Miller RK , Qadota H , Landsverk ML , Mercer KB , Epstein HF , Benian GM .

AR0500051 NIAMS NIH HHS , AR051466 NIAMS NIH HHS , AR052133 NIAMS NIH HHS

	Figure 1. Yeast two-hybrid analysis shows that aa 1–112 of UNC-98 interact specifically with aa 1771–1969 of MHC A, and not B, C, or D. (A) A yeast two-hybrid bait expressing the first 112 amino acids of UNC-98 excluding the four C2H2 Zn finger domains (pGDBU98-4c) was used to screen approximately four million yeast colonies. 33 confirmed interacting clones comprised 18 unique genes, including the C terminus of MHC A (isolated three times). (B) MHC A, B, C, and D have similar structures, including a myosin head domain (yellow), IQ domains (purple), and a coiled-coil domain (blue). In addition, MHC A and B have a nonhelical region (green). The prey proteins for the first experiment include ∼300 residues of the C termini of each MHC (A, B, C, and D). The prey proteins for the second experiment include the following portions of MHC A: aa 1636–1937, lacking the entire nonhelical region (MHC A (2)); aa 1636–1870, lacking a portion of the coiled-coil region and the nonhelical region (MHC A (3)); aa 1938–1969, including just the nonhelical region (MHC A (4)); aa 1871–1969, including a portion of the coiled-coil region and the nonhelical region (MHC A (5)); and aa 1771–1969, including a larger portion of the coiled-coil region and the nonhelical region (MHC A (6)). (C) Growth on media selecting for the maintenance of the bait (−URA) and the prey (−LEU) plasmids confirms that the yeast harbors both the N-terminal UNC-98 bait and the C terminus of each of the MHCs. (D) When the yeast shown in C were tested for their ability to grow on media excluding adenine (−ADE), growth only occurred when the N terminus of UNC-98 and the C terminus of MHC A, but not MHC B, C, or D were present. (E) Growth on media selecting for the maintenance of the bait and the prey plasmids confirms that the yeast harbors both the N-terminal UNC-98 bait and the preys containing deletion derivatives of the C terminus of MHC A. (F) When the yeast shown in E were tested for their ability to grow on media excluding adenine, growth only occurred when the N terminus of UNC-98 and either the C-terminal 330 (MHC A (1)) or 200 residues (MHC A (6)) of MHC A were present.
	Figure 2. UNC-98 shows saturable binding to nematode myosin in vitro and copurifies with nematode thick filaments. (A) The proteins used in the ELISA assay include total myosin II purified from C. elegans (MHC A, B, C, and D), bacterially expressed N terminus of UNC-98 (112 amino acids), and bacterially expressed UNC-98 (310 amino acids). These purified proteins (1 μg each) were visualized on an SDS-PAGE gel by Coomassie staining. (B) When increasing amounts (at concentrations from 0 to 1.0 μM) of the N-terminal portion of UNC-98 (100 μl) were exposed to a fixed amount of myosin (100 μl at 0.5 μM) in vitro, it bound increasingly until it reached a saturation level. The best-fit ligand binding curves were determined by plotting means and standard errors of three absorbance values (SigmaPlot 9.0). The dissociation constants were calculated from this data to be 0.037 μM for full-length UNC-98 and 0.295 μM for the N-terminal portion of UNC-98. (C and D) Native thick filaments were purified using established procedures from wild-type nematodes (Epstein et al., 1974; MacLeod et al., 1977). A fraction enriched in thick filaments (Fig. S1, B and C, available at http://www.jcb.org/cgi/content/full/jcb.200608043/DC1) was further fractionated on a sucrose density gradient. Gradient fractions S1–S14 were run on duplicate gels and immunoblotted. One blot was exposed to a combination of anti–MHC B, anti-paramyosin, and anti-actin antibodies (C). The other blot was exposed to anti–UNC-98 antibodies (D). UNC-98 is present in sucrose gradient fractions S3–S5 that contain myosin and paramyosin, markers for thick filaments.
	Figure 3. Loss of function of unc-98 affects the localization of MHC A but not UNC-97; the myosin binding N-terminal portion of UNC-98 but not the C-terminal portion of UNC-98 affects the localization of MHC A. (A) Immunofluorescence microscopy of unc-98(sf19) shows that the banding pattern of MHC A is not as sharply defined as in wild type, and the localization of UNC-97 is no longer in a sharply defined pattern. In fact, MHC A is no longer restricted to A-bands and can sometimes be seen to cross over rows of dense bodies. (B) When the N terminus of UNC-98 (lacking the Zn fingers) is overexpressed as a GFP fusion in a wild-type background, its localization is dispersed within the myofibril and results in aggregates of MHC A, whereas UNC-97 is normally localized to focal adhesions. (C) When the C terminus of UNC-98 (containing the Zn fingers) is overexpressed as a GFP fusion in a wild-type background, it localizes normally to M-lines and dense bodies, and both MHC A and UNC-97 are normally localized. M-lines are marked with arrows, and dense bodies are marked with arrowheads. Insets show threefold magnifications. Bars, 5 μm.
	Figure 4. The absence of MHC A or the knock down of UNC-97 affects the localization of UNC-98. (A) Within cells lacking MHC A (in a mosaic line expressing myo-3∷gfp to rescue the myo-3(st386) null mutation), UNC-98 localization is severely disrupted and found in aggregates at the ends of muscle cells. In comparison, UNC-97 appears to localize to focal adhesion structures, although in an irregular pattern, and is not present in aggregates. (Representative cells lacking MHC A expression are outlined in white.) (B) In adult body wall muscle cells in which unc-97 has been knocked down by RNAi, both UNC-98 and MHC A are mislocalized. Insets show threefold magnifications. Bars, 5 μm.
	Figure 5. UNC-98 as a molecular bridge between integrin-associated proteins and thick filaments at the M-line. In C. elegans body wall muscle, the myofibrils are closely apposed to the muscle cell membrane, and both the dense bodies (Z-disks) and M-lines are attached to the muscle cell membrane. At the base of both these focal adhesion–like structures are clustered UNC-52 (perlecan) in the ECM and the integrins in the muscle cell membrane. Associated with the cytoplasmic tail of PAT-3 is a complex of four proteins, including UNC-97 (PINCH). At the M-line, UNC-97 interacts with the four C2H2 Zn fingers of UNC-98, whereas the N terminus of UNC-98 interacts with the C-terminal portion of MHC A, but not MHC B. In nematode thick filaments, these MHCs are differentially localized, with MHC A in the middle and MHC B in the polar regions. An arrow points from UNC-97 to UNC-98 because, when the level of UNC-97 is reduced, UNC-98 is mislocalized. The arrow points in both directions between UNC-98 and MHC A because, in the absence of MHC A, UNC-98 is found in large aggregates. When UNC-98 levels are reduced, MHC A is not localized in its normal sharply defined pattern.

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