ODA16 in wild-type cells (Ahmed and Mitchell, 2005). These changes in heavy chain abundance can be observed directly in a Coomassie blue–stained gel (Fig. 3 A, top, HC) and by immunoblotting (Fig. 3 A, bottom). Probing this blot with anti-ODA16 shows that the ODA16 protein is expressed at wild-type levels in the oda4 cytoplasm, that no ODA16 is seen in the cytoplasm of oda16 cells, and that only the higher molecular weight HA-tagged ODA16 protein appears in the ODA16-1R(HA) strain. The increase in dynein abundance seen in oda16 cytoplasmic extracts correlates well with the increase seen by immunofluorescence in oda16 cell bodies (Fig. 1 B), and suggests that all of the dynein destined for flagellar assembly is synthesized in oda16 cells but accumulates in the cytoplasm because of its inefficient transport into the flagellar compartment.


ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery.

Ahmed NT, Gao C, Lucker BF, Cole DG, Mitchell DR - J. Cell Biol. (2008)

ODA16 is not needed for dynein preassembly in the cytoplasm. (A) Western blots demonstrate subunit abundance in wild-type and mutant cytoplasmic extracts. HCβ mutant oda4 lacks HCβ but retains normal levels of other dynein subunits and ODA16 protein. The oda16 extract lacks ODA16 and has abnormally high levels of dynein subunits. Expression of ODA16HA in the oda16 cells (ODA16HA) restores dynein subunits to wild-type levels. Note the increased size of ODA16 due to the HA tag. (B) Axonemal outer row dynein subunits are preassembled in oda16 cytoplasm. Immunoprecipitation of dynein with anti-HCβ coprecipitates all three heavy chains and both intermediate chains from both wild-type (WT) and oda16 cytoplasmic extracts. Mock precipitation from the wild type with anti-HA (Ig) and from oda4 with anti-HCβ failed to precipitate any subunits. (C) Axonemal dynein in oda16 cytoplasm is competent to bind to axonemes. Stained gels and blots of axonemes from the wild type (WT) or pf28 cells separated by SDS-PAGE show that pf28 axonemes completely lack outer row dynein (first two lanes). To test the ability of axonemal dynein in the cytoplasmic pool to bind to axonemes, pf28 axonemes and cytoplasmic extracts were incubated together at a 1:2 stoichiometric ratio for 1 h, and washed axonemes were analyzed (last four lanes). Outer arm dynein proteins in wild-type, oda16, and ift46 extracts, but not oda4 extracts, were competent to bind to pf28 axonemes and restore dynein to wild-type levels. The stained gel and IC1 blot in C used identical sample loads; the first two lanes were originally run on the same gel as other lanes, but intervening lanes have been spliced out (indicated by black vertical lines). Gels used to prepare blots of all three HCs and IC2 in C were prepared with fivefold less protein than is shown in the gel. In addition to the mass of size standards, the position of dynein heavy chains (HC) is indicated by an arrow next to the stained gel images in A and C. Numbers adjacent to gel blot panels indicate the estimated mass in kD of each detected band.
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Related In: Results  -  Collection

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fig3: ODA16 is not needed for dynein preassembly in the cytoplasm. (A) Western blots demonstrate subunit abundance in wild-type and mutant cytoplasmic extracts. HCβ mutant oda4 lacks HCβ but retains normal levels of other dynein subunits and ODA16 protein. The oda16 extract lacks ODA16 and has abnormally high levels of dynein subunits. Expression of ODA16HA in the oda16 cells (ODA16HA) restores dynein subunits to wild-type levels. Note the increased size of ODA16 due to the HA tag. (B) Axonemal outer row dynein subunits are preassembled in oda16 cytoplasm. Immunoprecipitation of dynein with anti-HCβ coprecipitates all three heavy chains and both intermediate chains from both wild-type (WT) and oda16 cytoplasmic extracts. Mock precipitation from the wild type with anti-HA (Ig) and from oda4 with anti-HCβ failed to precipitate any subunits. (C) Axonemal dynein in oda16 cytoplasm is competent to bind to axonemes. Stained gels and blots of axonemes from the wild type (WT) or pf28 cells separated by SDS-PAGE show that pf28 axonemes completely lack outer row dynein (first two lanes). To test the ability of axonemal dynein in the cytoplasmic pool to bind to axonemes, pf28 axonemes and cytoplasmic extracts were incubated together at a 1:2 stoichiometric ratio for 1 h, and washed axonemes were analyzed (last four lanes). Outer arm dynein proteins in wild-type, oda16, and ift46 extracts, but not oda4 extracts, were competent to bind to pf28 axonemes and restore dynein to wild-type levels. The stained gel and IC1 blot in C used identical sample loads; the first two lanes were originally run on the same gel as other lanes, but intervening lanes have been spliced out (indicated by black vertical lines). Gels used to prepare blots of all three HCs and IC2 in C were prepared with fivefold less protein than is shown in the gel. In addition to the mass of size standards, the position of dynein heavy chains (HC) is indicated by an arrow next to the stained gel images in A and C. Numbers adjacent to gel blot panels indicate the estimated mass in kD of each detected band.
Mentions: We next examined the role of ODA16 in cytoplasmic preassembly of outer arm dynein. Although data from dikaryon rescue experiments are consistent with the presence of intact, preassembled dynein in oda16 cytoplasm (Ahmed and Mitchell, 2005), we considered an alternative hypothesis. The ODA16 protein that is supplied to oda16 mutant cytoplasm during dikaryon formation might be rapidly converting dynein from a non-preassembled form into one that is preassembled and able to be transported into flagella. We previously demonstrated that soluble extracts of wild-type cells, made by glass bead disruption in the absence of detergent, contain outer row dynein proteins preassembled into a complex that includes all three heavy chains and both intermediate chains (Fowkes and Mitchell, 1998). In extracts from some dynein assembly mutants, such as oda7, these subunits fail to preassemble, and some individual subunits show reduced cytoplasmic abundance, whereas in other mutants, such as docking complex mutant oda1, all subunits appear at normal abundance and in a stable complex. To assess dynein subunit stability in oda16 cytoplasm, blots of cytoplasmic extracts were probed with antibodies to the five larger outer row dynein proteins. All five proteins show an increase in abundance in an oda16 extract compared with their levels in extracts of wild-type or oda4 (outer arm dynein heavy chain β [ODA-HCβ] mutant) cells (Fig. 3 A), and this increase in dynein subunit abundance is rescued back to wild-type levels by expression of an ODA16HA transgene. We previously showed that the HA-tagged ODA16 transgene phenotypically complements the oda16 assembly defect and that ODA16HA is expressed in ODA16-1R(HA) flagella at levels comparable to that of ODA16 in wild-type cells (Ahmed and Mitchell, 2005). These changes in heavy chain abundance can be observed directly in a Coomassie blue–stained gel (Fig. 3 A, top, HC) and by immunoblotting (Fig. 3 A, bottom). Probing this blot with anti-ODA16 shows that the ODA16 protein is expressed at wild-type levels in the oda4 cytoplasm, that no ODA16 is seen in the cytoplasm of oda16 cells, and that only the higher molecular weight HA-tagged ODA16 protein appears in the ODA16-1R(HA) strain. The increase in dynein abundance seen in oda16 cytoplasmic extracts correlates well with the increase seen by immunofluorescence in oda16 cell bodies (Fig. 1 B), and suggests that all of the dynein destined for flagellar assembly is synthesized in oda16 cells but accumulates in the cytoplasm because of its inefficient transport into the flagellar compartment.

Bottom Line: ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT.Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16.ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

ABSTRACT
Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

Show MeSH