Transformation Rescue

let-354(h934) dpy-5 unc-13/gaDp1 adult hermaphrodites were injected with a mixture of 2 ng/μl cosmid T21E12 DNA (linearized with SacII), 2 ng/μl cosmid ZK973 DNA (linearized with NotI), and 100 ng/μl pRF4 rol-6(su1006) DNA (linearized with SmaI). Cosmid DNA was prepared using the QIAGEN (Valencia, CA) maxiprep protocol, and plasmid DNA was prepared using the QIAGEN miniprep protocol. Two independent F1 lines of rescued let-354(h934) dpy-5 unc-13 worms were obtained. Those lines were Dpy Unc, which obscured the Rol-6 phenotype caused by the presence of a rol-6(su1006)-bearing extrachromosomal array, so the presence of an array was confirmed in two ways. Mating of rescued Dpy Unc hermaphrodites to N2 males resulted in the production of some Rol-6 outcross progeny. Rescued Dpy Unc hermaphrodites fixed and stained with Hoechst 33342 contained in their oocytes six bivalents and an extrachromosomal DNA mass.

Sequencing

Genomic DNA was extracted from let-354/unc-11 dpy-5 heterozygous hermaphrodites bearing each of three dominant ts alleles of let-354 (ct42, ct76, and ct77) by using a standard phenol/chloroform extraction method (Maniatis et al., 1982) followed by column purification using QIAGEN Genomic 100/G columns. DNA segments spanning the predicted coding region (~15 kb) of dhc-1 were polymerase chain reaction (PCR) amplified and sequenced by SeqWright DNA Sequencing (Houston, TX). We confirmed mutant changes by targeted sequencing of specific portions of PCR-amplified dhc-1 DNA from five to 10 heterozygous worms and from five to 10 N2 worms, by using an ABI 3700 prism automated fluorescent sequencer (Indiana University Molecular Biology Institute, Bloomington, IN).

Yeast Transformation

Yeast strains YEF473a and YEF473a dhc1Δ::HIS3 and plasmid CYDHC/GEM containing the full-length yeast dynein heavy chain gene DHC1 (a gift from K. Bloom, University of North Carolina, Chapel Hill, NC) were used to test whether the P-loop mutation found in let-354(ct77) and (ct42) would cause a dominant ts dhc1 mutant phenotype in yeast. The mutation was engineered into the yeast DHC1 gene by using PCR-based site-directed mutagenesis. A 1-kb fragment containing the third P-loop of DHC1 was cut from CYDHC/GEM, PCR amplified using primers 5′ GCG AAG AGT TCC GGA GTG CAT TAT TCA TAA TCA TTG TTT TAC CAG ATT CAG GTG GCC CAC AAA GG 3′ and 5′ ATA TGG TGG GTG CTT CTT CG 3′, and reintroduced back into the CYDHC/GEM plasmid to generate CYDHC^*/GEM. To allow expression in yeast, the CYDHC^* insert was transferred into pRS316 plasmid (a gift from A. Bender, Indiana University) and introduced into yeast by using either electroporation (protocol adapted from D. Gottschling, Fred Hutchinson Cancer Research Center, Seattle, WA; http://iprotocol.mit.edu/protocol/100.htm) or lithium acetate (Walhout and Vidal, 2001). Transformed yeast were selectively grown on URA^- plates, and then grown for 1–4 h at 16 or 30°C in YPD medium (BD Biosciences Clontech, Palo Alto, CA). Cells were fixed by standard methanol/acetone fixation, stained with 4,6-diamidino-2-phenylindole (DAPI), and scored for one nucleus or two or more nuclei on a Zeiss (Carl Zeiss, Thornwood, NY) Axioskop by using differential interference contrast (DIC) and fluorescence microscopy. To confirm the sequence change, plasmid was isolated from transformed yeast and the relevant region was sequenced.

Analysis of Embryonic Development by Time-Lapse DIC and Confocal Fluorescence Microscopy

Embryos were mounted in M9 buffer (Brenner, 1974) on 2% agarose pads containing a fine-Teflon PTFE-insulated probe connected to a Cole-Parmer Instrument (Vernon Hills, IL) Digisense thermocouple thermometer to monitor temperature and covered with a coverslip. DIC images were obtained on a Zeiss Axioplan microscope with a Hamamatsu C2400 camera and video controller with an Argus-10 image processor (Hamamatsu City, Japan). Images were captured every 3 s by using NIH Image (version 1.62b7, developed by Wayne Rasband, National Institutes of Health, Bethesda, MD and available at http://rsb.info.nih.gov/nih-image). Fluorescence images were obtained on a Nikon Optiphot microscope with a Bio-Rad MRC600 scanning confocal system (Hercules, CA) and collected as a time series by using MRC600 software. Images were captured every 6 s for green fluorescent protein (GFP)::histone time-lapse movies or every 10 s for GFP::tubulin time-lapse movies. Stacks of images were manipulated to generate figures in NIH Image.

