Dysferlin C2A Research Articles

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlinopathy.

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 also ineffective 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 alone or 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. Supported by the Jain Foundation, and NIH (RO1 AR064268).

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its β-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its β-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

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 10-50nM 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 10nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60nM, 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.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

Direct Evidence That Mutations within Dysferlin's C2A Domain Inhibit Lipid Clustering.

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

Effect of Bapta and Dysferlin's C2A Domain on Recovery of Ca2D Transients After Osmotic Shock in Dysferlin-Null Myofibers

Effect of Bapta and Dysferlin's C2A Domain on Recovery of Ca2D Transients After Osmotic Shock in Dysferlin-Null Myofibers

Conformational Consequences of Phosphoinositide Binding to Dysferlin C2A

Conformational Consequences of Phosphoinositide Binding to Dysferlin C2A

Structural Basis for the Distinct Membrane Binding Activity of the Homologous C2A Domains of Myoferlin and Dysferlin

Dysferlin has been implicated in acute membrane repair processes, whereas myoferlin's activity is maximal during the myoblast fusion stage of early skeletal muscle cell development. Both proteins are similar in size and domain structure; however, despite the overall similarity, myoferlin's known physiological functions do not overlap with those of dysferlin. Here we present for the first time the X‐ray crystal structure of human myoferlin C2A to 1.9 Å resolution bound to two divalent cations, and compare its three-dimensional structure and membrane binding activities to that of dysferlin C2A. We find that while dysferlin C2A binds membranes in a Ca2+-dependent manner, Ca2+ binding was the rate-limiting kinetic step for this interaction. Myoferlin C2A, on the other hand, binds two calcium ions with an affinity 3-fold lower than that of dysferlin C2A; and, surprisingly, myoferlin C2A binds only marginally to phospholipid mixtures with a high fraction of phosphatidylserine.

Role of Dysferlin's C2A Domain in Voltage-Induced Calcium Release After Osmotic Shock in Murine Skeletal Myofibers

Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2+-transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). We recently showed using OSI that dysferlin modulates the coupling of excitation to Ca2+ release (Lukyanenko et al., J. Physiol. 2017). Here we studied dysferlin-null A/J mouse FDB myofibers expressing mutated fluorescent dysferlin chimeras to examine the role of dysferlin's C2A domain in that coupling. We studied dysferlin missing C2A as well as C2A point mutations (pathogenic: W52R, V67D; polymorphisms: V68L; A84V), and Venus-C2A alone, expressed with and without dysferlin's transmembrane (TM) domain. Dysferlin-ΔC2A, -W52R and -V67D reduced recovery of VICTs after OSI (to 37%, 43% and 31% of controls, respectively). Dysf-V68L (72%) and -A84V (72%) were statistically identical to WT Venus-dysferlin (69%). Dysferlin localization to t-tubules, examined with C-terminal pHluorin constructs by reducing extracellular pH to 6.5 showed ∼60% reduction of fluorescence for WT Dysf-pHluorin fluorescence at pH6.5, 10-15% for the negative control (N-terminal pHluorin), and 10%, 35% and ∼60% for Dysf-W52R, -V67D, and -V68L, -A84V, and -ΔC2A, respectively. Thus, all mutants except Dysf-W52R were incorporated into t-tubules either partially or as efficiently as wild type. Venus-C2A alone, expressed with or without the TM domain, did not support the recovery of the VICT after OSI (both ∼50%). However, Venus-C2A lacking the TM domain significantly reduced Ca2+ waves after OSI (10% vs 83% of Venus- C2A-TM). We conclude that C2A modulates coupling of excitation to Ca2+ release in murine skeletal muscle, and that changes in coupling (e.g., with the Dysf-W52R and -V67D mutations) may be sufficient for pathogenicity. Supported by the Jain Foundation, and NIH (RO1 AR064268).

