Schematic diagrams (Left) and results of yeast two-hybrid analysis with deletion mutants (Right). Amino acid (aa) numbers at domain junctions are shown above schematics and amino acid residues contained in each deletion construct are indicated in clone name (Lower Left). Minimum critical regions necessary for interaction with binding partners are indicated by shaded bar. (A) Myozenin structure illustrating two putative α-helical domains (A1 and A2) and central glycine-rich domain (Gly). (B) α-Actinin-2 structure showing actin-binding domain (ABD), central repeats (R1–R4), spacer (S), and EF hand domain (EF). (C) γ-Filamin schematic illustrating ABD, repeats (numbered), repeats 19 and 20 interdomain insertion (I), and hinge region (H).

Northern blot analysis of MYOZ illustrating predominant expression in skeletal muscle on short exposure (A) and lower-level expression (and/or cross-hybridization to the related locus) in heart, placenta, and other tissues on long exposure (B).

Blot-overlay analysis of myozenin binding to α-actinins. ³⁵S-labeled probes are indicated below and nonradioactive IVTT products (A and B) or whole-tissue homogenates (C) loaded in each lane are indicated above A–C. (A) Myozenin binds to both α-actinin-2 and -3 but not to itself (Left), whereas α-actinin-2 and -3 both bind to myozenin as well as to themselves (Center and Right). Binding to α-actinin in myozenin lanes likely represents binding to α-actinin-1 and -4 produced from contaminating ACTN1 and ACTN4 mRNA in reticulocyte lysates. (B) Control experiment showing lack of myozenin binding to various partial dystrophin fragments: N828, N-terminal nos. 1–828; H12–R11, hinge no. 2–repeat no. 11; R7–R15, repeat nos. 7–15; R12–H13, repeat no. 12–hinge no. 3; and R19–H14, repeat no. 19–hinge no. 4. Parallel radiolabeled IVTT products of all clones are shown below to confirm production of expected size products. (C) Detection of myozenin-binding partners in human tissues by blot-overlay assay. Predominant binding is to a ≈100-kDa protein at expected size for α-actinin.

CoIP of IVTT myozenin, α-actinin, and γ-filamin. Presence (+) or absence (−) of ³⁵S-labeled IVTT products in each reaction is shown above and precipitating Abs are indicated below. Identity of each precipitated protein is indicated (Middle). A and B illustrate CoIP of full-length α-actinins-2 and -3, respectively, with FLAG-tagged full-length myozenin by using either anti-α-actinin or anti-FLAG Abs (lanes 2 and 3). Each Ab is specific (lanes 4 and 5); however, there is some degree of aggregation and nonspecific precipitation of IVTT myozenin (lanes 1 and 5). (C) CoIP of myozenin, α-actinin, and γ-filamin (FLNC_2191–2705) by using specific antimyozenin and antifilamin Abs. Myozenin coimmunoprecipitates both α-actinin-2 (lane 1) and γ-filamin (lane 3). When equal volumes of all three IVTT products are combined, α-actinin CoIP predominates (lane 4). Use of anti-γ-filamin-precipitating Abs results in CoIP of myozenin but not α-actinin (lanes 5–8). Similarly, use of anti-α-actinin Abs coimmunoprecipitated myozenin but not γ-filamin (not shown), indicating a lack of direct interaction between α-actinin and γ-filamin. (D) Competitive CoIP of α-actinin and γ-filamin (FLNC_2191–2705) by using specific antimyozenin Abs. Increasing relative amounts of α-actinin (lanes 1–6 contain 0-, 1-, 2-, 4-, 10-, and 20-fold levels, respectively) competitively decreased CoIP of γ-filamin. Similarly, increasing amounts of γ-filamin reduced CoIP of α-actinin (not shown).

Subcellular localization of myozenin in human skeletal muscle. (A) Confocal indirect immunofluorescence microscopy of human skeletal muscle by using antimyozenin antisera showing striated staining pattern characteristic of sarcomeric proteins. (B and C) Immunogold transmission electron microscopy for myozenin (B) and α-actinin-2 (C) in human skeletal muscle showing predominant labeling at Z lines. (D and E) Double-label indirect immunofluorescence microscopy for myozenin (red) and myosin (D, green) or tropomyosin (E, green) in cultured human myotubes. (F) Quantitative grain measurements showing distribution of α-actinin-2 centered in middle of Z line (Right) and myozenin slightly skewed toward edges of Z lines (Left).

Indirect immunofluorescence analysis of α-actinin and myozenin in nemaline rods. Longitudinal (A) and cross sections (B) stained with antimyozenin Ab. (C) Cross section stained with anti-α-actinin-2 Ab. All Abs stain both Z lines as well as nemaline rods.