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. 2003 May;14(5):2041-56.
doi: 10.1091/mbc.e02-10-0682. Epub 2003 Jan 26.

A Novel Dynein Light Intermediate Chain Colocalizes With the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly in Chlamydomonas and Mammalian Cells

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A Novel Dynein Light Intermediate Chain Colocalizes With the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly in Chlamydomonas and Mammalian Cells

Catherine A Perrone et al. Mol Biol Cell. .
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Abstract

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.

Figures

Figure 1.
Figure 1.
Identification of a novel LIC in Chlamydomonas. (A) Northern blot analysis. Total RNA isolated before (0) and 45 min after deflagellation (45) was size-fractionated on a gel, blotted to a nylon membrane, and hybridized overnight with a 367-base pair probe from the 5′ end of the LIC gene. Expression of the ∼2.3-kb LIC mRNA was enhanced by deflagellation, compared with a control probe (Cry1) for a ribosomal protein subunit. (B) The predicted LIC amino acid sequence contains 427 residues and corresponds to an ∼46.5-kDa polypeptide. The P-loop consensus motif is indicated in bold. (C). Schematic diagram of the LIC. Regions of homology with other dynein LICs include a P-loop motif near the amino terminus, a RAS signature motif, and a coiled coil domain near the carboxy terminus.
Figure 2.
Figure 2.
Association of the LIC with cDHC1b in flagellar extracts. (A) Whole cell extracts were made from wild-type and cDhc1b mutant cells and probed with an affinity purified cDHC1b antibody to demonstrate the specificity of the cDHC1b antibody. (B) Isolated flagella (F) from the outer arm mutant pf28 were first treated with a nonionic detergent to separate the soluble membrane plus matrix fraction (M) from isolated axonemes (A). Axonemes were then sequentially extracted with increasing concentrations of MgATP (1, 5, and 10 mM), followed by 0.6 M NaCl (HS). The outer doublets (OD) represent the axonemal proteins remaining after extraction. Gels were loaded stoichiometrically, blotted to polyvinylidene difluoride, and probed with the antibodies indicated. The LIC coextracts with cDHC1b. (C) Isolated axonemes prepared from an E8 strain lacking both outer arm and I1 inner arm dyneins were extracted with 10 mM MgATP. The ATP extract was then incubated with protein A-Sepharose beads containing either the affinity-purified LIC antibody or a control IgG. The ATP extract (lane 1) and the immunoprecipitates (lanes 2 and 3) were analyzed on Western blots probed with antibodies to cDHC1b, LIC, and LC8. Because the LIC is ∼46.5-kDa (see *), it is difficult to resolve from the IgG heavy chain and quantitate the extent of LIC immunoprecipitation. However, it is clear that the LIC antibody coimmunoprecipitates cDhc1b, whereas the control antibody does not. LC8, shown here on a 5–20% gradient gel, is present in the ATP extract, but not enriched in the LIC immunoprecipitate. (D) Western blots of equivalent numbers of wild-type, cDhc1b null (stf1-1), and LC8 null (fla14) cells were probed with antibodies to cDhc1b, LIC, and tubulin.
Figure 3.
Figure 3.
Copurification of the LIC with cDHC1b on sucrose density gradients. (A) A high salt extract of E8 axonemes (lacking the outer arm and I1 inner arm dyneins) was analyzed on a 5–20% sucrose density gradient. The resulting fractions were separated on a 5–15% PAGE, blotted to polyvinylidene difluoride, and probed with the antibodies indicated. (B) A similar Western blot containing sucrose gradient fractions from a 10 mM MgATP extract of E8 axonemes. The peak of cDHC1b and LIC has shifted to ∼19S.
Figure 4.
Figure 4.
Colocalization of the LIC with cDHC1b in wild-type and dynein mutant cells. In the top row, fixed wild-type cells were stained with antibodies to cDHC1b (A and B) or LIC (C and D) and imaged by differential interference contrast (DIC) microscopy (A and C) or epifluorescence (B and D). In the second row, cDhc1b mutant cells (stf1) were stained with antibodies to cDHC1b (E and F) or LIC (G and H). Arrowheads indicate the position of the flagellar stubs in DIC images (E and G). Note the absence of cDHC1b staining. LIC staining is also dispersed and reduced in cDhc1b mutant cells. In the third row, LC8 mutant cells (fla14) were stained with antibodies to cDHC1b (I and J) or LIC (K and L). Note that both cDHC1b and LIC accumulate in peribasal body region, but not in the flagellar stubs. In the fourth row, cDhc1b mutant cells were stained with antibodies to FLA10 kinesin (M and N) or the p172 subunit of IFT complex B (O and P). In the bottom row, LC8 mutant cells were stained with antibodies to the FLA10 kinesin subunit (Q and R) or p172 (S and T). Note the accumulation of both components in the short flagella of stf1 and fla14.
