Actin-ADF/cofilin rod formation in Caenorhabditis elegans muscle requires a putative F-actin binding site of ADF/cofilin at the C-terminus

Abstract

Under a number of stress or pathological conditions, actin and actin depolymerizing factor (ADF)/cofilin form rod-like structures that contain abnormal bundles of actin filaments that are heavily decorated with ADF/cofilin. However, the mechanism of actin rod formation and the physiological role of actin rods are not clearly understood. Here, we report that overexpression of green fluorescent protein-fused UNC-60B, a muscle-specific ADF/cofilin isoform, in Caenorhabditis elegans body wall muscle induces formation of rod-like structures. The rods contained GFP-UNC-60B, actin-interacting protein 1 (AIP1), and actin, but not other major actin-associated proteins, thus resembling actin-ADF/cofilin rods found in other organisms. However, depletion or overexpression of AIP1 did not affect formation of the actin-GFP-UNC-60B rods, suggesting that AIP1 does not play a significant role in the rod assembly. Truncation of the C-terminal tail, a putative F-actin binding site, of UNC-60B abolished induction of the rod formation, strongly suggesting that stable association of UNC-60B with F-actin, which is mediated by its C-terminus, is required for inducing actin-ADF/cofilin rods. This study suggests that C. elegans can be a new model to study functions of actin-ADF/cofilin rods.

Fig. 1

Formation of actin rods in the C. elegans body wall muscle by overexpression of GFP-UNC-60B. (A and B) Transgenic overexpression of GFP-UNC-60B in C. elegans. Worm lysates from wild-type, an unc-60B-null mutant (as a negative control), and a GFP-UNC-60B(WT)-expressing strains in wild-type background were subjected to Western blot using anti-UNC-60B (A) or anti-actin antibody (B). Positions of molecular weight markers are shown on the left of A. (A) Bands at ~18 kDa are endogenously expressed UNC-60B as this band is absent in the unc-60B-null mutant. In the transgenic strain, an additional 40 kDa band of GFP-UNC-60B was detected. (C and D) Localization of GFP-UNC-60B in the body wall muscle of live worms without any fixation. GFP-UNC-60B predominantly localized to needle-like rods. Faint striated localization was sometimes observed (C, arrowheads). (E–J) Fixed worms were stained with anti-actin antibody (F) or tetramethylrhodamine-phalloidin (I). Co-localization of GFP-UNC-60B and actin in rods was observed by staining with anti-actin antibody (E and F, arrowheads). However, phalloidin did not detect rods (I). Merged images of GFP-UNC-60B (green) and anti-actin or phalloidin (red) are shown in G and J. Bar, 10 μm.

Fig. 2

Investigation of other components of actin-GFP-UNC-60B rods. GFP-UNC-60B- expressing worms were stained for UNC-78 (AIP1) (B), myosin heavy chain A (MYO-3) (E), α-actinin (ATN-1) (H), vinculin (DEB-1) (K), tropomyosin (LEV-11) (N), Ce-kettin (Q), tropomodulin (UNC-94/TMD-1) (T), or UNC-87 (W). Localization of GFP-UNC-60B is shown in the left column (A, D, G, J, M, P, S, and V). Merged images of GFP-UNC-60B (green), actin associated proteins (red) are shown in the right column (C, F, I, L, O, R, U, and X). Only UNC-78 co-localized with GFP-UNC-60B to the rods (B). Bar, 10 μm.

Fig. 3

Knockdown of UNC-78 does not affect actin-GFP-UNC-60B rod formation. Worm lysates from control RNAi-treated and unc-78(RNAi)-treated GFP-UNC-60B-expressing strains were subjected to Western blot using anti-UNC-78 (A) or anti-actin (B) antibody. (C–H) Localization of UNC-78 (C and D) and GFP-UNC-60B (E and F) in the control RNAi-treated (C, E, and G) or unc-78(RNAi)-treated (D, F, and H) GFP-UNC-60B-expressing strain. Merged images of UNC-78 (red) and GFP-UNC-60B (green) are shown in G and H. Bar, 10 μm.

Fig. 4

Overexpression of GFP-UNC-78 does not induce rod formation. Worm lysates from wild-type and a GFP-UNC-78-expressing strains in wild-type background were subjected to Western blot using anti-UNC-78 (A) or anti-actin antibody (B). Positions of molecular weight markers are shown on the left of A. (A) Bands at ~65 kDa are endogenously expressed UNC-78. In the transgenic strain, an additional 90 kDa band of GFP-UNC-78 was detected. (C–E) Localization of GFP-UNC-78 (C), UNC-60B (D), and actin (E) in the body wall muscle of the GFP-UNC-78- expressing worms. None of these proteins form rod-like structures. Bar, 10 μm.

Fig. 5

Essential role of the C-terminal tail of UNC-60B for rod formation. (A and B) Transgenic overexpression of C-terminally truncated GFP-UNC-60B variants in C. elegans. Worm lysates from wild-type, an unc-60B-null mutant (as a negative control), and a GFP-UNC-60B(Δ150)- or GFP-UNC-60B(Δ152)-expressing strains in wild-type background were subjected to Western blot using anti-UNC-60B (A) or anti-actin antibody (B). Positions of molecular weight markers are shown on the left of A. In the transgenic strains, GFP-UNC-60B(Δ152) and GFP-UNC-60B(Δ152) were overexpressed at similar levels to GFP-UNC-60B(WT) in Fig. 1. (C–E) Localization of GFP-UNC-60B(WT) (C), GFP-UNC-60B(Δ150) (D) or GFP-UNC-60B(Δ152) (E) were observed in live worms without fixation. GFP-UNC-60B(WT) induced rod formation (C), but GFP-UNC-60B(Δ150) (D) and GFP-UNC-60B(Δ152) (E) localized to the diffuse cytoplasm with vague striation and the nucleus (asterisks) and did not induce rods. Bar, 10 μm.