Rapid temperature-upshift experiments were performed using a temperature-control microscope stage (Supplemental Figure s1) in a microscope room held at 16°C. Embryos were prepared and mounted on slides with agar pads at 16°C, placed on the microscope stage, observed, and shifted to 25°C just before an event of interest. Based on the read-out from a thermocouple probe mounted in the agar pad immediately adjacent to embryos, a shift from 16 to 25°C took 24–40 s.

Antibodies and Immunofluorescence Microscopy

To generate antisera against C. elegans DHC-1, a 17-amino acid peptide, MDSGNESSIIQPPNLKC, corresponding to the N-terminal 16 amino acids plus an additional cysteine, was synthesized and conjugated to maleimide-activated keyhole limpet hemocyanin (Research Genetics, Huntsville, AL). The peptide was used to immunize rabbits (Cocalico Biologicals, Reamstown, PA). Affinity-purified antibodies were prepared by passage over a column of DHC-1 peptide coupled to Sulfolink coupling gel (Pierce Chemical, Rockford, IL). Bound antibodies were eluted with 100 mM glycine, pH 2.5, dialyzed against phosphate-buffered saline, and concentrated (5-fold). Figure 1 demonstrates that the antibody is specific for DHC-1.

Open in a separate window

Figure 1.

Test of anti-DHC-1 antibody specificity. Rabbit antibodies were raised against the amino terminal 16 amino acids of DHC-1, affinity purified, and used to stain dhc-1(+) glh-1(gk100) embryos and dhc-1(RNAi) embryos. Both types of embryos were dissected, fixed, and stained together on the same slide to ensure identical treatment. glh-1 mutant embryos lack detectable staining by rat anti-PGL-3 (a marker for P granules) (Meyer and Strome, unpublished data), allowing control dhc-1(+) glh-1 embryos to be distinguished from dhc-1(RNAi) embryos. (A and B) Control 2-cell embryo containing DHC-1 (A) and lacking PGL-3 (B). (C and D) Nearby 1-cell RNAi embryo lacking detectable DHC-1 (C) and containing PGL-3 (D). Images are projections of Z-series from a spinning disk confocal fluorescence microscope. Embryos in this and subsequent figures are oriented with posterior to the right. Bar, 10 μm.

For immunofluorescent staining of embryos, adult hermaphrodites were cut, fixed, and stained as described previously (Strome and Wood, 1983). The primary antibodies used were anti-DHC-1 at 1:200, anti-DNC-1 at 1:200 (a gift from H. Zhang and J. White, University of Wisconsin), and mouse 4A1 anti-α-tubulin at 1:50 (a gift from M. Fuller, Stanford University School of Medicine, Stanford, CA; Piperno and Fuller, 1985). The secondary antibodies used were Alexa488 goat anti-mouse and Alexa594 goat anti-rabbit (Molecular Probes, Eugene, OR). Embryos were counterstained with DAPI to visualize DNA. Anti-DHC-1 and anti-tubulin images were collected as Z-series of 1.0-μm slices on a Bio-Rad MRC600 microscope, or on a PerkinElmer Ultraview LCI30E spinning disk confocal microscope by using Ultraview software (PerkinElmer Life and Analytical Sciences, Boston, MA). DNA images were collected with a Hamamatsu ORCA-ER digital camera on a Nikon (Tokyo, Japan) Eclipse E800 microscope with MetaMorph (Universal Imaging, Downingtown, PA) software. Images were processed using Photoshop 7.0 (Adobe Systems, Palo Alto, CA).

