LET-711, the Caenorhabditis elegans NOT1 ortholog, is required for spindle positioning and regulation of microtubule length in embryos

Abstract

Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.

Figure 1.

let-711 embryos have defects in centration and nuclear rotation. Time-lapse DIC microscopy series of wild-type (A) and let-711 mutant embryos (B). Each series consists of 1-cell embryos at pronuclear meeting, nuclear envelope breakdown, anaphase, telophase, and 2-cell prophase. Time is indicated (minutes:seconds) for each image, relative to nuclear envelope breakdown equals 0:00. Arrowheads indicate the position of centrosomes. Scale bar, 10 μm.

Figure 2.

let-711 embryos have robust asters and expanded centrosome morphology. Epifluorescence images of wild-type (A–D) and let-711 mutant embryos (E–H) stained for α-tubulin. One-cell embryos at prophase (A and E), metaphase (B and F), anaphase (C and G), and telophase (D and H) are shown. Although most let-711 embryos at metaphase and anaphase had more severely mispositioned spindles than these examples, these more clearly illustrate the altered centrosome size using a single focal plane image. The telophase embryo illustrates the abnormal extension of centrosomes along the cortex that occurs in anaphase and telophase let-711 mutants in which the spindle is posteriorly positioned. Note that exposure and gain settings were used that allowed visualization of the hollow center of centrosomes, and thus astral microtubules are less distinct compared with the images in Figure 3. Scale bar, 10 μm.

Figure 3.

Microtubules are longer in let-711 embryos. Confocal images of wild-type (A), let-711 (B), zyg-9(RNAi) (C), and let-711;zyg-9(RNAi) (D) embryos stained for α-tubulin. Each image is a projection of two optical sections through the middle of the centrosomes; exposure and gain settings were adjusted to allow visualization of microtubules extending to the cortex. Scale bar, 10 μm.

Figure 4.

Microtubules are more cold-stable in let-711 mutant embryos. Confocal images of wild-type (A–D), let-711 (E–H), and zyg-9(RNAi) (I–L) embryos mutant embryos after cold treatment and regrowth. Scale bar, 10 μm.

Figure 5.

Simultaneous reduction of let-711 and zyg-9 restores centration and rotation. Time-lapse videomicroscopy series of wild-type and mutant embryos during first division. A wild-type embryo (A) is shown from pronuclear meeting through early anaphase; a let-711 embryo (B), a zyg-9(RNAi) embryo (C), and a let-711;zyg-9(RNAi) (D) embryo at comparable stages are shown. Time is indicated (minutes:seconds) for each image, relative to nuclear envelope breakdown equals 0:00. Arrowheads indicate the position of centrosomes. Scale bar, 10 μm.

Figure 6.

Localization of ZYG-9 in wild-type and mutant embryos. Confocal images of embryos stained for ZYG-9 (A–D) or AIR-1 (E and F) or epifluorescence images of γ-tubulin:GFP expressing embryos (G and H). Genotypes are as shown; each pair of embryos is age matched (either late prophase or prometaphase). The positions of the centrosomes (white arrowheads) in zyg-9(RNAi) and let-711; zyg-9(RNAi) were determined from double-staining for tubulin. Note that only one centrosome is fully in the focal plane for A and C. For each protein analyzed, the images were taken at the same gain and exposure settings and thus intensities can be compared. Scale bar, 10 μm.

Figure 7.

Quantification of staining intensities of ZYG-9, AIR-1, and γ-tubulin::GFP. Each plot on the left shows the cytoplasmic and centrosomal staining intensities for a single antigen in wild-type and let-711 embryos at pronuclear meeting (Meet), nuclear envelope breakdown/prometaphase (NEB), metaphase (Meta) and anaphase (Ana). Values are mean intensities; error bars indicate the SEM at the 95% confidence level. Intensities for ZYG-9 and AIR-1 were determined from fixed samples: n = 6–22 centrosomes for each genotype at each stage (except n = 4 for let-711, ZYG-9 anaphase). Intensities for γ-tubulin were determined from living embryos expressing γ-tubulin::GFP (n = 2 centrosomes each from 7 let-711 and 5 wild-type embryos); the decrease in γ-tubulin::GFP intensities over time was due to bleaching. The ZYG-9 metaphase value is statistically different between let-711 and wild type at this confidence level, and calculation of the 90% confidence level gives significant differences for ZYG-9 during meeting and anaphase as well. All other values are not statistically different between let-711 and controls. For these measurements, the mean intensity of a circle of fixed size was used (see Materials and Methods for details) to compare centrosomes. Because this measure is proportional to the total intensity in a given area, this allows determination of whether a larger total amount is present or whether the wild-type amount is distributed in a larger area at let-711 centrosomes. This method includes some cytoplasmic signal, which could artificially lower the centrosomal value, especially for controls with smaller diameters of ZYG-9 staining. The right-hand panels show plots of the data where the cytoplasmic levels (Cyto) were subtracted from the centrosome levels (Cent). Using this measure, ZYG-9 intensities in let-711 are still significantly different from wild type (SEM, 95% confidence level) except at NEB.