tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells
Abstract Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocy...
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Wiley
2025-02-01
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| Series: | FASEB BioAdvances |
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| Online Access: | https://doi.org/10.1096/fba.2024-00131 |
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| author | Aishwarya A. Makam Shruti Sharma Prajwal Nagle Nandhini M. Sundaram Vidya Mangala Prasad Nikhil R. Gandasi |
| author_facet | Aishwarya A. Makam Shruti Sharma Prajwal Nagle Nandhini M. Sundaram Vidya Mangala Prasad Nikhil R. Gandasi |
| author_sort | Aishwarya A. Makam |
| collection | DOAJ |
| description | Abstract Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocytosis of insulin can occur via (1) single LDCVs fusing with the plasma membrane to release their content or (2) multiple vesicles are involved during a process called compound exocytosis. Compound exocytosis represents a specialized form of secretion in which vesicles undergo homotypic fusion either before (multi‐vesicular exocytosis) or continuous fusion in a sequential manner from (sequential exocytosis) within the same site at the plasma membrane. We see that the number of multi‐vesicles is few and not localized in the vicinity of the plasma membrane. Studying the kinetics of this process and correlating it with biphasic insulin secretion is not possible since there are no specific probes to detect them. It is challenging to identify compound exocytosis with probes that exist for simple exocytosis. To advance our understanding, we need a fluorescent probe that could detect secretory vesicles undergoing compound exocytosis and allow us to distinguish it from other modes of exocytosis. Here, we used two cargo proteins (NPY and tPA) labeled with different fluorescent proteins (mCherry GFP and eGFP) and employed total internal reflection fluorescence microscopy (TIRF‐M) to capture distinct single‐granule and multi‐granular fusion events. We identified tPA‐GFP as a better probe for studying compound exocytosis, as it can detect both simple and sequential exocytosis reliably. Using these probes, we have studied the kinetics of compound exocytosis in human β‐cells. These observations, with additional experiments, may open a whole new field to study the impact of compound exocytosis on biphasic secretion of insulin. Identifying targets to increase the compound exocytosis process can help potentiate insulin secretion in diabetics. |
| format | Article |
| id | doaj-art-4cbf9794003f4d16b92879ef303a6639 |
| institution | Kabale University |
| issn | 2573-9832 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Wiley |
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| series | FASEB BioAdvances |
| spelling | doaj-art-4cbf9794003f4d16b92879ef303a66392025-02-05T11:30:18ZengWileyFASEB BioAdvances2573-98322025-02-0172n/an/a10.1096/fba.2024-00131tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cellsAishwarya A. Makam0Shruti Sharma1Prajwal Nagle2Nandhini M. Sundaram3Vidya Mangala Prasad4Nikhil R. Gandasi5Department of Developmental Biology and Genetics (DBG) Indian Institute of Science (IISc) Bengaluru Karnataka IndiaMolecular Biophysics Unit Indian Institute of Science Bengaluru Karnataka IndiaDepartment of Developmental Biology and Genetics (DBG) Indian Institute of Science (IISc) Bengaluru Karnataka IndiaMolecular Biophysics Unit Indian Institute of Science Bengaluru Karnataka IndiaMolecular Biophysics Unit Indian Institute of Science Bengaluru Karnataka IndiaDepartment of Developmental Biology and Genetics (DBG) Indian Institute of Science (IISc) Bengaluru Karnataka IndiaAbstract Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocytosis of insulin can occur via (1) single LDCVs fusing with the plasma membrane to release their content or (2) multiple vesicles are involved during a process called compound exocytosis. Compound exocytosis represents a specialized form of secretion in which vesicles undergo homotypic fusion either before (multi‐vesicular exocytosis) or continuous fusion in a sequential manner from (sequential exocytosis) within the same site at the plasma membrane. We see that the number of multi‐vesicles is few and not localized in the vicinity of the plasma membrane. Studying the kinetics of this process and correlating it with biphasic insulin secretion is not possible since there are no specific probes to detect them. It is challenging to identify compound exocytosis with probes that exist for simple exocytosis. To advance our understanding, we need a fluorescent probe that could detect secretory vesicles undergoing compound exocytosis and allow us to distinguish it from other modes of exocytosis. Here, we used two cargo proteins (NPY and tPA) labeled with different fluorescent proteins (mCherry GFP and eGFP) and employed total internal reflection fluorescence microscopy (TIRF‐M) to capture distinct single‐granule and multi‐granular fusion events. We identified tPA‐GFP as a better probe for studying compound exocytosis, as it can detect both simple and sequential exocytosis reliably. Using these probes, we have studied the kinetics of compound exocytosis in human β‐cells. These observations, with additional experiments, may open a whole new field to study the impact of compound exocytosis on biphasic secretion of insulin. Identifying targets to increase the compound exocytosis process can help potentiate insulin secretion in diabetics.https://doi.org/10.1096/fba.2024-00131exocytosisinsulin granuleslarge dense core vesiclestotal internal reflection fluorescence microscopy |
| spellingShingle | Aishwarya A. Makam Shruti Sharma Prajwal Nagle Nandhini M. Sundaram Vidya Mangala Prasad Nikhil R. Gandasi tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells FASEB BioAdvances exocytosis insulin granules large dense core vesicles total internal reflection fluorescence microscopy |
| title | tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells |
| title_full | tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells |
| title_fullStr | tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells |
| title_full_unstemmed | tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells |
| title_short | tPA‐GFP is a reliable probe for detecting compound exocytosis in human pancreatic β‐cells |
| title_sort | tpa gfp is a reliable probe for detecting compound exocytosis in human pancreatic β cells |
| topic | exocytosis insulin granules large dense core vesicles total internal reflection fluorescence microscopy |
| url | https://doi.org/10.1096/fba.2024-00131 |
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