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Mechanobiology of Disease
Poster Abstracts
106
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.