Figure 1.
Identification of Mutations in Three Recessive dhc-1ts Mutant Alleles
(A) dhc-1(or195) and dhc-1(or283) both have a serine changed to leucine at codon 3200, which corresponds to the N-terminal coiled-coil domain of the microtubule-binding stalk. Other metazoans and S. pombe also have a serine at this position.
(B) dhc-1(or352) changes a glycine to an aspartic acid at codon 2158 within the Walker A region of the second AAA ATPase domain. Other organisms also have a glycine (or alanine for budding yeast) at this position. Organisms: Ce: Caenorhabditis elegans, Hs: Homo sapiens, Mm: Mus musculus, Dm: Drosophila melanogaster, Dd: Dictyostelium discoideum, Sp: Schizosaccharomyces pombe, and Sc: Saccharomyces cerevisiae.
(C) Model of the dynein heavy chain and location of three ts alleles. Numbered sectors represent the six AAA domains and “MT” denotes the microtubule-binding domain.
Figure 2.
Characterization of dhc-1ts Mutants and Overview of the Suppressor RNAi Screening Strategy
(A) Temperature versus viability plot for three recessive dhc-1 mutants and wild-type animals. L1 larvae were grown at the indicated temperatures on L4440 control dsRNA-expressing bacterial strains and progeny were scored as viable larvae or dead embryos. (B) Screening procedure used to isolate and characterize suppressor genes. dhc-1(or195) mutant animals were qualitatively screened with the RNAi E. coli library to isolate suppressing genes. Embryonic viability was quantified using consistently positive E. coli clones; clones producing embryonic viability greater than 3-fold above background were kept for further analysis. We assayed the specificity of suppression by using ts mutants with defects unrelated to dynein. Finally, the localization of the suppressor proteins was determined with GFP fusion proteins.
Figure 3.
(A) Effect of reducing the function of 49 genes on embryonic viability in the dhc-1(or195) mutant. E. coli containing the L4440 control vector produced 1.6% viable progeny at 23 °C, while the suppressor genes produced the indicated percent viable progeny when reduced in function with RNAi.
(B) Deletion and point mutation double mutants recapitulate the suppression of conditional dhc-1 alleles observed using RNA interference. Embryonic viability of wild type, dhc-1, and dhc-1 suppressor gene double mutants is displayed. The dhc-1 unc-29 strain serves as the negative control for the dhc-1 unc-29 dylt-1 strain.
Figure 4.
Specificity of the Dynein HC Suppressors As Assayed with Multiple ts Loci.
Increasing red brightness indicates greater embryonic viability (according to the scale at bottom). Fold suppression was calculated by dividing the percent viability observed with RNAi by the background viability observed with the L4440 control, for every RNAi experiment tested in the ts mutants. Protein descriptions are from Wormbase [29]. For the numbers of progeny produced and percent viability calculation, see Table S1. Three dsRNA-producing plasmids express an RNA molecule that overlaps two different genes; this is indicated by a forward slash between the gene names. Two different gene classes are observed: specific genes only suppress multiple dhc-1 mutant alleles while nonspecific genes suppress lit-1 and/or spn-4 mutants. One suppressor gene, Y40B1B.5, suppressed only one dhc-1 allele. The phenotypes of the genes (in a wild-type or rrf-3 background) are listed in the Phenotype column (data is from mutant or RNAi studies and collected from [29]). MRP, mitochondrial ribosomal protein, Mito, mitochondrial.
Figure 5.
Single Members of C. elegans Dynein Subunit Families Restore Embryonic Viability When Reduced in Function in dhc-1ts Mutants
(A) Genes encoding putative dynein subunits were tested for suppression in the three dhc-1 backgrounds, as described in Figure 4. CHE-3 is a cytoplasmic dynein 2 HC required for retrograde transport in sensory neurons and B0365.7 encodes a highly diverged HC fragment of unknown function. The other genes are listed according to their subunit family [3].
(B) RNAi targeted towards the suppressing ACs results in variable embryonic lethality in wild-type worms. Wild-type L4 larvae were transferred to plates containing bacteria expressing the indicated dsRNAs. The observed percentage of hatching embryos is indicated.
(C) Reduction of dhc-1 or dyci-1 function with RNAi in L4 larvae does not suppress the embryonic lethality of dhc-1(or195) embryos, but dylt-1(RNAi) does suppress embryonic lethality.
