Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein

Abstract

We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with alpha-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A-sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein-myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.

Figure 1.

Sequence alignment of human and mouse synaptopodin and myopodin. Deduced amino acid sequence (one letter code) of human and mouse synaptopodin cDNA (top lanes) and myopodin (bottom lanes). At the protein level, an overall identity of 46% is found between both proteins. Synaptopodin contains two PPXY motifs (bold), myopodin contains only one PPXY motif. Multiple PXXP motifs are found throughout both molecules. Myopodin contains two potential NLSs (bold and underlined) not found in synaptopodin. Synaptopodin, but not myopodin, contains two possible PEST sites (underlined in italics) between aa 44 and 88 (PEST score: 11.72) and between aa 327 and 342 (PEST score: 6.17). The sequence data of myopodin are available from GenBank/EMBL/DDBJ under accession nos. AJ010482 (human) and AJ306625 (mouse). Myopodin peptides used for immunization are bold and boxed (SRIB1) or boxed (SRIB2).

Figure 1.

Sequence alignment of human and mouse synaptopodin and myopodin. Deduced amino acid sequence (one letter code) of human and mouse synaptopodin cDNA (top lanes) and myopodin (bottom lanes). At the protein level, an overall identity of 46% is found between both proteins. Synaptopodin contains two PPXY motifs (bold), myopodin contains only one PPXY motif. Multiple PXXP motifs are found throughout both molecules. Myopodin contains two potential NLSs (bold and underlined) not found in synaptopodin. Synaptopodin, but not myopodin, contains two possible PEST sites (underlined in italics) between aa 44 and 88 (PEST score: 11.72) and between aa 327 and 342 (PEST score: 6.17). The sequence data of myopodin are available from GenBank/EMBL/DDBJ under accession nos. AJ010482 (human) and AJ306625 (mouse). Myopodin peptides used for immunization are bold and boxed (SRIB1) or boxed (SRIB2).

Figure 2.

Expression of myopodin in skeletal, heart, and smooth muscle cells. (a) A single 3.6 kB mRNA is detected in mouse skeletal muscle (S) and heart (H). The expression in the heart is weaker than in skeletal muscle. (b) Western blot analysis of myopodin and synaptopodin in cytosolic extracts of mouse skeletal (S) and heart (H) muscle. A 110-kD band corresponding to synaptopodin is found in both tissues using antisynaptopodin antibody NT. The myopodin antibody SRIB1 reveals a band of 80-kD in skeletal muscle and ∼95 kD in the heart (Fig. 2 b). Both signals were blocked with the corresponding peptide showing the specificity of the bands. Identical results were obtained for SRIB2 (Fig. 2 b). (c) Western blot analysis of myopodin (left) in colon (C), stomach (St), uterus (U), and lung (Lu) shows a single 80-kD band in all lanes. Interestingly the same tissues also express synaptopodin (right, arrow). (d) Immunofluorescence microscopy of myopodin in mouse kidney, colon, stomach, and uterus. In the kidney, myopodin is strongly expressed in arteriolar smooth muscle cells (arrowhead), but not in glomerular (G) podocytes. In colon, stomach, and uterus myopodin expression is restricted to smooth muscle cells. In the colon, the arrowheads mark the muscularis mucosae. Bars: (kidney) 40 μm; (stomach, colon, and uterus) 300 μm.

Figure 3.

Immunohistochemistry of myopodin in skeletal and heart muscle. (a) Immunofluorescence labeling of a rat skeletal muscle frozen section with antimyopodin SRIB1 shows a striated pattern. (b) In the human heart, a similar striated pattern is seen. (c and d) Double labeling of rat skeletal muscle with titin (c) and α-actinin (d). Myopodin is shown in green, titin and α-actinin in red. (c) Antititin BD6 localizes to the A-I junction and encircles the myopodin labeling. (d) Myopodin colocalizes with α-actinin at the Z-disk. The overlap of immunoreactivity results in a yellow staining of the Z-disks (d).

Figure 4.

Immunoelectron microscopic analysis of myopodin. Immunoelectron microscopy of ultrathin frozen sections from stretched rat psoas muscle (a and b) and rat heart (c). The arrows point to the localization of myopodin at the Z-disc of skeletal and heart muscle. Bars, 0.4 μm.

