A Region of UNC-89 (Obscurin) Lying between Two Protein Kinase Domains Is a Highly Elastic Spring Required for Proper Sarcomere Organization

Abstract

In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.

Keywords: C. elegans; UNC-89; giant polypeptides; obscurin; sarcomere.

Fig. 1.. Schematic representation of domain organization around kinase domains, and secondary structure predictions for the interkinase regions of nematode UNC-89, mouse obscurin and SPEG.

(A) Domain organization from PFAM predictions. Note that each protein, near its C-terminus consists of a protein kinase, a non-domain “interkinase” region (647–742 residues), Ig and Fn domains, and a second protein kinase domain. (B) Secondary structure predictions of the non-domain interkinase regions of UNC-89, obscurin and SPEG using JPred.

Fig. 2.. The interkinase region (IK) of UNC-89 is a highly elastic spring.

(A) Polyprotein construct encompassing the IK flanked on either side by two titin I27 domains used in single molecule force spectroscopy (SMFS) experiments. (B) SDS-PAGE after Coomassie Blue staining of ~2 μg of the polyprotein shown in (A). (C) Representative force curve of the mechanical unfolding of the polyprotein by SMFS. The dotted line is a worm-like-chain (WLC) fit to the force-extension curve with free contour length L_C and a fixed persistence length L_p = 0.36 nm. (D) Statistical distribution of the contour length of the first peak (L_C = 186 ± 3 nm, n=18). The solid line represents a Gaussian fit of the histogram.

Fig. 3.. Addition of HA tags at the N-termini of the largest UNC-89 isoforms does not affect the normal M-line localization of UNC-89 in body wall muscle sarcomeres, but does moderately reduce the level of UNC-89 giant isoforms in whole animal extracts.

(A) Total Laemmli-soluble proteins from wild type and unc-89(syb797) were separated on a 5% SDS-PAGE, transferred to membrane and reacted with antibody EU30, a well-established rabbit polyclonal antibody to UNC-89. On the right is shown Ponceau S staining of the blots before reaction with antibodies. Note the presence of HA tags in unc-89(syb797) but not in wild type, and a moderate reduction in the level of UNC-89 giant isoforms in unc-89(sy797) compared with wild type. The asterisk indicates the position of the UNC-89 giant isoforms. The thin upper-most band is likely to be some UNC-89 that could not enter the separating gel. The positions of size markers are shown on the left-hand side. (B) Co-immunostaining of parts of two body wall muscle cells in unc-89(syb797) with anti-HA and MH42, a well-established monoclonal that recognizes UNC-89. Note the co-localization of each antibody to the M-lines of these muscle sarcomeres (merged image). Scale bar, 10 μm.

Fig. 4.. Addition of HA tags at the N-termini of the largest UNC-89 isoforms does not affect the organization of sarcomeres in body wall muscle.

Each panel shows localization of the indicated sarcomere components using confocal microscopy. Scale bar, 10 μm.

Fig. 5.. unc-89(syb797 syb1257) is an in-frame deletion of most of the IK region and results in mis-localization of UNC-89 in the sarcomeres of body wall muscle.

(A) Representation of the exon (black boxes) and intron (lines) organization of the kinase-encoding region of the unc-89 gene. Blue bar, portion deleted in syb1257 by CRISPR/Cas9. This is a 4,614 bp genomic in-frame deletion which internally deletes 571 aa of the IK beginning 66 aa C-terminal of PK1 and continuing through 10 aa N-terminal of Ig53. (B) Western blots demonstrating that the large UNC-89 isoforms are expressed in unc-89(syb797) and unc-89(syb797 syb1257). Western blots and reactions conducted as in Fig. 3. The asterisk indicates the position of the UNC-89 giant isoforms. The thin upper-most band is likely to be some UNC-89 that could not enter the separating gel. (C) Co-immunostaining of parts of several body wall muscle cells in unc-89(syb797 syb1257) with anti-HA and the anti-UNC-89 antibody MH42. Note the disorganization of M-lines in this mutant. Scale bar, 10 μm.

Fig. 6.. unc-89(syb797 syb1257) animals that express HA-UNC-89 isoforms with an in-frame deletion of the interkinase region have disorganized myofibrils.

Each panel shows representative confocal images of several body wall muscle cells stained with antibodies to the indicated sarcomeric components or with rhodamine-phalloidin. Scale bar, 10 μm.

Fig. 7.. unc-89(syb797 syb1257) animals that express HA-UNC-89 isoforms with an in-frame deletion of the interkinase region show extra A-bands that are not full-depth.

After immunostaining with antibodies to MHC A (A) or antibodies to UNC-89 (B) multiple optical slices with a 0.2 μm interval were obtained by structured illumination microscopy (SIM). Yellow and blue arrows point to examples of short extra A-bands that lie between normally located A-bands and reside close to the outer muscle cell membrane. In comparison, unc-89(syb797) that expresses HA-UNC-89 shows parallel, normally spaced and full-depth A-bands. Scale bar, 5 μm.

Fig. 8.. Whole worm locomotion and muscle force measurements of wild type and various unc-89 mutant alleles.

(A) Swimming assays show that unc-89(syb797) and unc-89(ad539) swim faster than wild type, whereas all other unc-89 mutants, including unc-89(syb797 syb1257) swim slower than wild type. (B) Crawling assays show that unc-89(ad539) crawls faster than wild type, that unc-89(syb797) crawls at the same speed as wild type, and all other unc-89 mutants crawl more slowly than wild type. C) NemaFlex force measurements show that unc-89(tm752) develops the same muscle force as wild type, whereas all other unc-89 mutants develop less force than wild type, although, unc-89(syb797 syb1257), unc-89(e1460), and unc-89(su75) are more severely affected. The actual data, including sample sizes, means, and standard errors are shown in Supplementary Table 1 for swimming and crawling and Supplementary Table 2 for NemaFlex measurements.

Fig. 9.. Polarized light images of the myofilament lattice in the pharynges of wild type and various unc-89 mutant alleles.

Yellow arrows indicate mis-localized arc-like thick filaments at the periphery of the terminal bulb, and blue arrows indicate mis-localized arc-like thick filaments at the periphery of the anterior bulb. Note that unc-89(tm752) shows pharyngeal muscle like wild type, whereas all the other unc-89 mutants show defective organization. unc-89(su75) and unc-89(ad539) are the only alleles that show defects in both the posterior and anterior bulbs. Scale bar, 50 μm.

Fig. 10.. unc-89(syb797) and unc-89(ad539) have reduced levels of expression of UNC-89 in pharyngeal muscle.

The indicated strains were immunostained with antibodies to UNC-89 and the pharyngeal-specific myosin heavy chain C (MHC C), and representative confocal images of the pharynx are shown. Note that unc-89(syb797) shows reduced UNC-89 expression in the terminal bulb, whereas unc-89(ad539) shows reduced UNC-89 expression in both the terminal and anterior bulbs. Green arrowhead, anterior bulb; red arrowhead, posterior bulb; yellow arrow, arc-like thick filaments mis-placed at the periphery of the posterior bulb. Scale bar, 10 μm.