(D) Codepletion of the dynein HC and dynein ACs does not result in suppression of embryonic lethality. L4 wild-type larvae were transferred to plates seeded with equal amounts of bacteria expressing dhc-1 dsRNA and dsRNA for the second gene listed. These results correspond to the first 24 h after transferring the worms to the RNAi plates; on the second day only dead embryos were produced with all of the conditions.
(E) Model of the cytoplasmic dynein protein with suppressor subunits and the DYCI-1 intermediate chain labeled [adapted from reference 33].
Figure 6.
Localization of GFP-Tagged Dynein Suppressor Proteins in Wild-Type Embryos
Insets show 2× magnification of meiotic spindles.
(A–D) DYLT-1: oocyte nuclear envelopes (A), meiotic spindle poles (B), nuclear envelopes prior to mitosis (C), and centrosomes and mitotic spindle poles (D). Mitotic localization is most robust in embryos beyond the 12-cell stage; weaker localization to spindle poles was detectable in one- and two-cell stage embryos (see Figure 7).
(E–H) DYRB-1: similar to DYLT-1, except weaker subsequent to meiosis.
(I–L) K04F10.3: endoplasmic reticulum-like; meiotic spindle poles (I) and pericentrosomal during mitosis (I–L).
(M–P) NPP-22: nuclear envelope and pericentrosomal (also endoplasmic reticulum-like, except for absence of meiotic spindle localization).
(Q–T) EFA-6.c: nonpolarized cell cortex in oocytes and early one-cell zygotes (Q–R), anterior cell cortex in one-cell embryos subsequent to pseudocleavage (S) and present at the interface of the AB and P1 cells (T), undetectable by the four-cell stage (unpublished data).
(U–X) MOP-25.2: midbody after cytokinesis (arrows indicate the spot of localization) and weak localization to spindle poles (unpublished data).
(Y-B′) F10E7.8: pronuclear and nuclear (cytoplasmic signal is at least partially due to endogenous autofluorescence in this weakly expressing line).
(C′–F′) STAR-2 (a nonspecific suppressor gene): apparent localization to germline P-granules.
Figure 7.
Time-Lapse Images of GFP::DYRB-1 and GFP::DYLT-1 in Wild-Type and dhc-1(or195) Mutant Embryos
Images represent pronuclear migration to telophase in the first embryonic cell cycle. Identical conditions were used during microscopy and image manipulation so that images are directly comparable.
(A) Faint localization of GFP::DYRB-1 to the spindle in a wild-type embryo (top row of images), bright labeling of centrosomes and spindle poles in an embryo from a dhc-1(or195) heterozygous mutant worm grown at 15 °C (middle image sequence), and very strong labeling of centrosomes and this monopolar spindle in an embryo from a dhc-1(or195) homozygous mutant worm shifted to 26 °C for three h (bottom row).
(B) Faint localization of GFP::DYLT-1 to the spindle in a wild-type embryo (top row of images), bright labeling of centrosomes and spindle poles in an embryo from a dhc-1(or195) heterozygous mutant worm grown at 15 °C (middle image sequence), and very strong labeling of centrosomes and spindle poles in an embryo from a dhc-1(or195) homozygous mutant worm shifted to 26 °C for five h (bottom row).
Figure 8.
Characterization of Genes Encoding the dylt-1 and dyrb-1 Dynein Light Chains
(A) Alignment of DYLT-1 and DYRB-1 with the homologous human and Drosophila proteins. The following proteins were used to make the alignments: Dlc90F, DYNLT3, DYNLRB1, and robl.
(B) The genomic loci for dylt-1 and dyrb-1 are shown along with the locations of the deletions. Blue boxes represent exons, grey boxes are untranslated regions, and the deleted DNA is shown in red. Scale bar, in base pairs, is shown.
(C) Embryonic viability observed for the dynein heavy chain mutant shifted to 23 °C, and for wild type and the dynein light chain deletion mutants cultured at room temperature. We counted the following numbers of progeny for each strain: N2: 831, dhc-1(or195): 1539, dylt-1(ok417): 404, dyrb-1(tm2645): 273, and dyrb-1(2645) +GFP::dyrb-1: 366.
(D) Time-lapse Nomarski images for wild-type, dylt-1, and dyrb-1 embryos grown at room temperature. The dylt-1 embryo displays no obvious defects, but the dyrb-1 embryo exhibits failures in female meiotic divisions and pronuclear migration, and progression through mitosis is delayed. The time (in seconds) relative to pronuclear meeting is displayed in each panel. Red dots denote positions of spindle poles and the arrowheads point to extra maternal pronuclei in the dyrb-1 embryo.
Table 1.
Conserved and Specific dhc-1 Suppressor Genes