The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology.

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.

Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pf-5.

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allows producers to quickly amplify the production of antibiotics in response to environmental cues. Antibiotic autoinduction generally involves one pathway-specific transcriptional regulator that perceives an antibiotic as a signal and then directly stimulates transcription of the antibiotic biosynthesis genes. Pyoluteorin is an autoregulated antibiotic produced by some spp.

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain.

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.

Otoferlin C2F Domain-Induced Changes in Membrane Structure Observed by Sum Frequency Generation.

Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface.

Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish.

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otoferlin result in deafness and depending on the species, mild to strong vestibular deficits. While studies in mouse models suggest a role for otoferlin in synaptic vesicle exocytosis and endocytosis, it is unclear whether these functions are conserved across species.

Fer1l6 is essential for the development of vertebrate muscle tissue in zebrafish.

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development.

Emerging Functional Differences between the Synaptotagmin and Ferlin Calcium Sensor Families.

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels.

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate.

Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner.

Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE-mediated exocytotic events including cytokine release and acid sphingomyelinase secretion.

Characterization of the lipid binding properties of Otoferlin reveals specific interactions between PI(4,5)P2 and the C2C and C2F domains.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct.

The C2 domains of otoferlin, dysferlin, and myoferlin alter the packing of lipid bilayers.

Ferlins are large multi-C2 domain membrane proteins involved in membrane fusion and fission events. In this study, we investigate the effects of binding of the C2 domains of otoferlin, dysferlin, and myoferlin on the structure of lipid bilayers. Fluorescence measurements indicate that multi-C2 domain constructs of myoferlin, dysferlin, and otoferlin change the lipid packing of both small unilamellar vesicles and giant plasma membrane vesicles.

Mechanism and function of synaptotagmin-mediated membrane apposition.

Synaptotagmin-1 is a Ca(2+) sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca(2+). However, the mechanism and function of this process were poorly understood.

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion.

Otoferlin is a large multi-C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13-25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold.

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity.

Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating.

Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work.

Forced unfolding of proteins within cells.

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells.

Pathogenic proline mutation in the linker between spectrin repeats: disease caused by spectrin unfolding.

Pathogenic mutations in alpha and beta spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin alpha-betatetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of alpha-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5.

Structural studies of the neural-cell-adhesion molecule by X-ray and neutron reflectivity.

The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neural-cell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extracellular region.

Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion.

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion.

Engineered protein a for the orientational control of immobilized proteins.

This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A. The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports. The fragment was expressed at high yield in E.