Colin P Johnson | Oregon State University (original) (raw)

Papers by Colin P Johnson

The FASEB Journal, Apr 1, 2020

F1000 - Post-publication peer review of the biomedical literature, 2010

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its... more A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca 2þ-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca 2þ-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca 2þ-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape. METHODS Methods for PC12 cell culture and amperometry recording follow those described previously (6). PC12 cells were plated at 1-2 Â 10 5 cells in 35-mm dishes coated with 50 mg/mL poly-D-lysine plus 50 mg/mL collagen I, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 mg/mL glucose, 3.7 mg/mL NaHCO 3 , 5% horse serum, and 5% iron-supplemented calf serum at 37 C in a humidified 10% CO 2-90% air atmosphere. PC12 cells were loaded the day before experiments with 1.5 mM norepinephrine (NE) and 0.5 mM ascorbate. Chromaffin cells were prepared by collecting rat adrenal glands in ice cold Hanks' balanced salt solution (HBSS) (http://www.natureprotocols.com/ 2006/09/29/rat_chromaffin_cells_primary_c.php). Medullas were cut into small pieces and dissociated in HBSS with the additions of 2.6 mg/mL collagenase type I, 3 mg/mL bovine serum albumin, 0.15 mg/mL DNase I, and 0.15 mg/mL hyaluronidase IS at 37 C for 25-30 min. The tissue was triturated, and after adding 10 mL cold HBSS the cell suspension was centrifuged at 800 rpm for 10 min at 4 C. Cells were resuspended in 0.8 mL DMEM þ 10% fetal bovine serum and plated on glass coverslips coated with poly-D-lysine. After 1 h, 0.5 mL DMEM þ 10% fetal bovine serum was added and cells were maintained at 37 C in 5% CO 2-air. Culture medium was replaced every 48 h and cells were used within 1-4 days. NE release was measured with a VA-10 amperometry amplifier (ALA Scientific Instruments, Westbury, NY) and 5-mm carbon fiber electrodes polarized to 650 mV (7). Current was filtered at 1 kHz and digitized at 4 kHz with a digidata interface and a PC running PCLAMP 8 (Molecular Devices, Union City, CA). Recordings were carried out at room temperature (20-23 C) with cells bathing in a solution containing (in mM) 150 NaCl, 4.2 KCl, 1 NaH 2 PO 4 , 0.7 MgCl 2 , 2 CaCl 2 , 10 HEPES, pH 7.4. Exocytosis was triggered by pressure ejection of a depolarizing solution or by voltage steps under voltage clamp. The depolarizing solution was applied using pressure from a Picospritzer (Parker-Hannefin, Cleveland, OH) to puff a solution identical to the bathing solution but with 105 mM KCl and 5 mM NaCl. Puffs of 6 s were applied up to five times to a cell at 1-2 min intervals from a micropipette~10 mm away.

Proceedings of the National Academy of Sciences, 2004

The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly ... more The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for seque...

F1000 - Post-publication peer review of the biomedical literature, 2009

Microorganisms, 2021

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allo... more 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 Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing eff...

bioRxiv, 2021

C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the m... more C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the muscular dystrophy associated protein dysferlin is dependent on the C2A domain at the N-terminus of the protein, which couples calcium and PI(4,5)P2 binding through an unknown mechanism. Using solution state nuclear magnetic resonance spectroscopy we confirm the phosphoinositide binding site for the domain and find that calcium binding attenuates millisecond to microsecond motions at both in the calcium binding loops and the concave face of the C2A, including a portion of the phosphoinositide binding site. Our results support a model whereby increasing calcium concentrations shift the phosphoinositide binding pocket of C2A into a binding-competent state, allowing for calcium dependent membrane targeting. This model contrasts with the canonical mechanism for C2 domain-phosphoinositide interaction and provides a basis for how pathogenic mutations in the C2A domain result in loss of function...

