Two actin-interacting protein 1 isoforms function redundantly in the somatic gonad and are essential for reproduction in Caenorhabditis elegans

Abstract

The somatic gonad of the nematode Caenorhabditis elegans exhibits highly regulated contractility during ovulation, which is essential for successful reproduction. Nonstriated actin filament networks in the myoepithelial sheath at the proximal ovary provide contractile forces to push a mature oocyte for ovulation, but the mechanism of assembly and regulation of the contractile actin networks is poorly understood. Here, we show that actin-interacting protein 1 (AIP1) is essential for the assembly of the contractile actin networks in the myoepithelial sheath. AIP1 promotes disassembly of actin filaments in the presence of actin depolymerizing factor (ADF)/cofilin. C. elegans has two AIP1 genes, unc-78 and aipl-1. Mutation or RNA interference of a single AIP1 isoform causes only minor impacts on reproduction. However, simultaneous depletion of the two AIP1 isoforms causes sterility. AIP1-depleted animals show very weak contractility of the myoepithelial sheath and fail to ovulate a mature oocyte, which results in accumulation of endomitotic oocytes in the ovary. Depletion of AIP1 prevents assembly of actin networks and causes abnormal aggregation of actin as well as ADF/cofilin in the myoepithelial sheath. These results indicate that two AIP1 isoforms have redundant roles in assembly of the contractile apparatuses necessary for C. elegans reproduction.

Keywords: actin depolymerizing factor/cofilin; actin dynamics; myoepithelial cells; ovulation; spermatheca; sterility.

Fig. 1. Depletion of the two AIP1 isoforms causes accumulation of endomitotic oocytes in the ovary

Adult worms with genotypes and RNAi treatments as indicated on the left were fixed and stained with tetramethylrhodamine-phalloidin for F-actin (left column) and DAPI for DNA (middle column), and regions of the ovary and the spermatheca are shown. The spermatheca is on the right side of each micrograph, and its location indicated by “s” in merged images on the right column (red: F-actin; blue: DNA). Arrows in B indicate condensed chromosomes in normal oocytes. Asterisks in the left column show intense staining of F-actin in the body wall muscle. Arrowheads in N and T indicate abnormal accumulation of DNA in endomitotic oocytes. Bar, 100 µm.

Fig. 2. Depletion of the two AIP1 isoforms causes ovulation defects

Ovulation processes in live worms with genotypes and RNAi treatments as indicated on the left were recorded by time-lapse Nomarski microscopy. Snapshots at key steps are shown: before oocyte maturation (A, E, I and M), at oocyte maturation (nuclear envelope breakdown: NEBD) (B, F, J, and N), at entry of an oocyte into the spermatheca (C, G, K, and O), and after fertilization (D, H, L, and P). Arrows indicate the most mature oocytes being ovulated. Positions of the spermatheca are indicated by “s”. Note that the oocyte in unc-78(null);aipl-1(RNAi) was not ovulated (M-P), and snapshots at equivalent timing to wild-type worms are shown. Bar, 50 µm. See corresponding Supplemental Movies 1– 4 in supplemental materials.

Fig. 3. Depletion of the two AIP1 isoforms disrupts contractile actin networks in the myoepithelial sheath

Gonads from worms with genotypes and RNAi treatments as indicated on the left were dissected out and stained with tetramethylrhodamine-phalloidin for F-actin (left column) and DAPI for DNA (middle column), and the regions of the myoepithelial sheath are shown. Merged images are shown in the right column (red: F-actin; blue: DNA). Bar, 100 µm.

Fig. 4. Depletion of the two AIP1 isoforms disturbs actin organization in the spermatheca

Gonads from worms with genotypes and RNAi treatments as indicated on the left were dissected out and stained with tetramethylrhodamine-phalloidin for F-actin, and the regions of the spermatheca are shown. Note that the spermatheca is a highly flexible tissue, and the difference in the overall shape of the spermatheca is not a significant phenotype. Rather, parallel actin fibers seen in wild-type with control RNAi (A), wild-type with aipl-1(RNAi) (B), and unc-78(null) with control RNAi (C), were highly disorganized in unc-78(null) with aipl-1(RNAi) (D). Bar, 10 µm.

Fig. 5. UNC-78 is diffusely localized in the cytoplasm in the somatic gonad

Gonads from wild-type worms were dissected out and stained with anti-UNC-78 antibody (A) and DAPI for DNA (B). Merged image is shown in C (red: UNC-78; blue: DNA). UNC-78 was detected in oocytes, the myoepithelial sheath, and the spermatheca (also see Supplementary Fig. 1). A mature oocyte is impermeable and was present at the junction between the myoepithelial sheath and the spermatheca (A), providing a clear background to show diffuse localization of UNC-78 in both myoepithelial sheath and spermathecal cells. Bar, 10 µm.

Fig. 6. UNC-60A (ADF/cofilin) is mislocalized to actin aggregates in AIP1-depleted myoepithelial sheath

Gonads from worms with genotypes and RNAi treatments as indicated on the left were dissected out and stained with anti-UNC-60A antibody (left column) and anti-actin antibody (middle column), and the regions of the myoepithelial sheath are shown. Merged images are shown in the right column (red: UNC-60A; green: actin). UNC-60A was expressed in both oocytes and the myoepithelial sheath. Although the focuses were adjusted to the levels of the myoepithelial sheath, staining of oocytes were also visible. Bar, 10 µm.