SPD-3 is required for spindle alignment in Caenorhabditis elegans embryos and localizes to mitochondria

Abstract

During the development of multicellular organisms, cellular diversity is often achieved through asymmetric cell divisions that produce two daughter cells having different developmental potentials. Prior to an asymmetric cell division, cellular components segregate to opposite ends of the cell defining an axis of polarity. The mitotic spindle rotationally aligns along this axis of polarity, thereby ensuring that the cleavage plane is positioned such that segregated components end up in individual daughter cells. Here we report our characterization of a novel gene required for spindle alignment in Caenorhabditis elegans. During the first mitosis in spd-3(oj35) embryos the spindle failed to align along the anterior/posterior axis, leading to abnormal cleavage configurations. spd-3(oj35) embryos had additional defects reminiscent of dynein/dynactin loss-of-function possibly caused by the mislocalization of dynactin. Surprisingly, we found that SPD-3GFP localized to mitochondria. Consistent with this localization, spd-3(oj35) worms exhibited slow growth and increased ATP concentrations, which are phenotypes similar to those described for other mitochondrial mutants in C. elegans. To our knowledge, SPD-3 is the first example of a link between mitochondria and spindle alignment in C. elegans.

Figure 1.—

spd-3 is a novel gene with putative transmembrane domains and predicted coiled coils. (A) spd-3 is equivalent to the novel gene H34C03.1. The oj35 allele contains a single-base-pair change in the third exon. The ok1817 deletion allele removes the 5′-UTR through exon 3 of the spd-3 gene. (B) The predicted gene product contains two putative transmembrane domains and predicted coiled coils. (C) The y-axis represents the probability of coiled-coil conformations in the SPD-3 protein. A weak coil prediction is destroyed in the oj35 allele (arrow).

Figure 2.—

spd-3(oj35) is defective in meiosis. In wild-type embryos, two female pronuclei are faithfully extruded as polar bodies (a, arrow), leaving a single female pronucleus in the zygote (a, asterisk). oj35 embryos are defective in meiosis, often failing to extrude polar bodies resulting in extra maternal pronuclei in the zygote (b and c, asterisks). Often, the polar bodies that are extruded are very large (b, arrow) compared to wild type. Bar, 10 μm.

Figure 3.—

spd-3(oj35) embryos are defective in mitotic spindle alignment. In the single-cell embryo, the centrosomes are associated with the paternal pronucleus designating the posterior of the embryo, while the maternal pronucleus is usually located in the anterior (a and g). The pronuclei migrate toward each other, meeting near the posterior (b and h, centrosomes are marked by asterisks). In wild-type embryos, the pronuclear/centrosome complex then rotates 90°, aligning the mitotic spindle along the anterior/posterior axis to coordinate with the axis of polarity (c, centrosomes marked by asterisks). This rotation fails in the oj35 embryo, resulting in a mitotic spindle aligned transverse to the anterior/posterior axis (i, centrosomes marked by asterisks). The cleavage plane bisects the spindle (d and j), resulting in a proper asymmetric division in wild type (e) but not in oj35 embryos (k). In wild-type embryos, astral microtubules interact with the cell cortex and appear to be captured and stabilized (f). In oj35 embryos, astral microtubules appear to grow along the cortex (l) and are excessive in length. Bar, 10 μm. Tubulin is visualized with α-tubulin n357 (Amersham Pharmacia Biotech).

Figure 4.—

spd-3(oj35) embryos have defects in dynactin localization. In wild-type embryos, DNC-1 localizes to the centrosomes, the mitotic spindle, and the plus ends of astral microtubules (n = 12). oj35 embryos exhibit an additional, anomalous localization of DNC-1 to posterior structures resembling P granules (n = 12). DNC-1 signal is absent in dnc-1 RNAi embryos. DNC-2∷GFP in wild-type embryos is present throughout the cytoplasm, with enrichments in the pericentriolar region and along the mitotic spindle (n = 5). This enrichment is decreased in oj35 embryos, while the cytoplasmic signal is significantly increased (n = 6). DNC-2∷GFP images portrayed are snapshots taken from live imaging, while tubulin (n357) and DNC-1 antibodies were used with fixed embryos. Bar, 10 μm.

Figure 5.—

SPD-3∷GFP colocalizes with mitochondria. SPD-3∷GFP has a similar localization pattern throughout the cell cycle (top, interphase; bottom, mitosis) and colocalizes with MitoTracker CMXRosetta. Bar, 10 μm. There are no gross abnormalities in mitochondrial morphology or localization in oj35 embryos as visualized with Rhodamine 6G (Sigma) (n = 3).