Dysferlin C2A Domain Is Involved in Recovery of Voltage-Induced SR Calcium Release after Osmotic Shock in Murine Muscle Fibers

Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2+-transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). Using the OSI assay, we recently showed that dysferlin modulates the coupling of excitation to Ca2+ release in murine muscle fibers (Lukyanenko et al., J. Physiol. 2017). Here we studied dysferlin-null A/J mouse muscle fibers expressing mutated Venus-dysferlin chimeras to reveal the region of the dysferlin molecule responsible for that coupling. We examined the effects of deletion of the domain C2A and point mutations, V67D, R1022Q, I1298V, R1331L, I1298V, and K1598N, on VICT following OSI. Deletion of C2A and the V67D mutant, located in the C2A domain, inhibited recovery of VICTs after OSI (37% and 31%, respectively), while the R1022Q (77%), R1331L (68%), I1298V (61%), and K1598N (56%) mutations were statistically identical to wild type Venus-dysferlin chimera (69%). Dysferlin localization to transverse (t) tubules with the C2A deletants and each of the point mutations was examined with C-terminal pHluorin constructs by changing extracellular pH from 7.4 to 6.5. The positive control showed ∼60% reduction of fluorescence at pH=6.5, whereas negative controls showed 10-15% reduction; V67D and R1022Q values were about 35% and R1331L, I1298V, K1598N and C2A about 60%. Thus the mutants incorporated into T-tubules either partially (V67D, R1022Q) or as efficiently as wild type (R1331L, I1298V, K1598N, C2A). We conclude that domain C2A modulates the coupling of excitation to Ca2+ release in murine skeletal muscle, and that C2A deletion or mutation is pathogenic. Supported by the Jain Foundation, MDA and NIH (RO1 AR064268).

Inversion of Ligand Binding Preferences in Re-Engineered Dysferlin C2Av1

Dysferlin is a 237 kDa skeletal muscle protein that plays a key role in Ca2+-mediated membrane repair of muscle cells. The full length protein possesses seven tandem C2 domains and a single C-terminal transmembrane helix. Mutations in the dysferlin protein have been linked to Limb-Girdle Muscular Dystrophy in humans. The canonical C2A domain of dysferlin binds Ca2+ and phospholipid similar to other Ca2+-dependent phospholipid binding C2 domains; however, another co-expressed isoform of dysferlin C2A, C2A variant 1 (C2Av1), binds negatively changed phospholipid, but not Ca2+. To test whether we could modulate the binding behavior of the alternate domain with a simple mutation, we mutated two Arg residues (R20 and R21) in Loop 1 of C2Av1 to Asp, and tested the resulting protein for Ca2+ and phospholipid binding. Interestingly, the resulting domain (C2Av1 R20D,R21D) bound a single calcium ion but not phospholipid, a complete inversion of binding preference. Our result also supports the hypothesis that the C2A domains of dysferlin interact with negatively charged phospholipid surfaces using only electrostatic attraction. The 3D structure of the domain including ligand was obtained using X-Ray Crystallography.

Alternate Splicing of Dysferlin C2A Confers Ca2+-Dependent and Ca2+-Independent Binding for Membrane Repair

Alternate Splicing of Dysferlin C2A Confers Ca2+-Dependent and Ca2+-Independent Binding for Membrane Repair

Alternate Splicing of Dysferlin C2A Confers Ca2+-Dependent and Ca2+-Independent Binding for Membrane Repair

Dysferlin plays a critical role in the Ca²⁺-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²⁺ and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1). Interestingly, these isoforms have markedly different responses to Ca²⁺ and phospholipid. Structural and thermodynamic analyses are consistent with the canonical C2A domain as a Ca²⁺-dependent, phospholipid-binding domain, whereas C2Av1 would likely be Ca²⁺-independent under physiological conditions. Additionally, both isoforms display remarkably low free energies of stability, indicative of a highly flexible structure. The inverted ligand preference and flexibility for both C2A isoforms suggest the capability for both constitutive and Ca²⁺-regulated effector interactions, an activity that would be essential in its role as a mediator of membrane repair.

