A role for dynein in the inhibition of germ cell proliferative fate

Abstract

During normal development as well as in diseased states such as cancer, extracellular "niches" often provide cues to proximal cells and activate intracellular pathways. Activation of such signaling pathways in turn instructs cellular proliferation and differentiation. In the Caenorhabditis elegans gonad, GLP-1/Notch signaling instructs germ line stem cells to self-renew through mitotic cell division. As germ cells progressively move out of the niche, they differentiate by entering meiosis and eventually form gametes. In this model system, we uncovered an unexpected role for the dynein motor complex in promoting normal differentiation of proliferating germ cells. We demonstrate that dynein light chain 1 (DLC-1) and its partner, dynein heavy chain 1, inhibit the proliferative cell fate, in part through regulation of METT-10, a conserved putative methyltransferase. We show that DLC-1 physically interacts with METT-10 and promotes both its overall levels and nuclear accumulation. Our results add a new dimension to the processes controlled by the dynein motor complex, demonstrating that dynein can act as an antiproliferative factor.

FIG. 1.

(A) Fluorescence micrograph of a dissected, DAPI (4′,6-diamidino-2-phenylindole)-stained adult C. elegans hermaphrodite germ line, with schematic. In the distal region, proliferating germ cells (2) reside in close contact with the somatic distal tip cell (1). At the transition zone (3), germ cells enter meiosis and proceed through meiotic prophase (4) to give rise to sperm (6) and oocytes (5). (B) Scheme depicting the decision that C. elegans germ line stem cells make between maintaining a proliferative fate (green) and differentiating (red) to produce gametes.

FIG. 2.

dlc-1 functions to inhibit proliferative fate in the C. elegans germ line. (A) RNAi against dlc-1 and dhc-1 enhances tumor formation in the glp-1(oz264gf) background at 20°C, while RNAi against other light chains does not. dlc-2, paralog of dlc-1; dylt-1, Tctex-type dynein light chain; dyrb-1, Roadblock-type light chain. Two-tailed P values were determined using Fisher's exact test. (B, C) In wild-type germ lines (B), as well as in dlc-1 RNAi of control animals (C), a 3-h pulse of the nucleotide analog EdU, which incorporates during DNA synthesis, labels (green) only the very distal end (left) of the germ line, although dlc-1 RNAi causes abnormal nuclei containing large, dense DNA bodies (white arrows) and unpaired chromosomes in diakinesis to occur. Insets show chromosomal morphology of diakinetic oocytes, with six sets of bivalents in oocytes from control germ lines (B) but 12 univalents for dlc-1 RNAi (C). (D, E) dlc-1 RNAi causes tumor formation and EdU incorporation throughout the germ line in glp-1(oz264gf) (E), while the glp-1(oz264gf) mutant on its own has only a marginal increase in EdU incorporation (D). (F to H) Staining against DLC-1 (green) in glp-1(oz264gf) animals treated with double-stranded RNA targeting either gfp as a control (F) or dlc-1 (G, H). (G) A tumorous germ line with partial DLC-1 knockdown and unaffected nuclear morphology; (H) a tumorous germ line with stronger DLC-1 knockdown and abnormal nuclear morphology. (I) This inset from panel H highlights the large size of abnormal nuclei. Solid arrows indicate large, DNA-dense (abnormal) nuclei, and feather arrows indicate nuclei of relatively normal size. (J) Quantification of DLC-1 levels by pixel intensity for glp-1(oz264gf); gfp(RNAi) germ lines (white bar), glp-1(oz264gf); dlc-1(RNAi) tumorous germ lines with normal nuclear morphology (black bar), and glp-1(oz264gf); dlc-1(RNAi) tumorous germ lines with abnormal nuclear morphology (gray bar). Error bars indicate the standard errors of the means for the results. All scale bars = 20 μm. Tum, tumorous; wt, wild type.

FIG. 3.

DLC-1 promotes METT-10 nuclear accumulation. (A) METT-10::GFP expression in a wild-type germ line. (B) dlc-1 RNAi causes a decrease in METT-10::GFP accumulation, abnormal nuclear morphology (left), and unpaired chromosomes in diakinesis (right, yellow arrows). (C) METT-10::GFP nuclear accumulation is delayed in the dhc-1(or195) mutant background, with nuclear accumulation observed only in late pachytene. Solid and feather arrows indicate nuclei positive and negative for METT-10::GFP, respectively. (D) IP-Western blotting for METT-10::GFP levels in whole animals subject to both control (empty vector) RNAi and dlc-1(RNAi). CGH-1 is a germ line ubiquitous protein. (E) qRT-PCR for dlc-1 (top) and mett-10 (bottom) normalized to cgh-1 mRNA levels. RNAi was carried out in the rrf-1(pk1417) background, which retains RNAi only in the germ line.

FIG. 4.

METT-10 binds DLC-1 in vitro. (A) GST-DLC-1 point mutants (Coomassie) were assayed for binding to full-length His₆-METT-10 proteins. WT, wild type. (B) METT-10 can form multimers. Western blot (WB) analysis of bacterially expressed His₆-METT-10 fusion proteins reveals bands that run at the size of monomers (*) and dimers ($). The lowest band in lane 1 likely represents a METT-10 degradation product. (C) A dimeric band (feathered arrow) is observed when full-length His₆-METT-10 is pulled down with GST-DLC-1 and full-length GST-METT-10. (D) Fine mapping of the DLC-1 binding domain of METT-10 using GST-pulldown analysis analogous to that shown in panel C. The identity of individually mutated METT-10 residues are labeled on the left.

FIG. 5.

Dual mechanism ensures METT-10 nuclear accumulation. (A) Domain architecture of METT-10 marked with protein sequence change caused by mett-10(oz36) and location of the DLC-1 binding motif. Gray shading highlights the conserved methyltransferase-10 domain, and purple indicates the NLC sequence. (B to F) Confocal images (0.3 μm) of wild-type and mutant METT-10::GFP expression in gonadal sheath cells stained with antibodies against GFP and lamin to visualize nuclear membrane.

FIG. 6.

mett-10 mutants enhance dynein loss-of-function phenotypes of the dhc-1(js319) allele. (A, C, and D) Most dhc-1(js319) mutant germ lines are relatively normal at both 20 and 25°C, although they exhibit polyploidy (large DNA-dense nuclei) and univalents (unpaired chromosomes in diakinesis) at very low penetrance. (B, E, and F) Double mutations between dhc-1(js319) and any mett-10 allele lead to significant enhancement of both these phenotypes, and all double mutant animals are sterile at 20°C. This set of phenotypes closely phenocopies loss of dlc-1 function in the germ line.