1. Volume 23, Issue 7, Pages 816-826 (July 2016)
Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Regulates Ca2+-Influx and Insulin Secretion from Pancreatic β Cells 
Beichen Xie, Phuoc My Nguyen, Alenka Guček, Antje Thonig, Sebastian Barg, Olof Idevall-Hagren 
Cell Chemical Biology 
Volume 23, Issue 7, Pages (July 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 816-826DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1 Ca2+ and PI(4,5)P2 Are Interdependently Regulated
(A) TIRF microscopy recordings of PH-PLCδ1-GFP fluorescence from an MIN6 cell exposed to 30 mM K+ and 1 μM FCCP.
(B) Quantification of the response in (A). Data from 26 cells are presented as means ± SEM.
(C) TIRF microscopy recordings of PH-PLCδ1-GFP fluorescence in an α-toxin permeabilized MIN6 cell exposed to the indicated Ca2+ buffers.
(D) Quantification of the response in (A). Data from ten cells are presented as means ± SEM.
(E) TIRF microscopy recording of R-GECO (red) and PH-PLCδ1-GFP (green) fluorescence from an MIN6 cell exposed to 100 μM CPA, 30 mM K+, and 100 μM carbachol.
(F) TIRF microscopy recording of R-GECO (red) and PH-PLCδ1-GFP (green) fluorescence from an M1-receptor-overexpressing MIN6 cell exposed to 100 μM CPA, 30 mM K+, and 100 μM carbachol.
(G) Quantification (means ± SEM) of TIRF microscopy recordings of R-GECO (top) and PH-PLCδ1-GFP (bottom) fluorescence change in response to the indicated treatments. Data from 47 (control) and 22 (M1R overexpression) cells. ***p < compared with K+/CPA, ##p < 0.01 compared with K+, #p < 0.05 compared with K+, ¶p < compared with control (CPA/K+/carb).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Optogenetic Control of Plasma Membrane PI(4,5)P2
(A) Principle of blue-light-induced PI(4,5)P2 dephosphorylation.
(B) Confocal micrographs of MIN6 cells expressing mCh-CRY2-OCRL, iRFP-PH-PLCδ1, and CIBN-CAAX (not shown) in the absence of blue light. Magnifications show the time course of light-induced recruitment of mCh-CRY2-OCRL to the plasma membrane and the corresponding loss of iRFP-PH-PLCδ1.
(C) mCh-CRY2-OCRL and iRFP-PH-PLCδ1 fluorescence change at the plasma membrane following illumination.
(D) TIRF microscopy recording from an MIN6 cell expressing CIBN-CAAX, GFP-CRY2-OCRL, and RFP-PH-PLCδ1 during blue-light illumination of varying durations (blue bars). Bottom trace shows mRFP-PH-PLCδ1 fluorescence during continuous imaging and the top trace shows the GFP-CRY2-OCRL fluorescence at the interruption of blue-light illumination (circle) with extrapolated dotted lines.
(E) Schematic drawing of the principle for TIRF microscopy imaging and manipulation of islets.
(F and G) TIRF microscopy images (top) and recordings (bottom) from two cells within a mouse islet infected with Ad-CIBN-CAAX, Ad-GFP-CRY2-OCRL, and Ad-RFP-PH-PLCδ1 during repeated blue-light illumination.
(H) Scatterplot showing the correlation between evanescent wave blue-light illumination time and RFP-PH-PLCδ1 dissociation from the plasma membrane. Each dot represents one cell (n = 9–20 cells per time point). The data have been fitted to a single exponential function.
(I) TIRF microscopy recordings from an MIN6 cell exposed to blue-light evanescent wave illumination of varying duration. Stimulations have been aligned to the first frame of illumination.
(J and K) Quantification (means ± SEM) of the RFP-PH-PLCδ1 fluorescence decrease in response to illumination (J) and time to recovery following interruption of the illumination (K) in MIN6 cells (n = 40 cells). The response to 100 μM carbachol is shown for comparison.
See also Figure S1.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3 PI(4,5)P2 Regulates Depolarization-Induced Ca2+ Influx
(A and B) TIRF microscopy recordings of R-GECO (black), iRFP-PH-PLCδ1 (red), and GFP-CRY2-OCRL (green) fluorescence in a control MIN6 cell (A) and an MIN6 cell co-expressing CIBN-CAAX for plasma membrane recruitment of GFP-CRY2-OCRL (B).
(C and D) Scatterplots showing the correlation between light-induced changes in iRFP-PH-PLCδ1 (C) or GFP-CRY2-OCRL (D) fluorescence and the K+-induced R-GECO fluorescence change. Each dot represents one cell (n = 42 cells).
(E) Quantifications (means ± SEM) of the K+-induced R-GECO fluorescence changes in MIN6 cells co-expressing iRFP-PH-PLCδ1 and Opto-5ptase. The cells were divided into four groups based on the presence or absence of light-induced GFP-CRY2-OCRL plasma membrane binding or iRFP-PH-PLCδ1 plasma membrane dissociation, and the average R-GECO fluorescence change in the corresponding cells is shown (n = 42 cells; ***p < 0.001).
(F–H) TIRF microscopy recordings of R-GECO fluorescence from MIN6 (A), INS1 (B), and human islet (C) cells co-expressing GFP-CRY2-OCRL (control) or CIBN-CAAX and GFP-CRY2-OCRL (Opto-5ptase). The cells were stimulated with 0.5 mM tolbutamide (T) or 30 mM K+ in the absence or presence of blue-light illumination.
