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. 2004 Oct;15(10):4382-94.
doi: 10.1091/mbc.e04-05-0377. Epub 2004 Jul 21.

A Dynein Light Intermediate Chain, D1bLIC, Is Required for Retrograde Intraflagellar Transport

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Free PMC article

A Dynein Light Intermediate Chain, D1bLIC, Is Required for Retrograde Intraflagellar Transport

Yuqing Hou et al. Mol Biol Cell. .
Free PMC article

Abstract

Intraflagellar transport (IFT), the bidirectional movement of particles along flagella, is essential for flagellar assembly. The motor for retrograde IFT in Chlamydomonas is cytoplasmic dynein 1b, which contains the dynein heavy chain DHC1b and the light intermediate chain (LIC) D1bLIC. To investigate a possible role for the LIC in IFT, we identified a d1blic mutant. DHC1b is reduced in the mutant, indicating that D1bLIC is important for stabilizing dynein 1b. The mutant has variable length flagella that accumulate IFT-particle proteins, indicative of a defect in retrograde IFT. Interestingly, the remaining DHC1b is normally distributed in the mutant flagella, strongly suggesting that the defect is in binding of cargo to the retrograde motor rather than in motor activity per se. Cell growth and Golgi apparatus localization and morphology are normal in the mutant, indicating that D1bLIC is involved mainly in retrograde IFT. Like mammalian LICs, D1bLIC has a phosphate-binding domain (P-loop) at its N-terminus. To investigate the function of this conserved domain, d1blic mutant cells were transformed with constructs designed to express D1bLIC proteins with mutated P-loops. The constructs rescued the mutant cells to a wild-type phenotype, indicating that the function of D1bLIC in IFT is independent of its P-loop.