An Engineered Dominant Temperature-sensitive DHC1 in Budding Yeast

To determine whether the ct42-ct77 P-loop mutation has a dominant ts effect on dynein in other species, the equivalent G-to-E codon change was tested in the DHC1 gene of Saccharomyces cerevisiae. A deletion-disruption mutation of DHC1 (dhc1Δ) impairs translocation of the daughter nucleus to the bud-cell at the end of mitosis. It is not lethal but causes a significant percentage of mutant cells to carry two or more nuclei (Eshel et al., 1993; Li et al., 1993). Yeast cells were transformed with either a mutant dhc1(G-E) gene or a wild-type DHC1 gene in a low copy number plasmid. The effects of each on dynein function were tested in wild-type and dhc1Δ backgrounds by counting multinucleate cells after growth at 16 or 30°C (Table 1; statistical analysis is in Supplemental Table s1). At both temperatures, the wild-type transgene had little effect on wild-type cells and substantially reduced the multinucleate phenotype of dhc1Δ cells. In contrast, the dhc1(G-E) transgene in the wild-type background at 30°C caused multinucleate cells at a frequency equivalent to that observed in dhc1Δ (~17%). That effect was significantly reduced at 16°C but was not eliminated (3.2%). Interestingly, in the dhc1Δ background at 30°C, the dhc1(G-E) transgene caused a modest but significant increase in the percentage of multinucleate cells (23%) relative to that observed with dhc1Δ alone (17%), suggesting that the mutant motor has a slightly inhibitory effect on the dynein-independent mechanism for nuclear migration. These results demonstrate that the C. elegans dhc-1(ct42-ct77) G-E change, when placed in DHC of an evolutionarily distant organism, causes a dominant ts phenotype. This confirms in vivo that the third P-loop is essential for proper dynein function (Silvanovich et al., 2003; Reck-Peterson and Vale, 2004) and highlights the possibility that equivalent mutations can be used to create dominant ts versions of a variety of other P-loop proteins.

Processes Influenced by Inhibition of DHC-1

To assess which events in early embryogenesis are impaired by dhc-1 mutations, we compared wild-type, dhc-1(RNAi), and dhc-1 ts mutant embryos by time-lapse DIC microscopy (Figure 4, Videos 1–6, and Table 2). The progression of events in wild-type is illustrated in Figure 4A and in Video 1 (supplemental data). As described previously by Gönczy et al. (1999), RNAi depletion of DHC-1 from the maternal germline results in severe defects in 1-cell embryos (Figure 4B, Video 2, and Table 2). In embryos produced 24 h after dsRNA injection, the sperm pronucleus did not leave the posterior cortex, the oocyte pronucleus did not show a fast phase of migration, and hence pronuclei failed to meet before nuclear envelope breakdown (NEB) (Figure 4B, a–d). A bipolar spindle was not evident, and cytokinesis did not occur. Instead, RNAi embryos underwent dramatic cortical blebbing, and multiple nuclei formed in the posterior cytoplasm during telophase (Figure 4B, f and g). By 36 h after RNA injection, existing oocytes were aberrant and the gonad stopped producing new oocytes. This indicates that DHC-1 serves essential roles in the maternal germline and raises the possibility that defects in germline physiology contribute nonspecifically to the phenotypes observed in dhc-1(RNAi) embryos.

Open in a separate window

Figure 4.

Early development of wild-type, dhc-1(RNAi), and dhc-1 mutant embryos. Each image is a single frame from a time-lapse movie of a live embryo captured using DIC microscopy. Arrowheads indicate centrosomes, and arrows point to micronuclei. Oocyte pronuclei are marked with “o” and sperm pronuclei with “s” in the first frame of each series. In all cases (except C), embryos were dissected from hermaphrodites 24 h after a shift from 16 to 25°C, initiated when the mothers were L4 larvae. (A and Video 1) Wild-type embryo. (A, a and b) Pronuclear migration and meeting. (A, c) Rotation of the centrosome/nucleus complex. (A, d) Anaphase with a shift of the posterior spindle pole toward the posterior cortex. (A, e) Posterior spindle pole flattening. (A, g) P₁ centrosome axis alignment with the AP axis. (B and Video 2) dhc-1(RNAi) embryo. (B, a–c) Pronuclei failed to migrate. (B, d and e) Bipolar spindle assembly failed. (B, f and g) Chromosome separation failure produced multiple micronuclei. (B, e–g) Cytokinesis failed, although numerous transient membrane invaginations formed. (C and Video 3) Embryo from a dhc-1(ct76)/+ hermaphrodite raised at permissive temperature (16°C). All events resembled wild type, except that the posterior spindle pole did not flatten (C, e). (D and Video 4) Embryo from a dhc-1(ct76)/+ hermaphrodite. (D, a and b) Pronuclei met more centrally than in wild type. (D, c–e) Centrosome separation failed, and a bipolar spindle failed to form. (D, d and e) Posterior shift of the monopolar spindle. (D, f and g) Chromosome separation and cytokinesis failed. (E and Video 5) Embryo from a dhc-1(or195) hermaphrodite. (E, a and b) A minority case in which pronuclei met at a normal posterior position. (E, b–d) Centrosomes separated, but the centrosome axis failed to rotate and a stunted spindle formed on the transverse axis. (E, d and e) Posterior shift of the spindle. (E, e–g) Chromosome separation and cytokinesis failed. (F and Video 6) Embryo from a dhc-1(ct42)/+ hermaphrodite. (F, a) Occasionally two sperm pronuclei were observed in mutant embryos. (F, b) The pronuclei met near the center. (F, c and d) Centrosomes separated, but the centrosome axis did not rotate fully onto the AP axis. (F, d–f) A small spindle formed and moved toward the posterior cortex, chromosomes separated, and cytokinesis occurred. (F, g) The centrosome axis failed to rotate in P₁, and cytokinesis failed in AB. Bar, 10 μm.