Nature Structural & Molecular Biology, 2011

Synaptotagmin-I (syt) is a Ca 2+ sensor that triggers synchronous neurotransmitter release. The f... more Synaptotagmin-I (syt) is a Ca 2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca 2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca 2+-syt to aggregate vesicles and to stimulate SNAREmediated membrane fusion. Moreover, artificial aggregation of membranes-using non-syt proteins-also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca 2+-syt-regulated, SNARE-catalyzed fusion reactions. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Cell, 2009

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulti... more 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. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulationdefective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

Journal of Molecular Biology

Protein Science, 2000

The binding capacity of the l-leucine receptor from Escherichia coli was measured with l-phenylal... more The binding capacity of the l-leucine receptor from Escherichia coli was measured with l-phenylalanine and 4-fluorol-phenylalanine as substrates by fluorescence. The apparent dissociation constants~K D ! for l-leucine, l-phenylalanine, and 4-fluorol-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19 F NMR data show protein-induced shifts for the 4-fluoro-l-phenylalanine peak and 3-fluoro-l-phenylalanine when receptor is present. Evidence points to the binding of only the l-isomers of these fluorine analogs.

The Journal of General Physiology, 2010

Molecular Biology of the Cell

Using both in vitro and in vivo systems, the effect of truncation of the transmembrane domain of ... more Using both in vitro and in vivo systems, the effect of truncation of the transmembrane domain of otoferlin was investigated. We conclude that deficits in sensory hair cell signaling due to truncation stem from both low otoferlin expression levels and deficits in membrane docking.

Scientific Reports, 2019

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otof... more 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. To address this question, we characterized the impact of otoferlin depletion in zebrafish larvae and found defects in synaptic vesicle recycling, abnormal synaptic ribbons, and higher resting calcium concentrations in hair cells. We also observed abnormal expression of the calcium binding hair cell genes s100s and parvalbumin, as well as the nogo related proteins rtn4rl2a and rtn4rl2b. Exogenous otoferlin partially restored expression of genes affected by endogenous otoferlin depletion. Our results suggest that in addition to vesicle recycling, depletion of otoferlin disrupts resting calcium levels, alters synaptic ribbon arch...

Biophysical Journal, 2019

Proteins that contain C2 domains are involved in a variety of biological processes, including enc... more 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. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm À1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm À1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22 normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Molecular Biology of the Cell, 2018

The precise spatial and temporal expression of genes is essential for proper organismal developme... more 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. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein–based assays indicate that F...

Biochemistry, Jan 12, 2017

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membra... more 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.

Biophysical Journal, 2017

The FASEB Journal, Apr 1, 2020

F1000 - Post-publication peer review of the biomedical literature, 2010

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its... more A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca 2þ-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca 2þ-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca 2þ-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape. METHODS Methods for PC12 cell culture and amperometry recording follow those described previously (6). PC12 cells were plated at 1-2 Â 10 5 cells in 35-mm dishes coated with 50 mg/mL poly-D-lysine plus 50 mg/mL collagen I, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 mg/mL glucose, 3.7 mg/mL NaHCO 3 , 5% horse serum, and 5% iron-supplemented calf serum at 37 C in a humidified 10% CO 2-90% air atmosphere. PC12 cells were loaded the day before experiments with 1.5 mM norepinephrine (NE) and 0.5 mM ascorbate. Chromaffin cells were prepared by collecting rat adrenal glands in ice cold Hanks' balanced salt solution (HBSS) (http://www.natureprotocols.com/ 2006/09/29/rat_chromaffin_cells_primary_c.php). Medullas were cut into small pieces and dissociated in HBSS with the additions of 2.6 mg/mL collagenase type I, 3 mg/mL bovine serum albumin, 0.15 mg/mL DNase I, and 0.15 mg/mL hyaluronidase IS at 37 C for 25-30 min. The tissue was triturated, and after adding 10 mL cold HBSS the cell suspension was centrifuged at 800 rpm for 10 min at 4 C. Cells were resuspended in 0.8 mL DMEM þ 10% fetal bovine serum and plated on glass coverslips coated with poly-D-lysine. After 1 h, 0.5 mL DMEM þ 10% fetal bovine serum was added and cells were maintained at 37 C in 5% CO 2-air. Culture medium was replaced every 48 h and cells were used within 1-4 days. NE release was measured with a VA-10 amperometry amplifier (ALA Scientific Instruments, Westbury, NY) and 5-mm carbon fiber electrodes polarized to 650 mV (7). Current was filtered at 1 kHz and digitized at 4 kHz with a digidata interface and a PC running PCLAMP 8 (Molecular Devices, Union City, CA). Recordings were carried out at room temperature (20-23 C) with cells bathing in a solution containing (in mM) 150 NaCl, 4.2 KCl, 1 NaH 2 PO 4 , 0.7 MgCl 2 , 2 CaCl 2 , 10 HEPES, pH 7.4. Exocytosis was triggered by pressure ejection of a depolarizing solution or by voltage steps under voltage clamp. The depolarizing solution was applied using pressure from a Picospritzer (Parker-Hannefin, Cleveland, OH) to puff a solution identical to the bathing solution but with 105 mM KCl and 5 mM NaCl. Puffs of 6 s were applied up to five times to a cell at 1-2 min intervals from a micropipette~10 mm away.

