Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion
- Charles Perkins Centre, School of Medical Sciences, University of Sydney, Australia
- School of Physics, University of Sydney, Australia
- School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, Australia
- Sydney Nanoscience Institute, University of Sydney, Australia
Figures
Figure 1 with 1 supplement
Pancreatic slices have an intact capillary bed.
Integrin-β1, phosphorylated focal adhesion kinase (FAK), and liprin are enriched at the β-cell capillary interface. Pancreas slices and isolated islets were cultured overnight prior to fixation and …
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Figure 1—source data 1
pFAK area analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig1-data1-v2.xlsx
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Figure 1—source data 2
CD31 area analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig1-data2-v2.xlsx
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Figure 1—source data 3
Liprin distribution analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Immunostaining for E-cadherin and PAR3 in slices and isolated islets.
Immunostaining for E-cadherin and laminin in (A) slices and (B) isolated islets shows loss of laminin staining in isolated islets but enrichment of E-cadherin at cell–cell interfaces in both …
Figure 2
Glucose-stimulated insulin granule fusion in isolated islets and pancreas slices.
(A) Isolated islets and (B) pancreas slices, bathed in an extracellular dye (sulforhodamine B, SRB). This dye outlines each cell and is enriched in the islet capillaries (Low et al., 2013) which are …
Figure 3 with 1 supplement
Glucose-stimulated secretion in isolated islets and pancreas slices.
(A) Dose-dependent glucose-stimulated insulin secretion normalised to total cellular insulin content shows that isolated islets are less sensitive to low glucose concentration and secrete a lower …
Figure 3—figure supplement 1
Insulin secretion in pancreatic slices and isolated islets.
(A) Isolated islets, embedded in agarose showed no difference (Student’s t-test) from isolated islets alone in terms of basal and glucose-stimulated insulin secretion. (B) Measures of proinsulin …
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Figure 3—figure supplement 1—source data 1
Insulin secretion data.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig3-figsupp1-data1-v2.xlsx
Figure 4
GCaMP6 recording shows synchronous glucose-induced Ca2+ responses in slices and isolated islets.
GCaMP6 expressed in β cells shows rapid, synchronous Ca2+ responses in slices (A–D) and in isolated islets (E–H) in response to an increase of glucose concentration from 2.8 to 16.7 mM. Images (F) …
Figure 5 with 1 supplement
β-Cell Ca2+ responses in slices have short latencies to peak and higher glucose sensitivity compared to isolated islets.
In slices, (A) single example or (B) averaged responses of Ca2+ measured by changes in GCaMP6 fluorescence in β cells within slices showed large, sustained responses to an increase of glucose from …
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Figure 5—source data 1
GCaMP signal analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig5-data1-v2.xlsx
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Figure 5—source data 2
Time to peak analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig5-data2-v2.xlsx
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Figure 5—source data 3
Calcium wave analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
Example record showing use of fluorescent tracer to indicate addition of high glucose.
In experiments with isolated islets and with slices the high glucose solution contained sulforhodamine B as a fluorescent tracer which was recorded from a region of interest close to the responding …
Figure 6
Fast Ca2+ waves originate at the capillary interface of β cells in slices.
(A–C) β cells within the slices that adjoin the capillaries often showed glucose (16.7 mM)-induced Ca2+ responses that originated at the capillary interface and spread rapidly across the cell …
Figure 7
Focal adhesion kinase (FAK) activation regulates glucose-induced Ca2+ responses.
(A) As before, in the slice preparation, Ca2+ spikes were observed at 2.8 mM glucose, as measured with GCaMP6 fluorescence changes. (B) Pretreatment of slices with 2 µM Y15, an inhibitor of FAK, …
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Figure 7—source data 1
Fura calcium responses and effect of Y15.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig7-data1-v2.xlsx
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Figure 7—source data 2
Secretion and effect of Y15.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig7-data2-v2.xlsx
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Figure 7—source data 3
Secretion and Y15.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig7-data3-v2.xlsx
Figure 8 with 1 supplement
Integrin activation mediates β-cell orientation and glucose-dependent Ca2+ responses.
(A) Immunofluorescence staining of phospho-FAK, E-cadherin, and insulin showed that isolated β cells, cultured on bovine serum albumin (BSA)-coated coverslips, were disorganised. Cells were …
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Figure 8—source data 1
Scaffold protein distribution analysis.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Blockade of integrin activation disrupts β-cell structure.
Glucose-induced Ca2+ responses were recorded using live-cell two-photon microscopy in β cells expressing the Ca2+ indicator GCaMP. GCaMP fluorescence was recorded over 30 min following high glucose …
Figure 9
Focal adhesion kinase (FAK) regulates both Ca2+ responses and positioning of presynaptic scaffold proteins.
In Fura-2-loaded cells (cultured on laminin), we observed the typical robust response to high glucose followed by sustained oscillations in control (A). A smaller, delayed response was observed in …
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Figure 9—source data 1
Effect of Y15 on calcium responses.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig9-data1-v2.xlsx
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Figure 9—source data 2
Insulin secretion and effect of Y15.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig9-data2-v2.xlsx
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Figure 9—source data 3
Scaffold protein distribution and Y15.
- https://cdn.elifesciences.org/articles/76262/elife-76262-fig9-data3-v2.xlsx