Figure 5.
Figure 5.
Colocalization of the LIC with cDHC1b in a retrograde IFT mutant. fla15 cells have defects in raft complex A and retrograde IFT (Piperno et al., 1998). At the permissive temperature, these mutants develop one to two small blebs along the length of their flagella that can be detected by differential interference contrast microscopy (A, C, E, and G). The fla15 cells were stained with antibodies to cDHC1b (A and B), the LIC (C and D), the p172 subunit of IFT complex B (E and F), and the p139 subunit of IFT complex A (G and H). Note that the blebs do not stain with the p139 antibody, but they do stain with the cDHC1b, LIC, and p172 antibodies.
Figure 6.
Figure 6.
Redistribution of IFT motors and IFT particles in a length control mutant. lf1 cells assemble flagella that are 2 to 3 times wild-type length (Barsel et al., 1988; Aselson et al., 1998). Small bulges are often visible at the tips of the lf1 flagella by differential interference contrast microscopy. lf1 cells were stained with antibodies to cDHC1b (A and B), LIC (C and D), p172, (E and F), p139 (G and H), and the FLA10 kinesin (I and J). Note the accumulation of all components at the flagellar tips (cDHC1b staining of the peribasal body region is out of the plane of focus in B). (K and L) Staining of lf3 cells with antibodies to the LIC.
Figure 7.
Figure 7.
D2LIC colocalizes with primary cilia in MDCK cells. Confluent cultures of polarized MDCK cells were stained with antibodies to D2LIC (shown in red) and either tubulin or the Golgi marker p115 (shown in green). The cells shown in A–C were costained with anti-D2LIC and anti-tubulin and then viewed from above, at the level of the apical cytoplasm and primary cilia. D2LIC staining (A), merged image (B), and tubulin staining (C). The cells shown in D–F were costained with anti-D2LIC, anti-p115, and Hoescht. D2LIC staining (D), merged image (E), and p115 staining (F). The Golgi apparatus is often out of focus in optimal views of the primary cilia. D2LIC is abundant in the apical cytoplasm, but clearly enriched in the primary cilia.
Figure 8.
Figure 8.
The mammalian D2LIC colocalizes with DHC2 and the IFT particle subunit Polaris in the ciliated epithelium of the lung. Isolated mouse lungs were fixed, sectioned, and stained with an affinity-purified rat antibody to the mammalian D2LIC (shown in red on the left; A, D, and G). The sections were costained with a mouse monoclonal antibody to tubulin (C), or affinity-purified rabbit antibodies to DHC2 (F), or Polaris (I), all shown in green on the right. Merged images are shown in the middle (B, E, and H). Nuclei were stained with Hoechst and are shown in blue. The inset in each panel shows two to three cells at a higher magnification. Note that staining is restricted to ciliated cells and seems to be concentrated in the apical cytoplasm and the ciliary axonemes.
Figure 9.
Figure 9.
D2LIC colocalizes with DHC2 and the IFT particle subunit Polaris in the ciliated epithelium of the efferent duct. Isolated mouse efferent ducts were fixed, sectioned, and stained with an affinity-purified rat antibody to the mammalian D2LIC (shown in red on the left in A, D, G, and J). The sections were costained with antibodies to tubulin (C and F), DHC2 (I), or Polaris (L), all shown in green on the right. Merged images are shown in the middle (B, E, H, and K). (A–C) Low-magnification views displaying cross sections through an efferent duct; the remaining panels are higher magnification views of the ciliated cells of the ducts.
Figure 10.
Figure 10.
D2LIC does not colocalize with the Golgi apparatus in the efferent duct. Sections of efferent ducts were stained with the affinity-purified rat antibody to D2LIC (shown in red in A and D) and a rabbit antibody to the Golgi marker p115 (shown in green in C and F). Merged images are shown in B and E. (A–C) Low-magnification views of a single efferent duct and surrounding connective tissue. (D–F) higher magnification views of individual duct cells.

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