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

Therrien, Christian; Fulvio, Sabrina Di; Pickles, Sarah; Sinnreich, Michael

doi:10.1021/bi802242r

Cited by 89 publications

(136 citation statements)

References 30 publications

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“…Interestingly, biophysical studies on the C2A domain have determined that this is the only domain in otoferlin that does not bind calcium (12,53). However, studies on the otoferlin orthologues myoferlin and dysferlin have found that the C2A domains of these proteins do bind calcium, supporting the idea that the calcium binding activity of the C2A domain may have diverged among the ferlins (12,19,54,55).…”

Section: Discussionmentioning

confidence: 99%

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Chatterjee

Padmanarayana

Abdullah

et al. 2015

Molecular and Cellular Biology

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Sensory hair cells convert mechanical motion into chemical signals.Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.H air cells couple mechanical motion to neurotransmitter release at synapses (1). In contrast to conventional neural synapses, hair cell synapses release neurotransmitter continuously and, in a graded manner (2), possess synaptic ribbons (2-4), and lack synaptophysin (5), complexin (6-9), Munc13 (10), and the calcium sensors synaptotagmin I and II (11). In place of synaptotagmin, it is believed that otoferlin may confer calcium sensitivity to evoke neurotransmitter release (12, 13). Otoferlin is a six-C2 domain transmembrane protein expressed in inner, outer, and vestibular hair cells, as well as restricted regions of the brain (13-16). In humans, missense mutations in otoferlin have been linked to hearing loss (17,18), and biochemical studies have determined that otoferlin binds calcium and lipids (12,19), as well as membrane trafficking proteins (12,(20)(21)(22)(23). Further, in vitro assays have demonstrated that otoferlin accelerates SNARE-mediated membrane fusion (12). Based upon this evidence, it is hypothesized that otoferlin functions as a calcium-sensitive regulator of neurotransmitter release in sensory hair cells.However, the results of several studies have raised questions related to otoferlin's function. For instance, despite otoferlin expression in vestibular hair cells, knockout mice show no balance defects (24, 25) despite reduced exocytosis in vestibular type I hair cells (24,26). This raises questions as to the importance of otoferlin in this system. Further, otoferlin did not rescue synchronous neurotransmitter release in synaptotagmin I knockout cultured neurons, indicating that otoferlin and synaptotagmin are not functionally redundant (2...

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Section: Discussionmentioning

confidence: 99%

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Chatterjee

Padmanarayana

Abdullah

et al. 2015

Molecular and Cellular Biology

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“…Synaptotagmin 1 possesses two C2 domains, with each showing distinct calcium-regulated binding to different phospholipid membranes (28,29), and different SNARE proteins (30,31). Dysferlin has seven putative C2 domains, of which four show potential calcium sensitivity (32), suggesting even more modular complexity.…”

Section: Discussionmentioning

confidence: 99%

Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

Evesson

Peat

Lek

et al. 2010

Journal of Biological Chemistry

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Ferlins are an ancient family of C2 domain-containing proteins, with emerging roles in vesicular trafficking and human disease. Dysferlin mutations cause inherited muscular dystrophy, and dysferlin also shows abnormal plasma membrane expression in other forms of muscular dystrophy. We establish dysferlin as a shortlived (protein half-life ϳ4 -6 h) and transitory transmembrane protein (plasma membrane half-life ϳ3 h), with a propensity for rapid endocytosis when mutated, and an association with a syntaxin-4 endocytic route. Dysferlin plasma membrane expression and endocytic rate is regulated by the C2B-FerI-C2C motif, with a critical role identified for C2C. Disruption of C2C dramatically reduces plasma membrane dysferlin (by 2.5-fold), due largely to accelerated endocytosis (by 2.5-fold). These properties of reduced efficiency of plasma membrane expression due to accelerated endocytosis are also a feature of patient missense mutant L344P (within FerI, adjacent to C2C). Importantly, dysferlin mutants that demonstrate accelerated endocytosis also display increased protein lability via endosomal proteolysis, implicating endosomal-mediated proteolytic degradation as a novel basis for dysferlin-deficiency in patients with single missense mutations. Vesicular labeling studies establish that dysferlin mutants rapidly transit from EEA1-positive early endosomes through to dextran-positive lysosomes, co-labeled by syntaxin-4 at multiple stages of endosomal transit. In summary, our studies define a transient biology for dysferlin, relevant to emerging patient therapeutics targeting dysferlin replacement. We introduce accelerated endosomal-directed degradation as a basis for lability of dysferlin missense mutants in dysferlinopathy, and show that dysferlin and syntaxin-4 similarly transit a common endosomal pathway in skeletal muscle cells.

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“…1, A and B). However, C2 domains from several other proteins do not possess all five aspartate residues yet, in some cases, are still able to bind calcium (Therrien et al, 2009). Therefore, it is unclear from inspection of the sequence as to which domains of otoferlin, if any, bind calcium.…”

Section: Otoferlin Is a Calcium Sensormentioning

confidence: 99%

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

Johnson¹,

Chapman²

2010

J Gen Physiol

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

Cited by 89 publications

References 30 publications

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

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

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