Table 2.

Summary of defects observed in dhc-1 (RNAi) and dhc-1 mutant embryos.

Open in a separate window

Our analysis of events in dhc-1 mutant embryos was performed with six ts alleles: the dominant ct42, ct76, ct77, and sb42, plus the recessive or195 and h482 (Howell and Rose, 1990; Mains et al., 1990; Mitenko et al., 1997; Hamill et al., 2002). The 19 nonconditional recessive alleles result in larval lethality: homozygous progeny from heterozygous mothers undergo apparently normal embryo development, hatch, and arrest at a midlarval stage (Howell and Rose 1990). The different ts alleles of dhc-1 create different pools of DHC-1 dimer. Early embryos from mothers homozygous for a recessive ts allele contain fully mutant dimers, whereas early embryos from mothers heterozygous for a dominant ts allele are presumed to contain a mix of fully mutant dimers, fully wild-type dimers, and heterodimers with one mutant and one wild-type subunit. To simplify wording below, embryos derived from both types of mothers will be referred to as dhc-1 ts mutant embryos. dhc-1 ts embryos displayed low levels of embryonic lethality at permissive temperature (16°C) and 100% lethality at elevated temperature: 18.4°C in ct76 and 20°C in ct77-ct42 (Supplemental Table s2).

Our initial studies of the conditional mutants involved shifting mutant mothers (as L4 larvae) to a restrictive temperature (25°C) for 24 h and then analyzing their early embryos by time-lapse DIC microscopy at 25°C. The embryonic processes affected by this temperature regime were similar for all the ts alleles, but the severities of defects varied, indicating an allelic series as follows: strongest— ct76, or195, ct77-ct42, sb42, h482—weakest. To test the severity of a strong allele over a null, we examined embryos from worms carrying the recessive allele or195 over a deficiency of the region, hDf6 (Table 2). The phenotypes of embryos from dhc-1(or195)/hDf6 mutants at 25°C were slightly more severe than those of dhc-1(or195)/dhc-1(or195) and similar to those of dhc-1(ct76). This suggests that although neither or195 nor ct76 is a null allele, they do cause a nearly complete loss of function. We saw no evidence of a neomorphic gain of function here or in other tests.

Pronuclear Migration. Although dhc-1 ts mutant embryos at 25°C showed some defects in meiosis (our unpublished data), normal pronuclei usually formed (Figure 4, D and E, and Videos 4 and 5). In ct76 embryos (Figure 4D and Video 4), the female pronucleus seemed to migrate slowly and met the advancing male pronucleus near the midpoint of the embryo rather than in the posterior half (Figure 4D, b). To quantify this effect, the position of the center of the female pronucleus was measured as a function of time. In wild-type embryos (n = 10), an initial “slow phase” of migration (0.035 ± 0.004 μm/s) moved the female pronucleus to ~37% of embryo length (EL; anterior pole is 0%, posterior pole is 100%); a subsequent “fast phase” (0.26 ± 0.03 μm/s) moved it to the sperm pronucleus at 69 ± 4% EL. In ct76 embryos (n = 10), the slow phase was normal (0.035 ± 0.004 μm/s), but the fast phase was only 0.13 ± 0.07 μm/s and the pronuclei met at 58 ± 3% EL. Thus, the ct76 lesion reduces but does not eliminate the fast phase of migration. Other dhc-1 ts alleles also affect pronuclear migration, causing the pronuclei to meet more centrally than in wild type (Supplemental Table s3)

