Complex patterns of alternative splicing mediate the spatial and temporal distribution of perlecan/UNC-52 in Caenorhabditis elegans - PubMed (original) (raw)

Complex patterns of alternative splicing mediate the spatial and temporal distribution of perlecan/UNC-52 in Caenorhabditis elegans

G P Mullen et al. Mol Biol Cell. 1999 Oct.

Free PMC article

Abstract

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan. Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

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Figures

Figure 1

Figure 1

Structure of the unc-52 gene and protein products. The unc-52 gene consists of 37 exons and spans over 20 kb. Exons (boxes), introns (lines), and the three classes of protein products are shown. Mutant alleles used in this study are also indicated. The longest ORF encodes a protein of 3375 amino acids that is homologous to the mammalian heparan sulfate basement membrane proteoglycan perlecan. Like mammalian perlecan, this polypeptide can be divided into five domains (I–V). The first domain is unique, whereas the remaining four domains show similarity to the low-density lipoprotein receptor (domain II), α-laminin (domains III and V), and NCAM (domain IV). Additional isoforms are generated through alternative splicing of exons encoding alternative C termini, indicated on the gene as shaded regions. The various protein modules are indicated with shaded boxes or circles.

Figure 2

Figure 2

Complete amino acid sequence of UNC-52/perlecan and isoforms. Domains are indicated, and the signal peptide is italicized and underlined. The alternative C termini of the short and medium length isoforms are indicted (bold), and the threonine-rich region in domain V is also shown (bold and underlined).

Figure 3

Figure 3

Localization of UNC-52 isoforms during embryonic development. Embryos were visualized by confocal microscopy. Wild-type embryos were labeled with GM1 (domain III; A, C, E, and G) and GM3 (domain IV; B, D, F, and H). A and B show the dorsal view of a comma-stage embryo (∼350 min); these images are projections of the dorsal Z-sections. Arrows indicate positions of two adjacent muscle cells; the arrowhead indicates intracellular staining. Subsequent images are projections of the complete Z-series. C and D show the lateral view of late comma-stage embryo (350–400 min). Arrows indicate the position of a muscle cell, whereas the arrowheads indicate the basal face of the cell. E and F show the lateral view of a 1.5-fold embryo (∼420 min). The arrow in both panels indicates the basal face of the muscle cells in a dorsal quadrant. Note the pharyngeal staining (arrowhead) in E. G and H show a threefold embryo (∼800 min). Note that GM1 stains the pharynx (arrowhead in G), body-wall muscles, and anal muscles (arrow in G), whereas GM3 only stains the body-wall muscles. Bar, 10 μm.

Figure 4

Figure 4

Immunolocalization of UNC-52 in adult hermaphrodites. Wild-type hermaphrodites were labeled with GM1. A shows the head from a young adult. The arrow indicates the terminal bulb of the pharynx. Note the punctate pattern over this region. B shows a section of the body-wall muscles from a young adult. The large arrow indicates a dense body, the small arrow indicates an M-line, and the arrowhead indicates the margin of a body-wall muscle cell. C shows the uterine region from an older adult (dorsal view). The arrow indicates the base of the uterine muscles, whereas the arrowhead indicates the vulva. Bar, 10 μm.

Figure 5

Figure 5

unc-52(st560) mutant embryos have tissue-specific staining defects and lack a subset of UNC-52 isoforms. Embryos were visualized by confocal microscopy. Embryos in A–F were double-labeled with GM1 (green), which recognizes all UNC-52 isoforms, and DM5.6 (red), which recognizes myosin heavy chain A (MHC A). Small arrows indicate the pharynx, and large arrows indicate a body-wall muscle quadrant. A and D show both channels simultaneously, whereas B, C, E, and F show single-channel images. A–C show a wild-type embryo, whereas D–F show an arrested unc-52(st560) mutant embryo. Note the reduced staining of body-wall muscles with GM1 and disorganization of MHC A in the mutant (E and F; compare with B and C). The pharynx and anal muscles (arrowhead in E) in the mutant, however, exhibit a wild-type staining pattern (compare B and E). Embryos in G–L were double-labeled with GM1 (green, FITC) and MH3 (red, TRSC), which recognizes an epitope in domain IV of UNC-52. G–I show a wild-type embryo; J–L show a unc-52(st560) mutant embryo. G and J show both channels simultaneously. Note the absence of MH3 staining in the mutant embryo in L. Although the unc-52(st560) mutant embryos shown are arrested at the twofold stage, they are comparable to threefold wild-type embryos. Bar, 10 μm.

Figure 6

Figure 6

Phalloidin staining in wild-type and unc-52(null) embryos. Embryos were visualized by confocal microscopy. Wild-type (A–C) and unc-52(st549) (D–F) embryos were labeled with FITC-phalloidin. Images in A and D show staining from all focal planes. Computer-generated cross sections (B and E) and single focal plane images (C and F) are also shown. The arrowhead indicates the basal surface of the pharynx, whereas the arrow indicates the basal face of the body-wall muscles. Note the well organized thin filaments extending from the basal and apical faces of the pharynx in both wild-type and mutant embryos. Also note that actin in the body-wall muscles of the mutant is not associated with the basal cell membrane. Bar, 10 μm.

Figure 7

Figure 7

Immunolocalization of UNC-52 and myosin in unc-52(viable) mutants. Wild-type (A and B) and unc-52(e444) adults (C and D) were double-labeled with GM1 (UNC-52) and DM5.6 (MHC A). Note the reduced GM1 staining in C and disorganized myosin in D (compare with A and B). unc-52(viable) mutations disrupt the accumulation of UNC-52 and organization of myosin in late larval and adult animals. Bar, 10 μm.

Figure 8

Figure 8

Expression of UNC-52 isoforms in mec-8; unc-52 double mutants. A shows GM1 staining of a mec-8(u74); unc-52(e1012) double mutant. Note that pharyngeal staining appears to be normal, but body-wall muscle staining is greatly reduced, except over the anteriormost muscles. B shows MH3 staining of the same embryo. Note that body-wall muscle staining is restricted to the anteriormost muscle cells (indicated by arrows). Bar, 10 μm.

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