Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null skeletal muscle.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

µ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.

µ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6-147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, ß-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that µ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of µ-crystallin are associated with a shift toward greater fat metabolism.

Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle.

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Coupling of excitation to Ca2+ release is modulated by dysferlin.

KEY POINTS: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak. ABSTRACT: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm.

Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1).

SERCA1, the sarco(endo)plasmic reticulum Ca2+-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2+ levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca2+-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca2+-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity.

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle.

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation.

Myopathic changes in murine skeletal muscle lacking synemin.

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle.

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

Distinct regions within fibulin-1D modulate interactions with hemicentin.

Fibulins are evolutionarily conserved extracellular matrix (ECM) proteins that assemble in elastic fibers and basement membranes. Caenorhabditis elegans has a single fibulin gene that produces orthologs of vertebrate fibulin-1 C and D splice forms. In a structure-function analysis of fibulin-1 domains, a series of deletion constructs show that EGF repeats 4 and 5 are required for the hemicentin-dependent assembly and function of fibulin-1D in native locations. In contrast, constructs missing the second EGF repeat of fibulin-1D (EGF2D) assemble in ectopic locations in a hemicentin dependent manner. Constructs that contain EGF2D are cleaved into two fragments, but constructs with EGF2D missing are not, suggesting that a protease binds and/or cleaves fibulin-1D at a site that is likely within EGF2D. Together, the data suggests that EGF repeats 4 and 5 promote interaction with hemicentin while a region within EGF2D suppresses ectopic interactions with hemicentin and this suppression may be protease dependent.

Physiology, structure, and susceptibility to injury of skeletal muscle in mice lacking keratin 19-based and desmin-based intermediate filaments.

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.

Use of autologous platelet-rich plasma to treat muscle strain injuries.

BACKGROUND: Standard nonoperative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery. HYPOTHESIS: Local delivery of platelet-rich plasma to injured muscles hastens recovery of function. STUDY DESIGN: Controlled laboratory study. METHODS: In vivo, the tibialis anterior muscles of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which resulted in a significant injury. The tibialis anterior either was injected with platelet-rich plasma, was injected with platelet-poor plasma as a sham treatment, or received no treatment. RESULTS: Both injury protocols yielded a similar loss of force. The platelet-rich plasma only had a beneficial effect at 1 time point after the single contraction injury protocol. However, platelet-rich plasma had a beneficial effect at 2 time points after the multiple contraction injury protocol and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared with no treatment. CONCLUSION: Local delivery of platelet-rich plasma can shorten recovery time after a muscle strain injury in a small-animal model. Recovery of muscle from the high-repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why platelet-rich plasma was more effective in the high-repetition protocol, because platelet-rich plasma is rich in growth factors that can stimulate myogenesis. CLINICAL RELEVANCE: Because autologous blood products are safe, platelet-rich plasma may be a useful product in clinical treatment of muscle injuries.

Hemicentins: what have we learned from worms?

Hemicentins are conserved extracellular matrix proteins discovered in Caenorhabditis elegans, with orthologs in all vertebrate species including human and mouse. Hemicentins share a single, highly conserved amino-terminal von Willebrand A domain, followed by a long (>40) stretch of immunoglobulin repeats, multiple tandem epidermal growth factors and a fibulin-like carboxy-terminal module. C. elegans has a single hemicentin gene that has pleiotropic functions in transient cell contacts that are required for cell migration and basement membrane invasion and in stable contacts at hemidesmosome-mediated cell junctions and elastic fiber-like structures. Here, we summarize what is known about the function of hemicentin in C. elegans and discuss implications for hemicentin function in other species.

Selective assembly of fibulin-1 splice variants reveals distinct extracellular matrix networks and novel functions for perlecan/UNC-52 splice variants.

Fibulin-1C and fibulin-1D splice variants have been conserved throughout metazoan evolution and have distinct functions in Caenorhabditis elegans development. Both splice variants are required for the assembly of hemidesmosome-mediated mechanosensory neuron and uterine attachments, although the molecular associations that underlie their distinct functions at these locations are not known. Here, we show that the assembly of fibulin-1C and fibulin-1D splice variants at these anchorages is dependent upon distinct components of the extracellular matrix (ECM): Fibulin-1D assembly at uterine and mechanosensory neurons attachments is dependent upon a perlecan/ UNC-52 splice variant that includes alternately spliced IG8-IG10, whereas the assembly of fibulin-1C at mechanosensory neuron attachments is dependent upon laminin/ EPI-1. These data not only indicate that fibulin-1C and fibulin-1D are components of distinct networks of ECM but also demonstrates a novel function for a major class of perlecan splice variants found in C. elegans and mouse. In addition, we demonstrate that overexpression of another ECM protein, collagen XVIII, can suppress gonad morphogenesis defects associated with loss of fibulin-1C, suggesting that some genetic defects that result in a weakened basement membrane can be compensated by overexpression of genes for ECM components that stabilize basement membranes.

Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules.

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.

Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner.

Fibulins are a family of extracellular glycoproteins associated with basement membranes and elastic fibers in vertebrates. Conservation of the fibulin-1 gene throughout metazoan evolution includes fibulin-1C and fibulin-1D alternate splice variants, although little is known about variant specific functions that would justify this striking structural conservation. We have therefore investigated the structure, localization and loss-of-function phenotype specific to both fibulin-1 variants in C. elegans. We find that fibulin-1C has specific roles during pharynx, intestine, gonad and muscle morphogenesis, being required to regulate cell shape and adhesion, whereas fibulin-1D assembles in flexible polymers that connect the pharynx and body-wall-muscle basement membranes. The assembly of fibulin-1C and fibulin-1D in multiple locations is dependent upon the presence of hemicentin, a recently described extracellular member of the immunoglobulin superfamily. We suggest that the distinct developmental roles and hemicentin-dependent assembly for fibulin-1 splice variants demonstrated here may be relevant to fibulin-1 and possibly other fibulin family members in non-nematode species.

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape.

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.

Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.