Centrosome Movements and Spindle Formation. It was evident that restrictive temperature also affected the behavior of centrosomes in dhc-1 ts mutant embryos. In wild type (Figure 4A and Video 1), newly duplicated centrosomes on the male pronucleus move away from one another, separating to opposite sides, usually producing a centrosome-centrosome axis that lies transverse to the AP axis of the embryo. As male and female pronuclei meet, the centrosome/nucleus complex rotates 90° to align the centrosomes on the AP axis (Figure 4A, b–d). In embryos from dhc-1 ts mutant mothers cultured at permissive temperature, these processes looked normal (e.g., Figure 4C and Video 3). Of 16 dhc-1(ct76) embryos grown at restrictive temperature, centrosome separation failed completely in five, leading to formation of a monopolar first spindle (Figures ​(Figures4D,4D, b–d, and ​and5A).5A). In the remaining 11 embryos, centrosome separation was delayed until after the pronuclei met. In some cases, delayed separation was transverse to the AP axis, and in other cases it was along the AP axis. Rotation of the centrosome/nucleus complex failed or was partial in most embryos with transversely separated centrosomes (Table 2). In all cases with full separation, centrosomes collapsed toward one another after NEB, and abnormally small spindles formed. In or195, ct77, and ct42 embryos, initial centrosome separation was successful, centrosomes usually collapsed toward one another after NEB, and small spindles formed. Rotation onto the AP axis was often defective (Figure 4E, d and F, d; Videos 5 and 6; and Table 2), especially in or195, which is the strongest of the three alleles. Overall, our analysis of dhc-1 ts mutant and RNAi embryos as well as the RNAi studies of Gönczy et al. (1999) agree that loss of cytoplasmic DHC function inhibits centrosome separation, causes separated centrosomes to collapse toward one another after NEB, and inhibits rotation of the centrosome axis into alignment with the AP axis.

Open in a separate window

Figure 5.

Rotation of centrosomes fails in dhc-1 ts mutant embryos upshifted just before the rotation period. (A) Time-lapse confocal fluorescence images of an embryo from a dhc-1(ct76)/+ hermaphrodite expressing GFP::β-tubulin. Embryos generated at 25°C (Figure 4D) or shifted to 25°C early, in this case at the beginning of pronuclear migration (a), fail in centrosome separation (b–h). (B and Video 8) Time-lapse DIC microscopy images of a dhc-1(or195) embryo upshifted from 16 to 25°C after centrosome separation and pronuclear migration (b). The centrosome/nucleus complex failed to rotate from the transverse onto the AP axis, and after NEB the mitotic spindle formed transverse to the AP axis (c and d, arrowheads point to centrosomes in c and spindle poles in d) (see Figure 4, A and C, for normal rotation). Subsequent movement of one spindle pole toward the posterior cortex during anaphase eventually resulted in spindle orientation and position that resembled wild type (e and f). Chromosome segregation and cytokinesis occurred but were defective, as evidenced by the aberrant cleavage furrow (g) and the double nucleus in AB (h, arrow). (C and Video 9) Time-lapse confocal images of a two-cell embryo from a dhc-1(ct76)/+; GFP::β-tubulin hermaphrodite. The embryo was upshifted after the centrosomes in P₁ (b, arrowheads) had begun to separate. The centrosome/nucleus complex failed to rotate (c and d), and a mitotic spindle formed transverse to the AP axis (d–g). Bar, 10 μm.

Anaphase. Time-lapse DIC movies revealed that anaphase chromosome separation failed in the most severe dhc-1 ts mutants (Figure 4, D and E, and Table 2). In wild type (Figure 4A and Video 1), anaphase consists of spindle elongation (anaphase B) with no chromosome-to-pole movement (anaphase A) (Oegema et al., 2001). The anterior pole moves little, whereas the posterior pole moves toward the posterior cortex and oscillates dramatically from side to side, rocking the spindle. The posterior centrosome subsequently flattens, whereas the anterior centrosome remains spherical. In embryos from dhc-1 ts mutant mothers held at permissive temperature, these processes looked normal, except that posterior centrosome rocking and flattening did not occur in the five strongest ts alleles (Videos 3 and 7; our unpublished data). At restrictive temperature in severe ts mutants (ct76 and or195), stunted bipolar spindles rarely elongated and chromosomes did not separate (Figure 4, D and E; Videos 4 and 5; and Table 2). Notably however, during anaphase the monopolar or small bipolar spindles did migrate toward the posterior cortex, leading to formation of micronuclei in the posterior hemisphere. Subsequently, a cleavage furrow usually formed, but cytokinesis was not successful. Subsequent cell cycles continued and aberrant mitosis/cytokinesis resulted in multinucleate, sometimes multicellular, embryos. The less severe ts mutants often completed the first division cycle successfully and then failed in later cycles (Figure 4F, Video 6, and Table 2).

