Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans

Abstract

Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth.

Figure 1.—

Body size of mutant nematodes with reduced autophagy. The TGF-β genetic pathway component lon-1 encodes a negative regulator of cell size, whereas unc-51 and bec-1 are two autophagy genes. Bars, 0.1 mm.

Figure 2.—

The autophagic process appears to be defective in unc-51 mutant animals. (Left) Autophagic vacuole with normal double isolation membrane (arrow) in the hypodermis from a wild-type animal. (Right) Abnormal autophagic vacuole (arrow) with strongly myelinated isolation membrane is embedded in a region of the cytoplasm abundant in membrane whorls in a hypodermal seam cell from unc51(e369) mutant. c, cuticule; bars, 1 μm. For fixation and embedding of transmission electron microscopic samples, the nematodes were treated individually. They were cut open under a dissecting microscope in a drop of fixative composed of 0.2% glutaraldehyde and 3.2% formaldehyde in 0.15 m cacodylate buffer. After an overnight fixation at 4°, the fixative was changed to washing buffer (0.1 m cacodylate buffer), and the samples were embedded in agar, post-fixed with 0.5% cacodylate-buffered OsO₄, stained with 2% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in Durcupan (Fluka Chemical, Buchs, Switzerland). Thereafter the samples were cut along the longitudinal body axis with Reichert-Jung Ultracut-E type ultramicrotome, stained with lead citrate, and examined in a JEM100CX II electron microscope.

Figure 3.—

Visualization of intestinal cells in adult nematodes. (A) TRA-1∷GFP is expressed in the nucleus of gut cells from wild-type adult. (B) TRA-1∷GFP expression in unc-51(e369) mutant adult. (C) Expression of PRK-1∷GFP in the intestine of unc-51(e369) animal. (D) Expression of PRK-1∷GFP in individual intestinal cells of wild-type (top) and unc-51(e369) mutant (bottom) animals. Brackets indicate the longitudinal border of the first four individual intestinal cells. Both images were made with the same magnification.

Figure 4.—

UNC-51 and BEC-1 are required for male tail development. (A) Tail structure of wild-type adult male. (B) Tail phenotype of an unc-51(e369) male. In this animal the rays are completely missing. (C) pbec-1∷BEC-1∷GFP is expressed in wild-type male. Corresponding fluorescence (top) and Nomarski (bottom) images. (D) Male tail structure in a bec-1(ok691); Ex[pbec-1∷BEC-1∷GFP] adult. Arrowheads indicate the rays on one side. bec-1 mutants with affected tail always lost GFP expression in the tail.

Figure 5.—

Intracellular accumulation of LGG-1 in hypodermal seam cells is affected by TGF-β signaling. (A) Expression of GFP∷LGG-1 in the seam cells of wild-type animal. GFP foci are supposed to label autophagosomal structures. (B) Expression of the GFP∷LGG-1 reporter in the seam cells of sma-6(e1482) mutant L3 larva. (C) GFP∷LGG-1 accumulates in punctate areas in the seam cells of lon-1(e185) mutant L3 larva. (D) Quantification of GFP∷LGG-1-positive foci in individual seam cells of wild-type, sma-6(e1482), and lon-1(e185) mutant animals. The number of punctate area per seam cell is shown.

Figure 6.—

Two alternative models for how autophagy affects cell growth in C. elegans. (A) If autophagy genes mediate the effect of both insulin/IGF-1 and TGF-β signaling to control cell growth, i.e., insulin/IGF-1 and TGF-β signaling converge on autophagy to regulate cell size, unc-51 and bec-1 should function downstream of these two hormonal systems. (B) Because mutations in bec-1 and unc-51 do not completely suppress the Lon phenotype of daf-2 and certain TGF-β mutant strains, it is also possible that autophagy genes act in parallel to insulin/IGF-1 and TGF-β signaling in cell growth control. The question marks indicate whether the effect of the insulin/IGF-1 signaling pathway on body size is solely the result of changes in autophagy. Arrows indicate positive regulatory interactions, bars represent inhibitions. DAF-2, IGF-1 receptor; AGE-1, phosphatidylinositol-3-OH kinase; DAF-16, FOXO forkhead transcription factor; SMA-6, type I TGF-β receptor; DBL-1, TGF-β ligand for SMA-6; LON-1, cytoplasmic transducer of TGF-β signaling.