Figures

Figure 1.
Figure 1.
D1bLIC is the Chlamydomonas ortholog of mammalian D2LIC. (A) D1bLIC cDNA sequence and its predicted amino acid sequence. The P-loop from amino acids 47–54 is indicated by a box, and the predicted coiled-coil domain from amino acids 376–398 (predicted by COILS using the MTIDK matrix and a window of 21) is underlined. 5692 base pairs of genomic DNA, from 819 base pairs upstream of the D1bLIC start codon to 1354 base pairs downstream of the D1bLIC stop codon, were sequenced from a 7.3-kb SmaI-SacI cassette that was subcloned from the 01O10 BAC clone. The start of the 5′ untranslated region and the site of polyadenylation were determined by searching the Chlamydomonas EST database. The coding regions were determined by sequencing PCR products (containing sequences from nucleotides 58–1773) from a cDNA library. (B) Phylogenetic tree for dynein 1 and dynein 2 LICs from different species. The predicted D1bLIC sequence (in bold) was aligned with a subset of cytoplasmic dynein 1 and 1b/2 LIC sequences in GenBank using CLUSTAL W, and a phylogenetic tree was drawn by cluster algorithm. CrIC1 is Chlamydomonas outer arm dynein intermediate chain IC1. Branch lengths represent evolutionary relatedness. Numbers at branch points are bootstrap values. The Chlamydomonas D1bLIC groups closely with the mammalian and other presumptive D2LICs, but much less closely with cytoplasmic dynein 1 LICs. Cr, C. reinhardtii; Ce, C. elegans; Dm, D. melanogaster; Hs, Homo sapiens; Rn, Rattus novegicus; Mn, Mus musculus; Xl, Xenopus laevis; Gg, Gallus gallus. Sequences used for phylogenetic analysis are as follows: MnD2LIC (AAH39070); HsD2LIC (NP_057092); CeD2LIC (T20505); DmD2LIC (NP_609289); CrD1bLIC (AY616759); MnDLIC2 (XP_134573); HsDLIC2 (NP_006132); RnDLIC2 (I55514); RnDLIC1 (NP_665715); MmDLIC1 (NP_666341); HsDLIC1 (NP_057225); GgDLIC1 (I50637); XlDLIC1 (AAG42486); CrIC1 (Q39578).
Figure 2.
Figure 2.
An insertional mutant, TBD9–1, is defective in D1bLIC. (A) Schematic diagram of D1bLIC gene structure. The 10.0-kb fragment from SmaI to HindIII and the 7.3-kb fragment from SmaIto SacI were used for later rescue experiments. The D1bLIC gene is composed of 11 exons (shown as black boxes). The site where the polyA signal is added is represented by a black dot. PstI cuts the gene into six fragments of the indicated sizes. The regions corresponding to the 5′ cDNA probe and 3′ cDNA probe used in the Northern blotting, the full-length cDNA probe used in the Southern blotting, and the antigen region for antibody preparation are indicated by lines underneath. Sm, SmaI; K, KpnI; P, PstI; H, HindIII; Sa, SacI. (B) Southern blotting indicates the D1bLIC gene is disrupted in TBD9–1. Genomic DNA from wild-type cells and TBD9–1 cells was cut by PstI and probed with the full-length D1bLIC cDNA probe. The TBD9–1 DNA is lacking one 1.2-kb PstI band present in wild-type DNA and has two additional bands of ∼1.4 and 3.5 kb as indicated by arrowheads. This implies that there is an insertion in the 1.2-kb PstI region of the D1bLIC gene in TBD9–1 cells, whereas the rest of the gene is intact. (C) Northern blotting indicates D1bLIC mRNA is disrupted in TBD9-1. Total RNA from TBD9-1 cells and wild-type cells before (N) and 30 min after deflagellation (D) were probed with 5′ or 3′ D1bLIC cDNA probes. The D1bLIC mRNA is ∼2.2 kb and is up-regulated by deflagellation in wild-type cells. No signal was detected by the 3′ cDNA probe in RNA from TBD9-1. Two faint bands of ∼5 and 1.8 kb were detected by the 5′ cDNA probe in RNA from TBD9-1 cells; they are slightly down-regulated by deflagellation. The bottom panel was probed by fructose-bisphosphate aldolase (FBA) cDNA as a loading control. (D) D1bLIC protein is missing in TBD9-1. A Western blot of whole cell lysates from TBD9-1 cells and wild-type cells probed with the anti-D1bLIC antibody shows that D1bLIC migrates at ∼48.5 kDa in wild-type cells and that no protein is present in TBD9-1 cells. The bottom panel was probed with an anti–β-tubulin antibody as a loading control.
Figure 3.
Figure 3.
The phenotype of YH43 can be rescued by the cloned D1bLIC gene. (A) D1bLIC DNA is restored in cells rescued for the motility defect by transformation with the D1bLIC gene. Genomic DNA from wild-type cells (137c), YH43, and some of the cells rescued by the 10-kb D1bLIC genomic fragment (43R1, 43R5, 43R15, 43R17, 43R19, and 43R22) was cut by SmaI and analyzed on Southern blots probed with the full-length D1bLIC cDNA. In addition to the mutated D1bLIC gene, different bands were detected in the rescued cells, indicating the incorporation of the wild-type D1bLIC transgene at different sites. (B) D1bLIC protein is detected in rescued cells. Western blots of wild-type cells (137c), YH43, and some of the rescued cells (43R1, 43R5, 43R15, 43R17, 43R19, and 43R22) probed with the anti-D1bLIC antibody showed that the rescued cells express D1bLIC protein. β-tubulin was probed in the bottom panel as a loading control.
Figure 4.
Figure 4.
Immunofluorescence microscopy shows that the YH43 cell accumulates IFT-particle proteins in its short flagella. Wild-type cells, YH43 cells (d1blic), and rescued cells were stained with an anti-IFT172 antibody (green). Cell bodies are red due to auto-fluorescence. Most of the IFT172 protein is in the peri-basal body region with a lesser amount spread along the flagella in the wild-type cells. In the d1blic mutant cells, IFT172 is redistributed from the peri-basal body region to the flagella, which are usually short and stumpy but sometimes longer. In mutant cells rescued with the wild-type D1bLIC gene, IFT172 is distributed as in wild-type cells. Scale bar, 5 μm.
Figure 5.
Figure 5.
Electron microscopy shows that d1blic flagella accumulate IFT particles. In wild-type cells (a and b) and the rescued cells (g and h) the space between the flagellar membrane and doublet microtubules is usually devoid of material. In contrast, in the d1blic mutant cells (c, d, e, and f) the space between the doublet microtubules and the flagellar membrane is filled with electron-dense material identical in appearance to IFT particles (Kozminski et al., 1993, 1995; Pazour et al., 1998). Note that the d1blic mutant cells have an apparently normal axoneme. Scale bars, 100 nm.
Figure 6.
Figure 6.