To investigate whether DHC-1 contributes to the posterior spindle shift that ensures an asymmetric P₀ division in normal embryos, we measured changes in the position of the posterior centrosome as a function of time (Supplemental Table s3). After pronuclei met and moved to the center of the embryo, the position of the point of contact between the two pronuclei (the approximate position of both centrosomes) was not distinguishable in wild-type versus dhc-1 mutant embryos (Supplemental Table s3). During the ensuing mitosis in wild type, the posterior centrosome moved at a net rate of 0.067 ± 0.005 μm/s to a position 9.9 ± 0.6 μm from the posterior cortex. In dhc-1 ts mutant embryos, that rate was modestly reduced (0.056 ± 0.010 for ct76; 0.048 ± 0.013 for or195; Supplemental Table s3), but the final position of the posterior centrosome was similar to wild type. Thus, either residual DHC-1 function in dhc-1 ts mutant embryos is sufficient for the posterior spindle shift, or DHC-1 is not the primary motor for that movement.

Fast Temperature-Shift Analysis of DHC-1 Function in Rotation of the Centrosome/Nucleus Complex

Although the preceding tests of DHC-1 function were informative, the time delay between initiation of DHC-1 inhibition and analysis of events in embryos was 24 h. A process of interest might be influenced indirectly by defects in processes that preceded it. As noted above, RNAi depletion of DHC-1 can compromise the maternal germline sufficiently that it stops producing oocytes. The background physiology of embryos analyzed before that shutdown is probably abnormal to some degree. A more obvious problem is that spindle assembly defects preclude meaningful assessment of dynein's involvement in chromosome congression and segregation. In hopes of circumventing these problems, we tested how quickly phenotypes occurred in dhc-1 ts mutants after a temperature upshift. To allow direct observation during a temperature shift, a temperature-controlled microscope stage was built (Supplemental Figure s1). With two solid-state heat pumps controlling the slide temperature, it was possible to drive embryos from 16 to 25°C in ~30 s during observation with an oil immersion objective. dhc-1 ts mutant embryos (ct76, or195, and ct77-ct42) showed mutant phenotypes (e.g., cortical blebbing and spindle collapse) within 60 s of initiation of such an upshift. In additional tests of ct76, a fast upshift to 25°C that prevented spindle assembly and cytokinesis in P₀ followed by a downshift to 16°C allowed mitosis and cytokinesis to occur during the second cell cycle. Thus, with at least one ts allele, thermal inhibition of DHC-1 function could be reversed.

With the thermal stage, we first addressed the question of whether DHC-1 has a direct role in rotation of the centrosome/nucleus complex in the first two cell cycles. In all dhc-1 ts mutant embryos observed at 16°C, centrosomes separated normally and then established a mitotic spindle along the AP axis (n = 11) (Figure 4C and Videos 3 and 7). In ct76 embryos that showed normal transverse centrosome separation (n = 9), an upshift at the moment of pronuclear meeting prevented rotation of the centrosome axis onto the AP axis (our unpublished data). In or195 embryos (n = 10), an upshift at pronuclear meeting prevented rotation in eight cases (Figure 5B, d, and Video 8). In the other two cases, rotation was partial (~45°). Transverse spindles in or195 embryos eventually did rotate onto the AP axis, when one or the other spindle pole was pulled toward the posterior cortex during anaphase (Figure 5B, e and f). These results show that inhibition of DHC-1 has a direct effect on rotation of the P₀ centrosome-centrosome axis, consistent with a mechanism in which dynein-mediated pulling forces on microtubules emanating from one or both centrosomes drive that rotation.

The influence of dynein on rotation of the centrosome axis also was studied in P₁. The smaller size of P₁ and variations in the geometry of centrosome separation in wild type made observation of centrosomes and the timing of upshifts more difficult with DIC microscopy. Furthermore, in both wild-type and ct76 embryos held at 16°C, P₁ centrosome separation sometimes occurred directly along the AP axis. To facilitate well timed upshifts immediately after transverse centrosome separation, we generated a ct76 strain expressing GFP::β-tubulin (Figure 5, A and C). When ct76 embryos were shifted to 25°C immediately after transverse P₁ centrosome separation, rotation always failed (n = 12) (Figure 5C, c–e, and Video 9). Because the first mitotic midbody is thought to provide a cue for rotation of the centrosome axis in P₁, it is noteworthy that the ct76 embryos described above had completed cytokinesis and had a normally positioned midbody before the temperature shift. These results, along with the transient accumulation of DHC-1 observed along the AB-P₁ boundary (Figure 2D), support the hypothesis of Skop and White that dynein-dynactin anchored to the anterior cortex of P₁ generates the force for centrosome axis rotation (Skop and White, 1998).

