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. 1998 Mar 9;140(5):1137-47.
doi: 10.1083/jcb.140.5.1137.

Identification of the T Complex-Encoded Cytoplasmic Dynein Light Chain tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

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Free PMC article

Identification of the T Complex-Encoded Cytoplasmic Dynein Light Chain tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

A Harrison et al. J Cell Biol. .
Free PMC article

Abstract

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell-specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.

Figures

Figure 1
Figure 1
Tctex1 is present in both mouse sperm and Chlamydomonas axonemes. (a) Whole-cell extracts of equal numbers of sperm from +/+, tw 5/+, and tw 5/tw 32 mice were electrophoresed in a 15% acrylamide gel, blotted to Immobilon-P membranes, and probed with R5205 antibody (upper panel) or R5205 antibody that had been preadsorbed against recombinant Tctex1 (lower panel). Tctex1 is present in sperm from all genotypes. (b) 150 μg of Chlamydomonas axonemes were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (CBB) or blotted to nitrocellulose and probed with R5205 antibody. The locations of the relative molecular mass markers and the dye front (DF) are indicated on the left. A single 14,000-M r band was observed.
Figure 2
Figure 2
Distribution of Tctex1 in Chlamydomonas axonemes. 125 μg of axonemes were treated with 0.6 M NaCl. Equivalent amounts of axonemes (Ax), extracted axonemes (EA), and the high salt extract (HSE) were electrophoresed in 5–15% acrylamide gradient gels. One portion of the gel was stained with Coomassie blue (upper panel) while the other was blotted to nitrocellulose and probed with R5205 antibody (lower panel). The locations of the relative molecular mass markers and the dye front (DF) are indicated on the left. Approximately 90% of the Tctex1 band was extracted with 0.6 M NaCl, suggesting that it may be a component of axonemal dynein.
Figure 3
Figure 3
Intra-axonemal localization of Tctex1. Axonemes were prepared from wild-type Chlamydomonas (WT) and from strains lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), inner arms I2/I3 (ida4), radial spokes (pf14), and central pair complex (pf18). 100 μg of each preparation was electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose (lower panels) and probed with R5205 and R5391 antisera to detect Tctex1 and LC2 (Tctex2) of outer arm dynein, respectively. LC2 is missing only in the oda9 mutant, which lacks the outer arm (the smaller immunoreactive band is present in all strains tested as described previously [Patel-King et al., 1997]). Tctex1 is specifically absent in those strains that fail to assemble inner arm I1, suggesting that it may be a component of that complex.
Figure 4
Figure 4
Tctex1 is a component of inner arm dynein I1. Proteins extracted from oda9 axonemes (which completely lack the outer arm) by high salt were separated by sucrose density gradient centrifugation. Equal volumes of each fraction were electrophoresed in 5–15% gradient gels. One gel was stained with Coomassie blue (upper panel), while the other was blotted to nitrocellulose and probed with R5205 antibody (lower panel). The bottom of the gradient is at the left. After immunoblotting, the nitrocellulose was restained with Ponceau S to reveal the location of individual lanes and the relative molecular mass markers. Tctex1 comigrates with inner arm I1 components (IC140 and IC138) at ∼18 S, but it is clearly distinct from arms I2/3, which contain actin, p28, and centrin. (inset, upper panel) The I1 dynein–containing fractions from two gradients were pooled and concentrated in a Centricon-30 ultrafiltration unit and electrophoresed in a 5–15% acrylamide gel. The LC region of the Coomassie blue–stained gel is shown. Three bands are evident. The upper band was recognized by the R5205 antibody and is therefore Tctex1. The lower band was detected by the R4058 antibody, indicating that it represents an additional pool of the 8,000-M r LC (King and Patel-King, 1995b ). A third LC band of unknown origin is also present.
Figure 5
Figure 5
Peptide purification from Tctex1. The I1-containing fractions from seven gradients were pooled and concentrated in a Centricon 30 ultrafiltration unit. The concentrate was electrophoresed in a 5–15% acrylamide gradient gel and blotted to polyvinylidene difluoride. A strip was excised and probed with R5205 to localize the Tctex1 band. The remaining Tctex1 band was then excised from the unprobed PVDF and digested in situ with trypsin. Peptides eluting from the membrane were purified by reverse-phase chromatography on a C8 column (a). Peaks (i) and (ii) were sequenced. Mass spectrometry (b and c) revealed a single peptide of 1,825 D present in peak (i). Peak (ii) contained a single major peptide of 2,463 D and a minor amount of its methionine oxidation product at 2,479 D.
