Fig 3. Subcellular localization and molecular hierarchy of ATX-2. Immunostaining illustrates (A) a diffuse distribution of ATX-2 in the cytoplasm in one-cell (left) and two-cell (right) stage embryos, and (B) a reduced staining in
atx-2(
ne4297) embryo that exhibits cytokinesis failure. (C) Wild-type embryo co-stained for ATX-2 and SZY-20 illustrates that ATX-2 and SZY-20 partially coincide in the cytoplasm (S2B Fig). (D) Wild-type embryo stained for ATX-2 and GFP-PAB-1 illustrates that ATX-2 and PAB-1 partially coincide in the cytoplasm (S2C Fig). (E-I) Quantification (E) of cytoplasmic ATX-2 levels reveals that
szy-20(
bs52) embryos possess 50% (n = 22) of normal ATX-2 levels.
atx-2(
ne4297) embryos possess 18% (+- 9, n = 18) of cytoplasmicATX-2, while
zyg-1(
it25) embryos exhibit a normal level (96% +- 6, n = 17) of ATX-2, compared to N2 embryos (n = 15). (F-I) Embryos at first mitosis show that cytoplasmic expression of ATX-2 is affected by genetic mutations compared to N2 control.
atx-2(
ne4297) embryo exhibits a great reduction in ATX-2 staining (G). ATX-2 cytoplasmic staining is significantly reduced in
szy-20(
bs52) embryo (H), but no changes in
zyg-1(
it25)embryo (I). Shown are images from a single plane of the embryo. Bar, 5 um. (J-L) Immunoblot using embryonic lysates from
atx-2,
szy-20,
zyg-1 mutants and N2 worms grown at 22C. alpa-Tubulin was used as loading control. (K-L) Quantitative immunoblot analyses reveal (K)
szy-20(
bs52) embryos possess significantly reduced levels of ATX-2 (0.4 +- 0.1, n = 10) compared to N2 (n = 12), (L) whereas
atx-2(
ne4297) embryos contain normal levels of SZY-20 (0.98 +- 0.03, n = 12). Note that full-length protein levels arequantified for ATX-2 and SZY-20, as both
atx-2(
ne4297) and
szy-20(
bs52) embryos produce a truncated polypeptide. In contrast, no significant changes were found in the level of a centriole factor, SAS-6, (0.97 +- 0.1, n = 12) in any mutant embryos. Error bars are SD