Insights into Functions of DHC-1 during C. elegans Early Embryonic Development

Spindle Alignment. Because the position and orientation of the mitotic spindle dictate the pattern of cell division and segregation of components to daughter cells (reviewed in White and Strome, 1996), alignment of the spindle is tightly regulated in many developing organisms. Studies in yeast, Drosophila, C. elegans, and mammalian cells have implicated dynein in spindle alignment (Eschel et al., 1993; Li et al., 1993; McGrail and Hays, 1997; Skop and White, 1998; Gönczy et al., 1999; O'Connell and Wang, 2000). In C. elegans, RNAi depletion of DNC-1 or DNC-2, two components of dynactin, or partial RNAi depletion of DHC-1 impaired the 90° rotation of the centrosome/nucleus complex that is required to correctly align the mitotic spindle in the P₀ and P₁ blastomeres (Skop and White, 1998; Gönczy et al., 1999). Because of the gradual depletion caused by RNAi, the possibility of indirect effects was not eliminated.

Our studies of fast-acting ts dhc-1 mutations and DHC-1 location provide strong support for the direct involvement of dynein in rotation: rotation did not occur in ts mutant embryos upshifted just before rotation in P₀ or P₁. Hyman and White (Hyman and White, 1987; Hyman, 1989) demonstrated that the rotation in P₁ is mediated by an interaction of astral microtubules with a site on the anterior cortex. We observed a transient accumulation of DHC-1 along the anterior cortex of P₁ during the period of rotation, similar to that observed for DNC-1 (Skop and White, 1998), consistent with the hypothesis that dynein/dynactin tethered to the anterior cortex captures and reels in microtubules emanating from one centrosome. By analogy to P₁, dynein anchored to the anterior cortex of P₀ may capture astral microtubules and pull on them to mediate rotation of the centrosome/nucleus complex in that cell as well. An alternative model for P₀, suggested by analysis of let-99 mutant embryos (Tsou et al., 2002), is that dynein-generated pulling forces are distributed around the entire cortex but are attenuated at posterior-lateral sites by LET-99 protein. Our results do not discriminate between the “localized dynein” and “broadly distributed dynein with localized attenuation” models. They might be distinguished via spatially restricted thermal inactivation of ts DHC-1 by irradiation of specific cortical regions with a heat-generating microbeam of light.

Chromosome Congression. Dynein has been identified as a kinetochore component in numerous cell types (Pfarr et al., 1990; Steuer et al., 1990; Starr et al., 1998; Sharp et al., 2000b), consistent with a role in mitotic chromosome movement. Previous experiments have produced conflicting views of dynein function during prometaphase. Inhibition of dynein in Drosophila and mammalian cells by a variety of methods, including genetic mutations, RNAi, antibody injection, and overexpression of dynamitin, in some cases caused chromosome congression defects and in other cases did not. In Drosophila, congression defects included a reduced rate of poleward chromosome movement during prometaphase (Savoian et al., 2000) and a failure of kinetochore alignment at metaphase (Sharp et al., 2000b); congression defects were not observed by Starr et al. (1998) or Goshima and Vale (2003). In mammalian systems, Echeverri et al. (1996) observed defects in chromosome alignment in COS-7 cells, whereas Howell et al. (2001) did not observe congression defects in PtK1 cells. In our experiments, DHC-1 inhibition at NEB or later in prometaphase consistently caused disordered metaphase plates, revealing that in C. elegans DHC-1 does contribute to chromosome congression. The congression defects may be due to impaired dynein-dependent force generation at the kinetochore or to altered spindle microtubule dynamics and organization.