(I) Normalized Ca2+ increase in response to 0.5 mM tolbutamide or 30 mM K+ before and after blue-light illumination. The bars represent the average from all cells with detectable GFP-CRY2-OCRL plasma membrane binding after blue-light exposure (means ± SEM; n = 24, 49, and 53 cells; ***p < 0.001).
(J) Average K+-induced Ca2+ responses from cells within human islets expressing GFP-CRY2-OCRL (control) or GFP-CRY2-OCRL and CIBN-CAAX (Opto-5ptase) in the absence and presence of blue-light illumination (means ± SEM, n = 22 cells, **p < 0.01).
(K) TIRF microscopy recording of R-GECO fluorescence from an MIN6 cell co-expressing Opto-5ptase following depolarization with 30 mM K+ and subsequent short (0.4 s) and long (10 s) blue-light TIRF illumination. Trace is representative of 16 cells.
See also Figures S2 and S3.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4 PI(4,5)P2 Gates L-type Voltage-Dependent Ca2+ Channels
(A) TIRF microscopy image sequences for an mCherry-CRY2-OCRL-expressing INS-1E cell (CON) and an mCherry-CRY2-OCRL and CIBN-CAAX-expressing INS-1E cell (CIBN) with corresponding fluorescence changes (bottom). Increase in fluorescence (corrected for background and normalized to the first frame) was observed after stimulation with blue light only when both constructs were co-expressed.
(B) Ba2+-currents (normalized to cell size) for depolarized CON and CIBN cells from (A) before (black trace) and after (blue trace) blue-light activation. Only co-expression with CIBN-CAAX resulted in current decrease (blue trace on the right).
(C) Average Ba2+-currents (n = 6 cells per condition, normalized to cell size) for only CRY2-OCRL expressing INS-1E cells (CON; p = 0.73, U test) and CIBN-co-expressing cells (**p = , U test) before (black bars) and after blue-light stimulation (blue bars).
(D) Peak current-voltage (I-V) relations for voltage-activated Ba2+ currents in controls and CIBN-expressing cells before and after stimulation with blue light normalized to the maximum current. Points are means ± SEM (both n = 6).
(E) TIRF microscopy recording of R-GECO fluorescence from an MIN6 cell exposed to 30 mM K+ in the absence or presence of 2.5 μM nifedipine.
(F) Means ± SEM for the K+-induced R-GECO fluorescence change in (E) (n = 27 cells, ***p < 0.001).
(G) TIRF microscopy recordings of iRFP-PH-PLCδ1 (red) and R-GECO (black) fluorescence in an MIN6 cell co-expressing Opto-5ptase and treated as indicated.
(H) Means ± SEM for changes in R-GECO (top) and iRFP-PH-PLCδ1 (bottom) fluorescence in response to 30 mM K+ in the absence or presence of light-induced PI(4,5)P2 depletion and 10 μM BAY K8644 (n = 42 cells in the control group and 39 cells in the BAY-treated group; ***p < 0.001, *p < 0.05, NS, not significant).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
PI(4,5)P2 Regulates Glucose-Induced Ca2+ Influx and Insulin Secretion
(A) TIRF micrographs (top) and recordings (bottom) of GFP-PH-PLCδ1 fluorescence in MIN6 cells co-transfected with CIBN-CAAX, CRY2-OCRL, and R-GECO. Most cells showed robust dissociation of GFP-PH-PLCδ1 from the plasma membrane upon illumination, but some cells showed no response due to lack of CIBN-CAAX or CRY2-OCRL expression (control).
(B and C) TIRF microscopy recordings of R-GECO fluorescence from glucose-stimulated MIN6 (B) and primary dispersed mouse β cells (C) co-expressing CRY2-OCRL (control), CRY2-OCRL, and CIBN-CAAX (Opto-5ptase) or CRY2-OCRL (G523D), and CIBN-CAAX (Opto-5ptase(G523D)). Traces from mouse β cells are from two islet preparations (gray and black).
(D and E) Scatterplot showing linear correlation between glucose-induced Ca2+ influx and the GFP-PH-PLCδ1 plasma membrane dissociation in MIN6 cells (D; n = 51 cells) and between glucose-induced Ca2+ influx and GFP-CRY2-OCRL plasma membrane binding in mouse β cells expressing Opto-5ptase (E; n = 34 cells).
(F) TIRF micrograph of a human islet (outlined in red) infected with R-GECO (shown), GFP-CRY2-OCRL, and CIBN-CAAX (top). Trace below shows the Ca2+ response of the cell indicated by the green dotted line and arrow following glucose stimulation and illumination.
(G) Ca2+ increases in response to 20 mM glucose before and after illumination of the indicated cells expressing Opto-5ptase. The bars represent means ± SEM from all cells with detectable recruitment of GFP-CRY2-OCRL or dissociation of GFP-PH-PLCδ1 after blue-light illumination (n = 31, 12, 22 and 34 cells; ***p < 0.001, **p < 0.01).
(H) Measurements of hGH release from MIN6 cells expressing hGH and Opto-5ptase stimulated with 3 mM glucose, 20 mM glucose, and 20 mM glucose with blue-light illumination (n = 3; **p < 0.01, #p < 0.05).
See also Figure S4.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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