{"title":"Application of High-Throughput Assays to Examine Phospho-Modulation of the Late Steps of Regulated Exocytosis.","authors":"Prabhodh S Abbineni, Jens R Coorssen","doi":"10.3390/ht6040017","DOIUrl":null,"url":null,"abstract":"<p><p><b>Abstract</b><b>:</b> Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca<sup>2+</sup>-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine (e.g., to modulate Ca<sup>2+</sup> sensitivity). Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca<sup>2+</sup> sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca<sup>2+</sup> sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis.</p>","PeriodicalId":53433,"journal":{"name":"High-Throughput","volume":"6 4","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2017-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3390/ht6040017","citationCount":"2","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"High-Throughput","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/ht6040017","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 2
Abstract: Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca2+-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine (e.g., to modulate Ca2+ sensitivity). Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca2+ sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca2+ sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis.
High-ThroughputBiochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
3.60
自引率
0.00%
发文量
0
审稿时长
9 weeks
期刊介绍：
High-Throughput (formerly Microarrays, ISSN 2076-3905) is a multidisciplinary peer-reviewed scientific journal that provides an advanced forum for the publication of studies reporting high-dimensional approaches and developments in Life Sciences, Chemistry and related fields. Our aim is to encourage scientists to publish their experimental and theoretical results based on high-throughput techniques as well as computational and statistical tools for data analysis and interpretation. The full experimental or methodological details must be provided so that the results can be reproduced. There is no restriction on the length of the papers. High-Throughput invites submissions covering several topics, including, but not limited to: -Microarrays -DNA Sequencing -RNA Sequencing -Protein Identification and Quantification -Cell-based Approaches -Omics Technologies -Imaging -Bioinformatics -Computational Biology/Chemistry -Statistics -Integrative Omics -Drug Discovery and Development -Microfluidics -Lab-on-a-chip -Data Mining -Databases -Multiplex Assays
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