Figure 6
Figure 6
Sequence of Chlamydomonas Tctex1. Nucleotide and predicted amino acid sequence of the cDNA clone encoding Tctex1 are shown. The residues shown in bold type were obtained from peptide sequencing (33/33 residues correct) and confirm the identity of the clone. The 5′ untranslated region contains three in-frame stop codons. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF039437.
Figure 7
Figure 7
Southern and Northern blot analyses. (a) Southern blot of 10 μg genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI. Single bands were observed in all lanes, suggesting that there is only a single gene for this LC within Chlamydomonas. Standards are indicated on the left in kilobases. (b) Northern blot of 20 μg total RNA obtained from nondeflagellated cells (NDF) and from cells that had been deflagellated and allowed to regenerate flagella for 30 min (30′ post DF). Standards are shown on the left in kilobases. Two messages were observed. The message at ∼1.35 kb was detected only in the NDF sample, while a smaller message (∼1.30 kb) was greatly upregulated after deflagellation. (c) Enlargement of Fig. 7 b (upper panel) and comparison with both a deflagellation-regulated message (LC4 of outer arm dynein) and one that is present constitutively but is also upregulated (calmodulin).
Figure 8
Figure 8
Sequence analysis of Chlamydomonas flagellar Tctex1. (a) The secondary structure of Tctex1 was predicted using PHD (Rost and Sander, 1993). E, extended sheet; H, helix. Helical stretches (i) and (ii) are amphiphilic and displayed using HELICALWHEEL. Hydrophobic and hydrophilic residues cluster to opposite sides of the helix. (b) Sequence comparison between Chlamydomonas flagellar Tctex1 and human Tctex1 (D50663). The alignment was generated by GAP using the default parameters. These proteins share 62% identity (68% similarity) with the smallest Poisson probability P (n) = 6.3 × 10−45 (calculated by BLAST). (c) Phylogenetic analysis of the members of the Tctex1 protein family. The relationship was calculated with DISTANCES and plotted with GROWTREE (UPGMA option). Chlamydomonas flagellar Tctex1 is more closely related to mammalian and insect Tctex1 than it is to human rp3, which is an additional cytoplasmic dynein LC sharing 55% identity with Tctex1 (King, S.M., E. Barbarese, J.F. Dillman III, S.E. Benashski, K.T. Do, R.S. Patel-King, and K.K. Pfister. 1997. Mol. Biol. Cell. 8:163a).
Figure 9
Figure 9
A mechanism for flagellar dynein–mediated meiotic drive. In this model, the responder is hypothesized to be a “gatekeeper” or sorting mechanism that determines what may enter the growing flagellum during spermiogenesis. Because t mutations in both putative distorters Tctex1 and Tctex2 could lead to flagellar dynein dysfunction, their incorporation into + sperm by the wild-type responder (Tcr +) might result in defective motility. In contrast, the t mutant responder (Tcrt) is thought to protect the t sperm with which it associates by being unable to interact with (or having a lower affinity for) the mutant distorters. In the heterozygous case, this would lead to incorporation of only wild-type dyneins into t haplotype–bearing sperm and thus to normal motility. The molecular identities of all the distorters are unknown at the present time. In this model, it is hypothesized that Tctex1 and Tctex2 are the Tcd1 and Tcd3 distorters, respectively.

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References

    1. Altshul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic alignment search tool. J Mol Biol. 1990;215:403–410. - PubMed
    1. Bell CW, Fronk E, Gibbons IR. Polypeptide subunits of dynein 1 from sea urchin sperm flagella. J Supramol Struct. 1979;11:311–317. - PubMed
    1. Benashski SE, Harrison A, Patel-King RS, King SM. Dimerization of the highly conserved light chain shared by dynein and myosin V. J Biol Chem. 1997;272:20929–20935. - PubMed
    1. Cebra-Thomas JA, Decker CL, Snyder LC, Pilder SH, Silver LM. Allele- and haploid-specific product generated by alternative splicing from a mouse t complex responderlocus candidate. Nature. 1991;349:239–241. - PubMed
    1. Corthésy-Theulaz J, Pauloin A, Pfeffer SR. Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex. J Cell Biol. 1992;118:1333–1345. - PMC - PubMed

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