Linear Z-line-like alignment of capping protein in obliquely striated muscle of the nematode C. elegans suggests that dense bodies are not equivalent to Z-lines

Figures

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  Fig. 1.. F-actin and CAP-1 are not concentrated at the ATN-1-enriched dense bodies. A, B, Localization patterns of F-actin (left) and ATN-1 (middle) were examined by staining with Alexa 488-labeled phalloidin and mCherry-tagging of ATN-1. Merged images are shown (right). Approximate positions of the I-bands are indicated by vertical bars on the right. With this procedure, both relaxed (A) and contracted (B) states of muscle cells were observed. Rows of the dense bodies are oriented horizontally. C, D, Localization of GFP-CAP-1 (left) was compared with that of ATN-1 (C, middle) or actin (D, middle) by immunostaining. Merged images are shown (right). With the immunostaining procedure, only relaxed states of muscle cells were observed. Bar, 10 μm.

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  Fig. 2.. A model of organization of actin filaments and CP in the C. elegans body wall muscle. This model is a view from the top (the body surface). The axis of thin and thick filaments is oriented vertically. The oblique angle of the dense body alignment to the filament axis is termed “stagger angle”. The stagger angle is small at a relaxed state (6 ° in A) but large at a contracted state (30 ° in B). The scale of the illustration is based on the representative size of the dense bodies (0.2 μm) and distance between the nearest dense bodies (1.0 μm). CP (green dots) caps the barbed ends of F-actin (magenta) and is linearly arranged in a Z-line-like alignment. F-actin remains parallel at both states. For simplicity, only actin filaments in the top halves near the dense bodies in the center are shown.

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  Fig. 3.. Linear alignment of CAP-1 is maintained independently of ATN-1. A-D, Localization patterns of GFP-CAP-1 (left) and actin (middle) were examined in wild-type (+/+) and atn-1-null (atn-1) backgrounds. Merged images are shown (right). In low-magnification views (A, B; Bar, 20 μm), linear striated pattern of GFP-CAP-1 (A) was not disrupted in atn-1 (B). Actin aggregates were formed in atn-1 (B middle, arrows), but GFP-CAP-1 was absent from the aggregates (B left, arrows). In high-magnification views (C, D; Bar, 10 μm), the linear alignment of GFP-CAP-1 was detected at the middle of the I-band (approximate positions indicated by vertical bars on the right) in both wild-type (C) and atn-1 (D). E, F. Quantification of percentages of animals with actin aggregates (E) and with normal linear GFP-CAP-1 alignment (F). n = 3. ***, p < 0.001. NS, not significant.

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  Fig. 4.. RNA interference of genes encoding CP subunits, cap-1 and cap-2, causes embryonic or larval lethality. Worm cultures treated with control RNAi (A), cap-1(RNAi) (B), or cap-2(RNAi) (C) were observed on agar plates. Bar, 1.0 mm. At a time when most of control worms grew to adults (A), most of cap-1(RNAi) (B) and cap-2(RNAi) (C) animals were arrested at embryos or early larvae (insets in B and C, Bars, 0.1 mm). D, Lethality was quantified after 3 days of RNAi treatments when most of control worms reached the adult stage. Animals that were arrested at embryonic or early larval stages were counted as dead. n = 3. ***, p < 0.001. NS, not significant.

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  Fig. 5.. RNA interference of cap-1 or cap-2 causes disorganization of actin filaments in embryonic and larval body wall muscle. Worm cultures treated with control RNAi, cap-1(RNAi), or cap-2(RNAi) were stained with rhodamine-phalloidin. A-D, Representative images of two-fold embryos (~450-min-old) (A), three-fold embryos (~520-min-old) (B, enlarged images shown in C), and L1 larvae (D) are shown. The heads are oriented to the left. Asterisks indicate fluorescence from the pharyngeal muscle. E, Quantification of percentages of animals with abnormal F-actin organization. n = 4. ***, p < 0.001. NS, not significant. Bars, 20 μm (A, B, and D) and 5 μm (C)