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	Figure 1. Release of intracellular Ca21 from RyR stores is necessary for myofibrillogenesis. (A) Myocytes grown 48 h in 0-Ca21 medium are indistinguishable from controls with respect to bipolar morphology, parallel myofibril alignment, and number and regularity of sarcomeres. Image is a Z-series maximum projection of sections encompassing the full thickness of the cell. In the central region, a loose meshwork of myofibrils surrounds normal cell inclusions including numerous yolk platelets. Trace below shows Ca21 transients that occur in normal and 0-Ca21 media up to 15 h in culture (Ferrari et al., 1996). (B) Myocytes grown in the presence of ryanodine (100 mM) at early times show disruption of myofibrils and reduced sarcomere numbers; cell shown was treated with ryanodine from 6 to 48 h in culture. Major phenotypic characteristics are myofibril misalignments, diffuse myosin immunoreactivity throughout the cell, fewer sarcomeres, and localized patches of dense myosin accumulation. Trace below shows Ca21 transients are blocked by ryanodine (n 5 109 cells examined for 30–60 min at 3–9 h in culture; Ferrari et al., 1996). (C) Resting Ca21 levels are not significantly altered in 0-Ca21 medium or with the application of 100 mM ryanodine when measured during 3–9 h in culture. Means are from $20 cells per condition, boxes enclose 50% of the data, and lines indicate median values. (D) Mean steady-state current– voltage relations of the inward rectifier potassium current at 24–28 h in culture are normal in control and 100 mM ryanodine-treated (6–24 h in culture) cells. Number of myocytes is $12 for each condition. Bars, 20 mm.
	Figure 2. Time course and ryanodine sensitivity of A band assembly in Xenopus myocytes. (A) Mean number of sarcomeres per myocyte (n $ 30 myocytes per time point) versus time in culture. The period of spontaneous Ca21 transient production is shown along the x axis (gray bar). After experimental perturbations, sarcomeres were assayed at 24 or 48 h in culture (arrows). (B) Normalized mean sarcomere numbers per myocyte from chronic 0-Ca21 and ryanodine- treated cultures. 0-Ca21 cultures were assayed at 48 h; ryanodine was applied at 100 mM in control saline, with 3–6, 3–15, and 6–24 h treatments assayed at 24 h. Note that the early period of ryanodine sensitivity corresponds to the period of Ca21 transient production shown above. Asterisks, significantly different from controls in this and subsequent figures.
	Figure 3. Pharmacological inhibition of MLCK disrupts A band formation. All data are from 24 h cultures. (A) Staurosporine (100 nM), a general kinase inhibitor, inhibits thick filament assembly without disrupting bipolar morphology. Myosin is diffusely distributed throughout the cells, with regions of dense accumulation. (B) ML-7 (1 mM), a specific MLCK inhibitor, disrupts myosin incorporation into A bands without affecting bipolar morphology. Myosin is distributed diffusely throughout the cells with localized dense patches. (C) Summary of kinase inhibitor effects on A band assembly. Inhibitors were applied at the concentration indicated (mM) to block the listed kinase(s). Note that KT5926, which inhibits CaMK II with high specificity (10 nM), has no effect, whereas a higher concentration (100 nM) of this agent inhibits MLCK as well, producing the same effects as ML-7 and ML-9. KT, KT 5926; Stauro, staurosporine; Bis I, bisindolylmaleimide 1. (D) Development of the inward rectifier potassium current is unaffected by 6–24 h, 1 mM ML-7 when assayed at 24 h in culture. Bars, 20 mm.
	Figure 4. Activation of PKC disrupts A band assembly. (A) Phorbol 12-myristate, 13-acetate (PMA; 10 nM), a potent activator of PKC, disrupts myofibrillogenesis in the same manner as MLCK inhibitors. Myosin is diffusely distributed throughout these cells, with dense punctate staining located centrally. (B) Co-application of the PKC inhibitor Bis I (100 nM) blocks the action of PMA. Myofibril and sarcomeric structure are normal. (C) PMA application from 6 to 24 h, but not 24 to 48 h, disrupts A band assembly and is rescued by Bis I. Bars, 20 mm.
	Figure 5. Molecular inhibition of MLCK blocks A band assembly. (A) Intracellular delivery via antennapedia peptide (pANT) of a pseudosubstrate inhibitory peptide (MLCKi) results in disruption of myofibrillogenesis. (B) Later application of MLCKi at 24 h in culture has no effect on sarcomere assembly. (C) Effects of treatment with synthetic peptides. In addition to late application of MLCKi, pANT alone or pANT-scrambled MLCKi have no significant effect on myofibrillogenesis. Bars, 20 mm.
	Figure 6. Detection and developmental regulation of an embryonic MLCK isoform. (A) An embryonic isoform of MLCK is developmentally upregulated during the period of Ca21 transient production. Western blot of Xenopus embryonic and adult skeletal muscle tissue with R57 antiserum shows that a single isoform running z225 kD is upregulated in embryonic myotomal tissue from stage 15 (lane 2) to stage 27 (lane 3), corresponding to 0 and 15 h in culture, respectively. This isoform is absent in adult skeletal muscle (lane 4), where a strong single band at 130 kD is detected. MLCK isoforms at 130, 208, and 220 kD are recognized in the rat A10 cell line (ATCC CRL1476) lysate (lane 1). (B) Striated pattern of labeling with the K36 smooth muscle MLCK mAb is visible in myocyte endfeet at 24 h in culture. The width and spacing of these bands matches the normal A band geometry. (C) Schematic model of Ca21 transient–dependent MLCK activation and A band assembly in embryonic skeletal muscle. Inhibiting this cascade (dashed lines) at multiple points (boxes) disrupts formation of thick filaments. Bar, 20 mm.

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