Louise Cottle1, Wan Jun Gan1,2, Ian Gilroy1, Jaswinder S Samra3,4, Anthony J Gill3,5,6, Thomas Loudovaris7, Helen E Thomas7,8, Wayne J Hawthorne9,10, Melkam A Kebede1, Peter Thorn11. 1. Charles Perkins Centre, Discipline of Physiology, School of Medical Sciences, University of Sydney, Camperdown, NSW, Australia. 2. Temasek Life-Science Laboratory, Singapore, Republic of Singapore. 3. The University of Sydney Northern Clinical School, Sydney, NSW, Australia. 4. Upper Gastrointestinal Surgical Unit, Royal North Shore Hospital, St Leonards, NSW, Australia. 5. Department of Anatomical Pathology, Royal North Shore Hospital, St Leonards, NSW, Australia. 6. Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research, St Leonards, NSW, Australia. 7. St Vincent's Institute, Fitzroy, VIC, Australia. 8. The University of Melbourne, Department of Medicine, St Vincent's Hospital, Fitzroy, VIC, Australia. 9. Centre for Transplant and Renal Research, Westmead Hospital, Sydney, NSW, Australia. 10. Westmead Clinical School, Faculty of Health and Medicine, University of Sydney, Sydney, Australia. 11. Charles Perkins Centre, Discipline of Physiology, School of Medical Sciences, University of Sydney, Camperdown, NSW, Australia. p.thorn@sydney.edu.au.
Abstract
AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/ INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.
AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/ INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.
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Authors: Ivar Noordstra; Cyntha M van den Berg; Fransje W J Boot; Eugene A Katrukha; Ka Lou Yu; Roderick P Tas; Sybren Portegies; Bastiaan J Viergever; Esther de Graaff; Casper C Hoogenraad; Eelco J P de Koning; Françoise Carlotti; Lukas C Kapitein; Anna Akhmanova Journal: J Cell Sci Date: 2022-02-03 Impact factor: 5.285
Authors: Louise Cottle; Ian Gilroy; Kylie Deng; Thomas Loudovaris; Helen E Thomas; Anthony J Gill; Jaswinder S Samra; Melkam A Kebede; Jinman Kim; Peter Thorn Journal: Metabolites Date: 2021-06-07