Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Poster Abstracts

21-POS Board 21 Control of Insulin Secretion by Basement Membrane Proteins Wan Jun Gan 1 , Elena Kosobrodova 2 , Marcela Bilek 2 , Peter Thorn 1 . 1 School Medical Sciences, University of Sydney, Camperdown, NSW, Australia, 2 School of Physics, University of Sydney, Camperdown, NSW, Australia. Cell-based therapies such as islet transplantation or engineering of stem cells, to restore insulin secretion, are promising treatments for type-1 diabetic patients. However, these cells typically secrete less insulin than native beta cells. Our recent work, in intact islets, shows that beta cells are structurally polarised and that insulin secretion is targeted towards the vasculature. This led us to hypothesize that correct orientation of secretory machinery may be important in normal glucose-induced response. We are currently investigating whether signals from the vascular basement membrane provide cues for the establishment and maintenance of beta cell orientation. We isolate mouse islets and then break them down into single cells. Immunostaining for insulin shows that 85% of the isolated cells are beta cells. When these cells are cultured on gelatin (denatured collagen) coated coverslips, focal adhesion proteins such as talin and synaptic proteins, such as liprin, are selectively enriched at the interface of the beta cells with the coverslip. To investigate secretory function, we use live-cell two photon imaging to identify each insulin granule as it fuses with the cell membrane, in response to 15 mM glucose stimulus. Sequential Z stack imaging (2um/steps, 6 steps/stack), through the cells, enables us to determine the 3D distribution of fusion events. Our data show that 45% of granules fuse in the limited area of the membrane-coverslip interface, indicating enrichment of fusion. Currently experiments are underway to understand the mechanisms that orientate the beta cells and targeting secretion. To this end we are printing micropatterns, of different basement membrane proteins, using plasma immersion ion implantation treated polystyrene to covalently immobilize dense monolayers of protein directly from PDMS stamps. In conclusion, evidence indicates that beta cells are specifically orientated in the islets and this affects their structure and function.

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