Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A

Abstract

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.

Figure 1

Comparison of the functional UNC-45::GFP protein distribution and the muscle filament pattern under polarized light microscopy in the body wall muscles of the rescued unc-45 (r450). a is the GFP signal associated with the UNC-45::GFP fusion protein. b is the same field showing the muscle filament pattern as visualized by polarized light microscopy. The arrows indicate the thick filaments containing A-bands. Bar, 10 μm.

Figure 2

The 7N5 antiserum generated against an NH₂-terminal fragment of UNC-45 reacts specifically with a polypeptide of about 105 K (MWr) on Western blots of wild-type worms. The polypeptide detected is consistent with the predicted size of UNC-45 (∼107 kD). The same amount of protein extract was used for each lane. The 7N5 antiserum was used in dilutions of 1:5,000 and 1:10,000, respectively, in lanes A and B. The preimmune serum used at 1:5,000 dilution did not show any specific reaction on Western blots (data not shown).

Figure 3

UNC-45 colocalizes with MHC B in the thick filaments of body wall muscles of wild-type adult worms. a, b, and c are the same field of body wall muscle labeled with anti–UNC-45 (7N5), anti–MHC B (DM 5-8), or double-labeled with 7N5 and DM 5-8, respectively. d, e, and f are the same field of body wall muscle labeled with 7N5, anti–MHC A (DM 5-6), or double-labeled with 7N5 and DM 5-6, respectively. g, h, and i are the same field of body wall muscle labeled with DM 5-8, DM 5-6, or double-labeled with DM 5-8 and DM 5-6, respectively. Note that DM 5-8 reacts with MHC B, which is localized on the two polar regions of the A-bands, and DM 5-6 reacts with MHC A, which is localized in the middle of the A-bands. c shows the overlapped pattern of UNC-45 and MHC B at the two polar regions of the thick filaments. f and i show a similar pattern with a central labeled strip when double-labeled with UNC-45 and DM 5-6, or DM 5-8 and DM 5-6, respectively. The arrows indicate the central unlabeled strip for UNC-45 (a and d) and for MHC B (b and g), the central unlabeled strip for double-staining (c) as shown in Fig. 4 B, panel b, and the central labeled strip for double-staining (f and i) in Fig. 4 B, panel c. Bars, 10 μm.

Figure 4

Schematic diagram of muscle thick filaments. (A) Thick filament, depicting the localization of UNC-45, MHC A, and B. (B, panel a) Schematic view of thick filaments as visualized by polarized light microscopy, showing A-band and the central dark strip in the A-band that corresponds to the region containing only the thick filaments but not thin filaments. (B, panel b) Schematic view of thick filaments when labeled with both UNC-45 (green) and MHC B (red) antibodies as shown in Fig. 3 c. The color appears as light yellow, since the UNC-45 and MHC B are completely overlapped. The central unlabeled strip corresponds to the region containing only MHC A, but not MHC B or UNC-45. (B, panel c) Schematic view of thick filaments when labeled with both UNC-45 (green) and MHC A (red) antibodies as shown in Fig. 3 f. The central labeled strip corresponds to the whole region containing MHC A and appears as dark yellow due to the overlapping parts of MHC A and UNC-45 and the central part containing only MHC A.

Figure 6

Motility assay for RW2329 (unc-54 [e190]; sup-3 [e1407st90st92]) and RW2665 (unc-54 [e190]; unc-45 [m94]; sup-3 [e1407st90st92]) mutants. a, b, c, and d are plates showing the traces of worms after crawling for 1 h at 25°C for RW2665 (unc-54 [e190]; unc-45 [m94]; sup-3 [e1407st90st92]), RW2329 (unc-54 [e190]; sup-3 [e1407st90st92]), N2, and unc-45 (m94), respectively. The dots in the centers of the plates are the start points and the others are the end points. N2 and unc-45 (m94) were used as controls. Five young adults were assayed for each strain and all showed similar motility. There is no apparent difference for the motility between RW2329 and RW2665.

Figure 5

UNC-45 and MHC A staining in mutant worms. a and b are the same field of body wall muscle from unc-45 (r450) mutant animals grown at the restrictive temperature (22°C) labeled with 7N5 and DM 5-6, respectively. c and d are the same field of body wall muscle from unc-54 (e190) mutants labeled with 7N5 and DM 5-6, respectively. e and f are the same field of body wall muscle from unc-54 (e190); sup-3 (e1407st90st92) mutants labeled with 7N5 and DM 5-6, respectively. g and h are the same field of body muscle from unc-54 (e190); unc-45 (m94); sup-3 (e1407st90st92) mutants labeled with 7N5 and DM 5-6, respectively. Note that UNC-45 is localized to thick filaments in unc-45 (ts) mutants (a), but not in unc-54 (0) mutants (c) in which MHC B is absent and unc-54 (0); sup-3 mutants (e and g) in which MHC B is absent, but the level of MHC A is increased. Bars, 10 μm.

Figure 7

UNC-45 may be added into thick filaments after MHC isoforms. a, b, and c are the same part of a wild-type worm at the L1 larval stage labeled with 7N5, DM 5-8, or double-labeled with 7N5 and DM 5-8, respectively. d, e, and f are enlarged for the boxed areas of a, b, and c, respectively, showing one muscle cell. Note that UNC-45 is diffuse in the cytoplasm (d), whereas MHC B has been assembled into thick filaments near the cell membrane along the face of the muscle cell that is adjacent to the hypodermis (e). Bars, 10 μm.

Figure 8

A model for UNC-45 function. UNC-45 is required for the initiation of thick filament assembly in the early stages of development. It may function as a catalyst to modify MHC B (and/or MHC A) before assembly or directly participate in the assembly itself. In the case of UNC-45 amber mutations, UNC-45 cannot function properly and causes embryonic lethality. Later, UNC-45 is localized to thick filaments in the same patterns as MHC B. In the adult, UNC-45 normally functions as a stabilizer for MHC B. In animals carrying UNC-45 missense mutations in the CRO1/SHE4 domain at the restrictive temperature, MHC B turnover is increased, either due to inherent MHC B instability on the thick filament or an inability to properly assemble MHC B into the thick filament, followed by increased turnover of unassembled MHC B.