Spatial and Temporal Analysis of Active ERK in the <em>C. elegans</em> Germline

Abstract

The evolutionarily conserved extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Functional genomic analysis identifies miRNA repertoire regulating C. elegans oocyte development

“…Gonads were dissected 37 from indicated genotypes as described above, and fixed in 3% paraformaldehyde/0.25% glutaraldehyde/0.1 M K 2 HPO 4 for 2 h at room temperature. Fixed gonads were washed three times in 1x PBS with 0.1% Tween-20 and stored overnight in Methanol at −20 °C.…”

ERK phosphorylates chromosomal axis component HORMA domain protein HTP-1 to regulate oocyte numbers

“…After each experiment, worms (n = 80 to 100) were dissected as de scribed (51). Dissected worms were fixed in 3% formaldehyde with 100 mM K 2 HPO 4 (pH 7.2) for 10 min at room temperature washed with phosphatebuffered saline (PBS) and 0.1% Tween 20 (PBST) and postfixed with 100% methanol (−20°C) for 1 hour [for diphosphorylated (dp) ERK, GLD1, RME2, XND1, pSUN1(S8), and pHTP1(S325) antibodies] or overnight (for HIM3, phistone H3, SYP1, HTP1/2, and RAD51 antibodies).…”

“…Dissected worms were fixed in 3% formaldehyde with 100 mM K 2 HPO 4 (pH 7.2) for 10 min at room temperature washed with phosphatebuffered saline (PBS) and 0.1% Tween 20 (PBST) and postfixed with 100% methanol (−20°C) for 1 hour [for diphosphorylated (dp) ERK, GLD1, RME2, XND1, pSUN1(S8), and pHTP1(S325) antibodies] or overnight (for HIM3, phistone H3, SYP1, HTP1/2, and RAD51 antibodies). Fixed gonads, in batches, were washed with PBST and blocked with 30% normal goat serum for 1 hour at room temperature (for dpERK, GLD1, RME2, XND1, and pSUN1 antibodies) or 3 hours [HIM3, phistone H3, SYP1, pHTP1(S325), HTP1/2, and RAD51 antibodies] before incubation with the desired primary antibody as described (51). For phistone H3 (1:400), HIM3 (1:800), and SYP1 (1:200) staining, germ lines were incubated in primary antibody for 4 hours at room temperature.…”

“…For phistone H3 (1:400), HIM3 (1:800), and SYP1 (1:200) staining, germ lines were incubated in primary antibody for 4 hours at room temperature. dpERK (1:400)-, GLD1 (1:200)-, RME2 (1:600)-, XND1 (1:800)-, pSUN1(S8) (1:800)-, pHTP1(S325) (1:400)-, HTP1/2 (1:400)-, and RAD51 (1:2000)-treated germ lines were incubated in primary antibody overnight at 4°C and processed as described (51) (see the antibodies in Table 3). Images were acquired either with Zeiss Axio Imager M2 equipped with an AxioCam MRm camera (Zeiss) or with an in verted laser scanning confocal microscope (Zeiss LSM 800).…”

Antitumoral Drug Potential of Tryptophan-Betaxanthin and Related Plant Betalains in the Caenorhabditis elegans Tumoral Model

“…Gonad sizes were measured from the loop region to the proximal region, including uterus area when it was filled with tumorous cells. In addition, to characterize gonad tumor morphology of the JK1466 strain, gonads were dissected and DAPI (4 ,6-diamidino-2-phenylindole) -stained following the protocol by Barth Grant, adapted from R. Francis (Schedl Lab) and Sarah Crittenden (Kimble Lab) [7,17], with some modifications. Briefly, worms were transferred to depression slides and they were anesthetized with 10 mM sodium azide.…”