Figure 5.

Differentiation-dependent nuclear cytoplasmic redistribution of myopodin in C2C12 cells. C2C12 cells were analyzed by immunofluorescence microscopy at different time points of differentiation using SRIB2 antibody. (a) In proliferating myoblasts, myopodin was strongly expressed in the nucleus, but only weakly in the cytoplasm. (b) Soon (6 h) after induction of myocyte differentiation, myopodin was found in the nucleus and in the cytoplasm along actin filaments. (c) Later myopodin was seen in a dotted pattern along the actin filaments. (d) In differentiated myotubes, it was found at the Z-disc in a pattern resembling that found in adult muscle fibers (compare with Fig. 3 a), and was no longer in the nucleus (arrow). (e) Northern blot analysis (top lanes) detected a 3.6-kB transcript in myoblasts (Mb) and myotubes (Mt). Western blot analysis (middle) revealed a strong up-regulation of protein expression in Mt. Equal protein loading was confirmed by reprobing with α-tubulin (bottom). Bars: (a) 40 μm; (d) 20 μm.

Figure 6.

Myopodin directly binds to actin. (a) Detection of radio-labeled myopodin by actin cosedimentation in the 100,00 g pellet (P; left). In the presence of cold myopodin as a competitor (right), most of the labeled protein remains in the supernatant (S). (b) Densitometric quantification reveals that >65% of radio-labeled myopodin is found in the pellet (P). In the presence of unlabeled myopodin as competitor, >70% of radioactive-labeled protein remains in the supernatant (S).

Figure 7.

Myopodin contains a novel actin binding site. C2C12 myoblasts were transfected with myopodin-GFP constructs of variable length. In addition to full-length myopodin (MP full), constructs were generated which contained various fragments of the ORF. With this approach, a single actin binding site of myopodin was defined that corresponds to fragment MP7. pEGFP-C1 alone did not bind to the actin filaments. Bar, 30 μm.

Figure 8.

Overexpression of myopodin reveals actin bundling activity. (a) Immunofluorescence of A7 cells transfected with a myopodin full-length GFP (MPfl-GFP). Actin fibers are labeled with rhodamine-conjugated phalloidin and nuclei are stained with DAPI. Expression of myopodin induces actin bundles in the cytoplasm (arrows) and actin loops in the nucleus (arrowheads). Note the absence of actin bundles in nontransfected cells. (b) High power view of transfected cells seen in panel a. In the triple-labeling (merge), intranuclear actin loops appear blue-white (arrowheads) and cytosolic actin bundles yellow (arrows). (c) C2C12 myoblasts transfected with GFP-myopodin develop giant nuclear loops. Myopodin-induced loops contain actin as revealed by double labeling with rhodamine-conjugated phalloidin, but not α-actinin or titin (unpublished data). (d) Myopodin-induced actin loops (arrows) are sensitive to lat-A. Bars: (a) 80 μm; (b) 30 μm; (c and d) 10 μm.

Figure 9.

The two potential NLSs are not required for nuclear import of myopodin. (a) Inactivation of two potential NLSs present in myopodin by site directed mutagenesis. (b) Myopodin is transported to the nucleus and induces nuclear actin bundling after inactivation of the COOH-terminal NLS (mut 2) alone or after inactivation of both the COOH-terminal NLS and the NH₂-terminal NLS (mut 1+2). Bar, 20 μm.

Figure 10.

Stress-induced nuclear actin rod formation and redistribution of myopodin. (a–c) Formation of myopodin (a) and actin (b) containing intranuclear rods (arrows) in myoblasts after a 150 min heat shock. (d–f) Nuclear accumulation and cytoplasmic depletion of myopodin after 90 min heat shock in the presence of 10 μm LMB (e). (f) In some cells, a punctuated pattern of nuclear myopodin redistribution was noted. (g–i) After heat shock of differentiated myotubes, myopodin (g) shuttles from the Z-disk (arrows; see Fig. 5 d for control) visualized by α-actinin staining (h) to the nucleus. Bars: (a–e) 40 μm; (f–i) 20 μm.