Anaphase. Studies of dynein contributions to anaphase in other systems have often been complicated by the requirement for dynein in spindle assembly (Robinson et al., 1999) and have produced conflicting results. For example, injection of dynein inhibitors into Drosophila embryos disrupted both poleward chromosome movement (anaphase A) and spindle pole separation (anaphase B) (Sharp et al., 2000a,b). Also in Drosophila, mutations in zw10, which disrupt dynein localization to kinetochores but do not otherwise impair spindle structure, caused reduced rates of poleward chromosome movement, lagging chromosomes, and segregation defects (Starr et al., 1998; Savoian et al., 2000). In contrast, RNAi depletion of dynein from Drosophila S2 cells caused a delay in the metaphase-to-anaphase transition, but did not impair chromosome segregation (Goshima and Vale, 2003). Similarly, inhibition of dynein function in PtK1 cells affected spindle checkpoint inactivation but did not impair chromosome segregation during anaphase (Howell et al., 2001). Thus, whether dynein contributes directly to anaphase chromosome separation remains controversial.

Two aspects of anaphase in C. elegans are unusual and intriguing. First, chromosome segregation is accomplished solely by spindle pole separation (anaphase B) (Oegema et al., 2001). Thus, a role for dynein in anaphase A chromosome-to-pole movement is not an issue. Second, anaphase B spindle pole separation seems to be driven primarily by cortical pulling forces on astral microtubules (Grill et al., 2001) with little or no contribution from pushing forces by antiparallel microtubules in the spindle interzone. In fact, the C. elegans interzone may actually resist pole separation (Grill et al., 2001; Saunders and Saxton, unpublished data). The cortical pulling forces are stronger toward the posterior than toward the anterior (Grill et al., 2001, 2003). Thus, anaphase B is accomplished primarily by movement of the posterior spindle pole toward the posterior cortex. This movement also positions the spindle asymmetrically, leading to an unequal first division, which is critical for subsequent normal development.

Dynein, tethered at the posterior cortex, has been a prime candidate for the posterior cortical pulling motor. Our studies of spindle movements in upshifted dhc-1 ts mutant embryos do not support such a role for dynein. All upshifted dhc-1 embryos that we analyzed displayed robust posterior movement of the spindle during anaphase. Mutant embryos containing a bipolar spindle displayed posterior migration of one or both spindle poles. Monopolar spindles, too, underwent posterior movement. Furthermore, anaphase chromosome separation was achieved by ts mutant embryos upshifted after they had well-formed bipolar spindles. Thus, either dynein is not the posterior pulling motor, or adequate pulling forces can be generated by residual dynein function in the upshifted ts mutants. The accumulation of DHC-1 on centrosomes and spindle poles in ts mutant embryos suggests that mutant DHC-1 might be capable of translocating along microtubules to their minus ends and fail to release. So, mutant DHC-1 tethered to the cortex could retain the ability to capture and reel in astral microtubules during anaphase. Arguing against retention of the ability to generate cortical pulling forces, at least by the more severe ts mutants, is the fact that they are unable to pull on astral microtubules during rotation of the centrosome/nucleus complex.

In wild-type embryos, anaphase B movement of the posterior spindle pole is accompanied by dramatic lateral oscillations and followed by flattening of the centrosome. Gotta and Ahringer (2001) observed that all three events require G protein function, consistent with the general assumption that posterior-directed movement, oscillations, and flattening of the posterior spindle pole are mechanistically linked, perhaps by a single force-producing mechanism. Our analysis of ts dhc-1 mutants suggests otherwise. The five most severe dhc-1 ts mutations caused a marked reduction or complete absence of oscillations and centrosome flattening at both 16 and 25°C, but posterior-directed anaphase movement of the spindle occurred (e.g., compare Videos 3 and 7 with Video 1). Severson and Bowerman (2003) observed similar results in embryos partially depleted of DHC-1 by RNAi. This demonstrates that the lateral oscillations and flattening are not causally linked to posterior-directed spindle migration. This suggests different force-generating mechanisms; oscillations and centrosome flattening require normal DHC-1 activity, whereas the posterior spindle shift requires little or none.

We are especially grateful to Paul Mains, Danielle Hamill, Bruce Bowerman, and Ann Rose for sharing dhc-1 alleles and information with us. We thank Alan Bender, Kerry Bloom, Arshad Desai, Margaret Fuller, Pierre Gönczy, Tony Hyman, Yoji Kohara, Chris Malone, John White, and Haining Zhang for reagents; Adam Saunders for help with some strain constructions; Aaron Pilling for the NIH Image tracking macro; Lynda Delph for assistance with statistics; Jon Henry for building the temperature-controlled stage; and members of the Strome and Saxton laboratories for discussions and ideas. This research was supported by National Institutes of Health grants GM58811 (to W.M.S. and S. S.) and GM34059 (to S. S.)