Different localizations and cellular behaviors of leiomodin and tropomodulin in mature cardiomyocyte sarcomeres

Abstract

Leiomodin (Lmod) is a muscle-specific F-actin-nucleating protein that is related to the F-actin pointed-end-capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.

Figure 1.

The expression and sarcomere localization of Lmod are enhanced during myofibril maturation. (A) Western blot analysis showing lower levels of Lmod protein in newly plated neonatal rat cardiomyocytes (1 d of culture; 1D) compared with cells 3 (3D) and 5 (5D) d after plating (top). Actin was used as a loading control (bottom). Comparison of Lmod localizations in (B) nonmature and (C) mature cardiomyocytes. One-day-old cells (nonmature) display punctate cytoplasmic Lmod staining, whereas mature (3D) cells display striated Lmod localization along myofibrils. Cells were costained with phalloidin to visualize F-actin (middle), and with antibodies against the Z-disk protein α-actinin (left). Insets show enlarged views on selected cell regions (rotated 90° counterclockwise). Bars, 10 μm.

Figure 2.

Localization of Lmod in sarcomeres. (A) Schematic presentation of the domain structures of Tmod and Lmod constructs used in these experiments. (B and E–H) Immunostaining of endogenous Lmod (green) relative to other proteins (red) in chicken cardiomyocytes (B and F–H) or zebra fish skeletal muscle myocytes (E). (C) Localization of expressed Lmod_1-342-EGFP (green) relative to α-actinin (red) in chicken cardiomyocytes. (D) Localization of expressed full-length Lmod–EGFP (green) relative to endogenous Lmod revealed by immunostaining (red). Boxed regions in each panel are enlarged below the main panels as individual channels and as a merged image. Line scans through the middle of the selections are shown at the bottom of each panel. Red and green arrowheads in G indicate position of individual bands within doublets of Tmod and Lmod, respectively. Lmod localizes at both sides from myomesin in M-lines, is well separated from α-actinin in Z-disks, frequently flanks single or double bands of Tmod, and partially overlaps with the myosin thick filaments. Bars, 10 μm.

Figure 3.

Dynamics of Lmod association with sarcomeres. (A) Images of cells expressing GFP-tagged Lmod_FL, Lmod_1-342, and Tmod before photobleaching (pre-bleach) and during the fluorescence recovery period. Enlarged views from selected areas of the frames (left panels) are shown. (B) Recovery profiles of bleached zones over the time course of the FRAP experiments. (C) Quantitative analysis of the Lmod and Tmod mobile fraction and fluorescence recovery half-lives in myofibrils. Bars, 10 μm.

Figure 4.

The inhibition of myosin II–based contractility leads to the delocalization of Lmod, but not Tmod. (A) In control cells, Lmod and Tmod display striated localization along myofibrils. (B) After blebbistatin treatment (50 μM for 15 min), Lmod displayed mainly diffuse cytoplasmic localization, whereas Tmod still localized to myofibrils in a striated pattern. Selected enlarged areas of the cells are shown below each picture. Bars, 10 μm. (C) Fraction of cells with detectable myofibril localization of Tmod and Lmod. Inhibition of contractility gradually displaced Lmod from sarcomeres, whereas the localization of Tmod was not affected. Values represent the mean percent of the cells displaying clear myofibril localization of the two proteins; error bars, ±SEM. Data represent averages from three independent experiments. In each experiment >150 cells were analyzed.

Figure 5.

The absence of polymerization-competent actin monomers leads to the delocalization of Lmod, but not Tmod. (A). Representative immunolocalization of Lmod (left panel) and Tmod (right panel) in cardiomyocytes after 15-min treatment with 20 μM LatB. Cells were costained with phalloidin to visualize F-actin and with an antibody against the Z-disk protein α-actinin. Bars, 10 μm. (B) Analysis of Tmod and Lmod localization 5/15/30 min after treatment with 20 μM LatB. Incubation of cardiomyocytes with LatB induces rapid dissociation of Lmod from sarcomeres, whereas the localization of Tmod was not affected. For each experiment, >200 cells were analyzed. Data represent averages from three independent experiments; error bars, ±SEM. (C) LatB treatment induces Lmod delocalization to the perinuclear region of cardiomyocytes, as well as to rods inside the nucleus (arrowheads). Bars, 10 μm.

Figure 6.

Modulation of Lmod nucleation activity by interaction with TM. (A) Effect of TM on the nucleation activities of Lmod constructs. TM inhibits polymerization of 2 μM actin induced by 0.5 μM F-actin seeds. However, low TM concentrations stimulate polymerization by 25 nM Lmod_FL, but not Lmod_162-495 nor Lmod_44-495, which lack the TM-binding site. Polymerization rates were determined from the slope of the curves at 50% polymerization and expressed as percentage of the rates without TM. Data reported are mean of three or more experiments; error bars, ±SD. (B) Polymerization rates of 2 μM actin with increasing concentrations of Lmod_FL, Lmod_44-495, or Lmod_1-342 in the absence (dashed lines) or the presence (solid lines) of 1 μM TM. Rates are expressed as percentages of the polymerization rate of actin alone in the absence or the presence of 1 μM TM. Data reported are mean of three experiments; error bars, ±SD. (C) Time course of fluorescence increase upon polymerization of 2 μM actin (6% pyrene-labeled) alone (black) or with addition of 25 nM Lmod constructs, and in the presence (solid lines) or the absence (dashed lines) of 1 μM TM. Each measurement was performed at least three times (one representative curve is shown).

Figure 7.

Subcellular localization of Lmod mutants lacking the tropomyosin-binding site and the basic patch. (A) Schematic representation of the GFP-Lmod constructs used in this study. Localization in cardiomyocytes of GFP-tagged Lmod_FL (B), Lmod_44-495 (C), and Lmod_44-495(5xGS) (D). Insets display selected regions of the cells at higher magnification, highlighting the localization pattern of each protein in myofibrils. Lmod_FL shows striated localization along myofibrils, with distinguishable accumulation near M-lines. Lmod_44-495 shows diffuse localization along myofibrils, with a large fraction localizing to the nucleus (E). Lmod₄₄(5xGS) displays a striated pattern along myofibrils but, in contrast to Lmod_FL, it does not accumulate near M-lines. The cells were also stained with rhodamine-phalloidin to visualize F-actin. Bars, 10 μm.