Thermodynamic Comparison of Dysferlin C2A Wild Type, and C2A V-1 Domains, to the Synaptotamin I C2A and C2B Domains

Through regular use the plasma membrane or, sarcolemma, of myocytes undergoes a number of physical disruptions which in order for the cell to survive must be effectively and rapidly repaired. Currently, it is believed that small lesions in the cellular membrane are repaired through a calcium dependent mechanism which patches the lesion through the rapid exocytosis of intracellular vesicles. Synaptotagmin I is a transmembrane protein which has been shown to play an integral role in vesicular fusion in neurons. Dysferlin is highly homologous to Synaptotamin I which has led to speculation that it plays a critical role in the final steps of vesicular fusion leading to the formation of a membrane patch over the lesion. Dysferlin consists of a single transmembrane region linked to seven C2 domains and two DYSF domains. A C2 domain is a structural motif that consists of an eight strand β-sandwich; these domains are involved in binding both calcium and phospholipids. In the present study, the stability of the wild type Dysferlin C2A domain and an isoform of the C2A domain, V-1, are compared to the stabilities of the C2A and C2B domains of Synaptotagmin I. By comparing these domains we have gained valuable insight into the physiological function of Dysferlin. In the absence of ligand the free energy of denaturation at 37°C of the Dysferlin C2A domains was found to be 0.17±0.02 and 0.51±0.02 in (kcal/mol) for the wild type and V-1 isoform respectively, compared to 2.32±0.05 and 1.74±0.9 in (kcal/mol) for the Synaptotagmin I C2A and C2B domains.

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5Å resolution X-ray structure shows that a single region from the AHNAK Cterminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane.

Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production--an important step that mediates membrane raft clustering--was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation.

Characterization of Lipid Binding Specificities of Dysferlin C2 Domains Reveals Novel Interactions with Phosphoinositides

Dysferlin is a type II transmembrane protein implicated in Ca(2+)-dependent sarcolemmal membrane repair. Dysferlin has seven C2 domains, which are lipid and protein binding modules. In this study, we sought to characterize the lipid binding specificity of dysferlin's seven C2 domains. Dysferlin's C2A domain was able to bind to phosphatidylserine (PS), phosphatidylinositol 4-phosphate [PtdIns(4)P], and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in a Ca(2+)-dependent fashion. The remainder of the C2 domains exhibited weaker and Ca(2+)-independent binding to PS and no significant binding to phosphoinositides.

Calcium-sensitive Phospholipid Binding Properties of Normal and Mutant Ferlin C2 Domains

Mutations in dysferlin, a novel membrane protein of unknown function, lead to muscular dystrophy. Myoferlin is highly homologous to dysferlin and like dysferlin is a plasma membrane protein with six C2 domains highly expressed in muscle. C2 domains are found in a variety of membrane-associated proteins where they have been implicated in calcium, phospholipid, and protein-binding. We investigated the pattern of dysferlin and myoferlin expression in a cell culture model of muscle development and found that dysferlin is expressed in mature myotubes. In contrast, myoferlin is highly expressed in elongated "prefusion" myoblasts and is decreased in mature myotubes where dysferlin expression is greatest. We tested ferlin C2 domains for their ability to bind phospholipid in a calcium-sensitive manner. We found that C2A, the first C2 domain of dysferlin and myoferlin, bound 50% phosphatidylserine and that phospholipid binding was regulated by calcium concentration. A dysferlin point mutation responsible for muscular dystrophy was engineered into the dysferlin C2A domain and demonstrated reduced calcium-sensitive phospholipid binding. Based on these data, we propose a mechanism for muscular dystrophy in which calcium-regulated phospholipid binding is abnormal, leading to defective maintenance and repair of muscle membranes.