Immunohistochemical analysis reveals variations in proteasome tissue expression in C. elegans

Abstract

The ubiquitin-proteasome system (UPS) plays a crucial part in normal cell function by mediating intracellular protein clearance. We have previously shown that UPS-mediated protein degradation varies in a cell type-specific manner in C. elegans. Here, we use formalin-fixed, paraffin-embedded C. elegans sections to enable studies on endogenous proteasome tissue expression. We show that the proteasome immunoreactivity pattern differs between cell types and within subcellular compartments in adult wild-type (N2) C. elegans. Interestingly, widespread knockdown of proteasome subunits by RNAi results in tissue-specific changes in proteasome expression instead of a uniform response. In addition, long-lived daf-2(e1370) mutants with impaired insulin/IGF-1 signaling (IIS) display similar proteasome tissue expression as aged-matched wild-type animals. Our study emphasizes the importance of alternate approaches to the commonly used whole animal lysate-based methods to detect changes in proteasome expression occurring at the sub-cellular, cell or tissue resolution level in a multicellular organism.

Fig 1. Proteasome tissue expression in wild-type (N2) C. elegans sections.

(A) Schematic picture of the body plan and tissues of an adult hermaphrodite C. elegans. (B) Proteasome immunostaining in a longitudinal whole animal section (4μm) of 1-day old adult C. elegans. Specific cell types, rachis, and extracellular cuticle are indicated by arrows. Scale bar: 100 μm. (C) Large magnification images of different areas of the same C. elegans as presented in (B) showing proteasome immunoreactivity in an intestinal cell (upper left panel, outlined with a black dash line), body-wall muscle cell (upper right panel, indicated by a black arrow), oocytes (upper right panel, outlined with white dash lines), germ cells (lower left panel, indicated by a white arrow), rachis (lower left panel, indicated by a white arrowhead), and embryos (lower right panel, outlined with red dash lines). Black arrowhead points to the nucleus. Scale bars: 10 μm. (D) Quantification of immunoreactivity. Graph shows the mean staining intensity of three independent experiments (n = number of animals). Error bars: ± SEM. ***p < 0,001.

Fig 2. Targeted pas-5 RNAi results in SKN-1–dependent intestinal upregulation of proteasome α-subunits.

(A) Lysates of wild-type animals treated with control or pas-5 RNAi separated on SDS-PAGE prior to immunoblotting with anti-20S α-subunits antibody (upper panels). Black arrow points to a band expected to correspond to proteasome α-subunit 5. Lower panels show α-tubulin expression. The samples were run on the same gel. (B) Quantification of the immunoblots corresponding to the total intensity of the displayed bands. Graph shows mean relative fold change in levels of 20S α-subunits, when normalized against α-tubulin. Error bars ± SEM of three independent experiments. *p < 0.05. (C) Images of an adult wild-type animal fed control (left panel) or pas-5 RNAi bacteria (right panel) presenting proteasome immunoreactivity in intestinal cells (outlined with black dash lines), oocytes (indicated by white arrows), and body-wall muscle cells (indicated by black arrows). Black arrowhead points to nucleus. Scale bars: 10 µm. (D) Quantification of immunoreactivity. Graph shows the mean staining intensity of three independent experiments (n = number of animals). Error bars ± SEM. ***p < 0,001, **p < 0,01, *p < 0,05. (E) Images presenting proteasome immunoreactivity in the cytoplasm of intestinal cells (outlined with black dash lines) of wild-type animal fed with control (upper left panel), diluted pas-5 (upper right panel), diluted skn-1 RNAi bacteria (lower left panel) or a culture containing both pas-5 and skn-1 RNAi bacteria in 1:1 volume ratio (lower right panel). Scale bars: 10 μm. (F) Quantification of cytoplasmic proteasome immunoreactivity in the intestinal cells. Graph shows the mean staining intensity of three independent experiments (n = number of animals). Error bars ± SEM, ***p < 0,001, n.s. = non-significant.

Fig 3. Proteasome tissue expression is similar in long-lived daf-2(e1370) mutants and wild-type animals.

(A) Large magnification images of a long-lived daf-2(e1370) mutant showing proteasome immunoreactivity in an intestinal cell (left panel, outlined with a black dash line), body-wall muscle cell (left panel, indicated by a black arrow), germ cells (middle panel, indicated by a white arrow), rachis (middle panel, indicated by a white arrowhead), oocytes (middle panel, outlined with a white dash line), and embryos (right panel, outlined with red dash lines). Black arrowhead points to nucleus. Scale bars: 10 μm. (B) Quantification of immunoreactivity. Graph shows the mean staining intensity of three independent experiments (n = number of animals). Wild-type staining intensity data are the same as used in Fig 1D. Error bars ± SEM. No clear differences (≥10%) were detected in immunoreactivity.