Proceedings of the National Academy of Sciences, 2004

The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly ... more The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for seque...

F1000 - Post-publication peer review of the biomedical literature, 2009

Microorganisms, 2021

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allo... more 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 Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing eff...

bioRxiv, 2021

C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the m... more C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the muscular dystrophy associated protein dysferlin is dependent on the C2A domain at the N-terminus of the protein, which couples calcium and PI(4,5)P2 binding through an unknown mechanism. Using solution state nuclear magnetic resonance spectroscopy we confirm the phosphoinositide binding site for the domain and find that calcium binding attenuates millisecond to microsecond motions at both in the calcium binding loops and the concave face of the C2A, including a portion of the phosphoinositide binding site. Our results support a model whereby increasing calcium concentrations shift the phosphoinositide binding pocket of C2A into a binding-competent state, allowing for calcium dependent membrane targeting. This model contrasts with the canonical mechanism for C2 domain-phosphoinositide interaction and provides a basis for how pathogenic mutations in the C2A domain result in loss of function...

Nature Structural & Molecular Biology, 2011

Synaptotagmin-I (syt) is a Ca 2+ sensor that triggers synchronous neurotransmitter release. The f... more Synaptotagmin-I (syt) is a Ca 2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca 2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca 2+-syt to aggregate vesicles and to stimulate SNAREmediated membrane fusion. Moreover, artificial aggregation of membranes-using non-syt proteins-also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca 2+-syt-regulated, SNARE-catalyzed fusion reactions. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Cell, 2009

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulti... more 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. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulationdefective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

Journal of Molecular Biology

Protein Science, 2000

The binding capacity of the l-leucine receptor from Escherichia coli was measured with l-phenylal... more The binding capacity of the l-leucine receptor from Escherichia coli was measured with l-phenylalanine and 4-fluorol-phenylalanine as substrates by fluorescence. The apparent dissociation constants~K D ! for l-leucine, l-phenylalanine, and 4-fluorol-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19 F NMR data show protein-induced shifts for the 4-fluoro-l-phenylalanine peak and 3-fluoro-l-phenylalanine when receptor is present. Evidence points to the binding of only the l-isomers of these fluorine analogs.

The Journal of General Physiology, 2010

Molecular Biology of the Cell

Using both in vitro and in vivo systems, the effect of truncation of the transmembrane domain of ... more Using both in vitro and in vivo systems, the effect of truncation of the transmembrane domain of otoferlin was investigated. We conclude that deficits in sensory hair cell signaling due to truncation stem from both low otoferlin expression levels and deficits in membrane docking.

Scientific Reports, 2019

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otof... more 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. To address this question, we characterized the impact of otoferlin depletion in zebrafish larvae and found defects in synaptic vesicle recycling, abnormal synaptic ribbons, and higher resting calcium concentrations in hair cells. We also observed abnormal expression of the calcium binding hair cell genes s100s and parvalbumin, as well as the nogo related proteins rtn4rl2a and rtn4rl2b. Exogenous otoferlin partially restored expression of genes affected by endogenous otoferlin depletion. Our results suggest that in addition to vesicle recycling, depletion of otoferlin disrupts resting calcium levels, alters synaptic ribbon arch...

Biophysical Journal, 2019

Proteins that contain C2 domains are involved in a variety of biological processes, including enc... more 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. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm À1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm À1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22 normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Molecular Biology of the Cell, 2018

The precise spatial and temporal expression of genes is essential for proper organismal developme... more 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. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein–based assays indicate that F...

Biochemistry, Jan 12, 2017

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membra... more 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.

Biophysical Journal, 2017