The d1blic mutant is normal in cell growth rate and localization and morphology of the Golgi apparatus. (A) Growth curves for wild-type cells and d1blic mutant cells. Wild-type cells and d1blic cells were grown in liquid M medium with aeration with 5% CO2. Every 24 h a sample was removed and the cells counted with a hemocytometer. On day 3, a second set of cultures was inoculated by diluting cells from the first series to 105 cells/ml. (B) The Golgi apparatus of d1blic mutant cells is morphologically normal. Like the wild-type cells, the d1blic cells have a pair of Golgi apparatuses on the opposite side of the nucleus from the basal bodies. The Golgi apparatus is composed of 4–8 cisternae and is adjacent to a large membrane-bound vesicle on the trans side.
Figure 7.
Figure 7.
The level of DHC1b is reduced in d1blic mutant cells and vice versa. Western blots of whole-cell lysates from wild-type cells, d1blic mutant cells, and dhc1b mutant cells were probed with anti-DHC1b or anti-D1bLIC antibodies. The DHC1b level is reduced in the d1blic mutant; the D1bLIC level is greatly reduced in the dhc1b mutant. β-tubulin was probed with an anti–β-tubulin antibody as a loading control.
Figure 8.
Figure 8.
D1bLIC and DHC1b have a similar cellular localization. Wild-type cells (left panels) were stained with anti-D1bLIC antibody or anti-DHC1b antibody. The null mutants (right panels) served as controls and were stained in the same way. The antigens are in green and the cell bodies are in red. Both DHC1b and D1bLIC are located primarily in the peri-basal body region and sparsely distributed along the flagella.
Figure 9.
Figure 9.
D1bLIC is in the same complex as DHC1b. (A) D1bLIC and DHC1b have a similar distribution in flagellar fractions. Wild-type flagella were fractionated into detergent-soluble membrane plus matrix (M+M), ATP wash 1 (ATP1), ATP wash 2 (ATP2), ATP wash 3 (ATP3), salt extract (Salt), and salt-extracted axonemes (Axo). Extracts from an equal number of flagella were probed with anti-DHC1b, anti-D1bLIC, anti-IFT172, anti-IFT139, and anti-IC1 antibodies. As reported by Pazour et al. (1999), DHC1b is contained primarily in the first ATP wash, with a lesser amount in the membrane plus matrix fraction and very little in the other fractions. In contrast, IFT172 and IFT139 are concentrated primarily in the membrane plus matrix fraction, whereas outer dynein arm intermediate chain IC1 is located primarily in the salt extract. Like DHC1b, D1bLIC also is contained primarily in the first ATP wash. (B) D1bLIC and DHC1b cosediment with each other. The matrix fraction from wild-type flagella was fractionated by centrifugation in a 5–20% sucrose gradient. Fractions were probed with anti-D1bLIC and anti-DHC1b antibodies. D1bLIC and DHC1b comigrate in a sharp peak at ∼12 S. (C) DHC1b is coimmunoprecipitated by anti-D1bLIC antibody. Anti-D1bLIC antibody (IP) or preimmune serum (Control) was used for the immunoprecipitation experiments. The resulting supernatants (Sup) and pellets (Pel) were probed with anti-D1bLIC, anti-DHC1b, and anti-IC1 antibodies. Both D1bLIC and DHC1b were immunoprecipitated by the anti-D1bLIC antibody but not by preimmune serum. IC1 remained in the supernatant from both the anti-D1bLIC antibody and preimmune serum immunoprecipitations.
Figure 10.
Figure 10.
DHC1b has a normal localization in d1blic mutant cells. Wild-type (a and b) and d1blic mutant cells with near full-length flagella (c and d) or shorter flagella (e and f) were stained with the anti-DHC1b antibody and were imaged by fluorescence microscopy (a, c, and e) or DIC microscopy (b, d, and f). Arrowheads indicate the bulges, shown in Figure 4 to be filled with IFT particles, in d1blic mutant flagella. DHC1b protein is in green and cell bodies in red. The DHC1b protein was present as punctae along the length of both wild-type and d1blic mutant flagella. No obvious accumulation of DHC1b protein was observed in the bulges on the d1blic flagella. Scale bar, 5 μm.
Figure 11.
Figure 11.
The P-loop is not required for D1bLIC's function in IFT. (A) PCR amplification shows that the mutated exogenous D1bLIC genes were incorporated into the cell lines transformed with mutated P-loop constructs K and KS. The P-loop region was amplified using genomic DNA from wild-type cells (W), d1blic mutant cells (M), and cells transformed with mutated P-loop construct K (1, 2, and 3) or construct KS (1, 2, and 3). As expected, a ∼700-base pair product obtained from wild-type cells was cut by NaeI into two ∼350-base pair products, but was not cut by SalI. No specific product was obtained from mutant cells. The ∼700-base pair products obtained from cells transformed with the K construct contained a SalI site and thus could be cut by SalI into two ∼350-base pair fragments. The ∼700-base pair products obtained from cells transformed with the KS construct lack the NaeI site and thus remained ∼700 base pairs in length after NaeI treatment. (B) Western blotting shows that the transformed cells express the 3HA-tagged D1bLIC protein. Whole-cell lysate from wild-type cells (W), cells transformed with the 3HA-tagged wild-type D1bLIC gene (43R), cells transformed with the K construct (1, 2, and 3), and cells rescued with the KS construct (1, 2, and 3) were probed with the anti-HA antibody or the anti-D1bLIC antibody. The anti-HA antibody did not recognize any band in the wild-type cell lysate, but recognized a doublet of ∼ 56–57 kDa in the transformed cell lysates. Western blotting using the anti-D1bLIC antibody on an equivalent blot confirmed that these doublets represented HA-tagged D1bLIC protein, which migrated at a higher position than the endogenous D1bLIC protein in the wild-type cells. (C) DIC images of wild-type cells, d1blic cells, and d1blic cells transformed with the K (K) and KS (KS) constructs. Flagellar length is completely restored in cells transformed with the K or KS constructs. Scale bar, 5 μm. (D) IFT172 is normally distributed in d1blic cells transformed with the K and KS constructs. Wild-type cells, d1blic cells, and d1blic cells transformed with the K (K) and KS (KS) constructs were stained with an anti-IFT172 antibody (green). Cell bodies are red due to auto-fluorescence. Scale bar, 5 μm.

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