Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns

Abstract

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.

Keywords: alternative splicing; denervation; nemaline myopathy; neurogenic atrophy; protein isoforms; smooth muscle; striated muscle; synaptopodin-2/SYNPO2/myopodin; synemin; α-actinin.

Figure 1

Tissue-specific expression of SYNPO2 isoforms. (A) Schematic presentation of the structure of the SYNPO2 gene and splice patterns. Isoforms a/d, b/e and c/f are alternatively spliced and have different 3’ exons. Isoforms d–f start from an alternative transcription start site. Calculated molecular masses of the respective isoforms are given at the right. (B) Summary of the RT-PCR experiments and the SYNPO2 isoforms amplified by the respective primer pairs. (C–E) Analysis of SYNPO2 expression at the RNA level in different human tissues by RT-PCR. Positions of primer pairs I–IX are indicated in (A), except primer pair VIII, since exon 0 is not shown in (A). Ht: heart; Sk: skeletal muscle; Ut: uterus (smooth muscle); Pr: prostate. (C) The PDZ domain-containing 5’ end (I, II, III) is strongly expressed in the heart, uterus and prostate, but only very weakly in skeletal muscle. The 3’ end encoding the carboxy-terminus of isoforms a and d (V) is expressed in the uterus and prostate, but only weakly or not in both striated muscles. + and − show PCR reactions with and without addition of cDNA, respectively. (D) Weak expression of the exon 5-containing isoform (VII) and the isoform lacking the PDZ-domain (VIII) in prostate tissue. (E) An isoform in which exon 2 is directly spliced to exon 6 (IX) is detected in all tested tissues. (F) Graphical representation of the results of the alternative splicing events at the protein level. Two alternative transcriptional start sites (arrows) result in two distinct protein amino-termini, and inclusion of a PDZ domain only in the variants derived from the more upstream start site. Additional complexity is created at the carboxy-terminus by alternative splicing. H1, H2 and H3 indicate parts of the protein with high homology to SYNPO and SYNPO2L. The positions of epitopes of the different antibodies are indicated by the black bars below the sketch. (G,H) Analysis of SYNPO2 expression by Western blotting confirms the expression of different isoforms in different human (Pr, Ht, Sk) or mouse (Ut) muscle types and tissues. Blots were double stained with HH9 (green bands) and (G) SYNPO2a-NT (isoforms a/d) or (H) SYNPO2a-CT (isoforms a–c) as indicated (red bands). In the merged pictures, bands stained by both antibodies are marked by yellow arrowheads. Note that probably due to the high proline content of all isoforms, the proteins migrate slower than expected.

Figure 2

The PDZ domain of SYNPO2 interacts with synemin. (A) Schematic illustration of SYNPO2 isoforms illustrating the position of the PDZ domain in isoforms a, b and c (red box). (B) A yeast-two-hybrid screen identifies synemin isoforms A (prey 1) and B (preys 2, 3, 4) as novel binding partners of this domain. “GHWF” are the amino acids at the C-terminus of the preys. (C,D) Mutagenesis of the carboxy-terminus of synemin, reveals specific interaction of SYNPO2 with the carboxy-terminus of wildtype synemin (---GHWF), but not with the mutant variants (---GHAF, and ---GHWR). (E) Western blot overlay assays indicate interaction of SYNPO2 with GST-synemin amino acids 1464-1565, but not with carboxy-terminally truncated synemin (GST-Synm-CTΔ, amino acids 1464-1533) and GST. Left panel: localization of the recombinant proteins revealed by staining with anti-GST antibody; center panel: overlay with SYNPO2-PDZ showing interaction with GST-Synm-CT only; right panel: incubation with T7-antibody without overlay with SYNPO2-PDZ. (F,G) Immunofluorescence localization of SYNPO2 and binding partners in sections of smooth and striated muscle samples. (F) SYNPO2 and synemin partially colocalize in dense bodies in the muscularis externa of human colon (arrowheads). (G) SYNPO2 and synemin colocalize with filamin C (FLNc) in the Z-disc region of human cardiomyocytes (arrowheads). Insets in (F,G) show a magnification of the respective boxed areas. Bars: 10 μm. 5 μm (insets).

Figure 3

The carboxy-terminal extension of SYNPO2a and d localizes to dense bodies in smooth muscle cells and interacts with α-actinin. (A) Schematic illustration of SYNPO2 isoforms illustrating the position of the carboxy-terminal extension of isoforms a and d (red box). (B) Transient expression of the carboxy-terminal extension of SYNPO2 isoforms a and d (SYNPO2a-CT) fused to GFP in A7r5 smooth muscle cells shows targeting of the fusion protein to dense bodies that were identified by staining for α-actinin (arrowheads). The boxed area in the upper panel is shown enlarged in the lower panel. Bars: 20 μm. (C,D) Co-immunoprecipitation experiments reveal binding of the carboxy-terminal extension of isoforms a and d to α-actinin-2 (C) and α-actinin-1 (D). The weak signal marked with an asterisk indicates the light chain of the T7-tag antibody used for the immunoprecipitation.

Figure 4

SYNPO2 is expressed in smooth muscle cells of the uterus. (A–C) Immunolocalization of SYNPO2 and α-actinin in cryosections of mouse uterus. DAPI was used to stain nuclei. (A) SYNPO2 is strongly expressed in the myometrium (mm), but not in the mucosa of the endometrium (em) that contains uterine glands and arteries embedded in connective tissue stroma. (B) Higher magnification of the boxed area in (A) reveals strong expression of SYNPO2 in smooth muscle (sm) cells of the myometrium, but not in surrounding connective tissue (con) and the outer connective tissue layer, the perimetrium (pm). (C) Enlargement of the boxed area in B shows specific and exclusive SYNPO2 staining of smooth muscle cells. Also note that nuclei (n, arrows) are not stained. Bars: 200 μm (A), 50 μm (B), 10 μm (C).

Figure 5

SYNPO2 is mainly localized in smooth muscle cells and not in epithelial cells of the human prostate. (A–C) Immunolocalization of SYNPO2 and keratin (A,B) or smooth muscle actin (sm-actin, C) in cryosections of human prostate. DAPI was used to stain nuclei. (A) Low magnification reveals strong expression of SYNPO2 in stromal cells, but no detectable staining in epithelial cells, which in turn are identified by staining for keratin (arrowheads). (B) Higher magnification confirms the absence of SYNPO2 in keratin-positive glandular epithelial cells (closed arrowheads). Instead, stromal cells are stained (open arrowheads). (C) Staining for sm-actin unequivocally clarifies that it is smooth muscle cells that are strongly stained by the SYNPO2 antibody (open arrowheads), whereas epithelial cells are not stained (closed arrowheads). Bars: 200 μm (A), 20 μm (B,C).

Figure 6

SYNPO2 is a component of nemaline bodies and the central and intermediate zone of target fibers. (A) Skeletal muscle cryosections of two patients with nemaline myopathy stained with antibodies against SYNPO2 and α-actinin. SYNPO2 is found together with the verified rod component α-actinin in nemaline bodies (arrowheads). (B) Skeletal muscle cryosections of two patients with neurogenic muscular atrophy stained with antibodies against SYNPO2 and filamin C (FLNc). SYNPO2 localizes together with filamin C in the center of the targets that occur in the muscle fibers of these patients (arrowheads). Wheat germ agglutinin (WGA) was used to visualize the borders of the muscle fibers. Bars: 50 μm (A), 20 μm (B).