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Imaging of fixed cells was carried out on a Leica AOBS SP2 microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with a 63x oil immersion objective with a 1.4 numerical aperture and in most cases the pinhole was set to airy1. Images of STIM1-EYFP were obtained with 514 nm excitation light and emission fluorescence selected with a 520-595 nm bandpass filter. For excitation of DyLight 649, a 633 nm excitation laser line was used and light was collected at 660 – 800 nm.

2.5.2 Live cell imaging

Live cell imaging was performed using a Leica AOBS SP2 microscope equipped with a 63x oil immersion objective with a 1.4 numerical aperture and a 63x water objective with a 1.2 numerical aperture. Cells were maintained at room

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temperature throughout each experiment. The wavelengths used for excitation of fluorophores and the emission ranges collected are listed for different combinations of fluorophores in Table 2.3 below.

Fluorophores Excitation wavelengths (nm) Emission specta (nm) Cerulean alone 405 460-492 ECFP alone 405 450-500 EGFP alone 488 490-525 EYFP alone 514 520-580 Fluo4 alone 488 495-545 mRFP or mCherry alone 594 610-730 ECFP & RFP/mCherry 405 594 450-530 610-730 EGFP & EYFP 476 514 490-505 580-615 EGFP & RFP/mCherry 488 594 490-510 610-730 EYFP & RFP/mCherry 514 594 520-560 610-730 Fluo4 & RFP/mCherry 488 594 495-545 710-730 Cerulean & EYFP & RFP/mCherry 405 514 594 460-490 520-560 610-730 EYFP & Dylight649 514 633 520-595 660-800

Table 2.3 Fluorophores used in live cell imaging and the excitation and emission wavelengths used.

2.5.3 Anti-His6 antibody staining of live cells

In order to determine whether STIM1 inserts into the plasma membrane following translocation, thereby exposing its N-terminus at the cell surface, HeLa cells were

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transfected with a STIM1-EYFP containing an N-terminal tag of 6 consecutive histidine residues (His6). Antibody staining was carried out to determine whether

this tag became exposed on the cell surface following store-depletion induced translocation of His6-STIM1-EYFP. Cells were maintained in sodium hepes (NaCl 140

mM, KCl 4.7 mM, MgCl2 1.13 mM, Hepes 10 mM, glucose 10 mM) containing 2 mM

Ca2+. To deplete intracellular stores, cells were treated with 2 µM thapsigargin (Calbiochem, San Diego, California, USA) for 10 minutes. Control cells were kept in sodium hepes only for 10 minutes. Cells were then incubated with 500 µl of a 1 in 40 dilution of mouse monoclonal anti-His6 primary antibody (Sigma) for 15 minutes,

washed with 6 ml of Sodium Hepes and incubated with a 1 in 200 dilution of DyLight649 secondary antibody for 15 minutes. Cells were washed with a further 6 ml Sodium Hepes before being imaged.

2.5.4 ATP depletion of live cells

To examine whether ATP depletion inhibits the translocation of STIM1 to the plasma membrane, HeLa Cells overexpressing STIM1-EYFP alone or in combination with mCherry-Orai1 or GFP-PH-PLCδ, were washed three times in Sodium Hepes

and mounted in a perfusion chamber on the stage of an inverted microscope. During experiments a gravity-fed perfusion system was used to exchange extracellular solutions. To induce ATP depletion cells were perfused with a combination of 5µM Oligomycin (Calbiochem) and 2mM Iodoacetate (Sigma) or 5µM Oligomycin and 10mM 2-Deoxy-D-glucose (Sigma). In experiments using 2- Deoxy-D-glucose, normal glucose was omitted from the extracellular solution. In

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some cases in cells expressing GFP-PH-PLCδ, cells were pretreated with 20 µM

wortmannin before perfusion with ATP inhibitors.

2.5.5 Lipid Kinase Inhibition

Several methods were used to inhibit lipid kinases for the depletion of various phosphoinositide species in this study. To inhibit PI3 kinase alone, cells were treated with 50 µM LY294002 (Calbiochem) for 10 minutes. To simultaneously inhibit PI3 kinase and PI4 kinase, cells were treated with either 300 µM LY294002 for 10 minutes or with 20 µM wortmannin (Calbiochem) for 30 minutes. To specifically deplete PtdIns(4,5)P2 a previously described rapamycin inducible 5-

phosphatase system was used (Korzeniowski et al., 2009). For this, cells were cotransfected with PM-FRB-CFP or PM-FRB-RFP and the cytosolic 5-ptase-RFP. Cells were treated with 1 µM rapamycin (Calbiochem) for 2 minutes to activate the 5- phosphatase.

2.5.6 Quantification of fluorescence distribution

In order to quantify the effects of phosphoinositide depletion on the peripheral distribution of mCherry-Rit1 or GFP-PH-PLCδ fluorescence, the following means of

quantification was used. Regions of interest were drawn around the outside of each cell, immediately beneath the cell periphery (~1 µm in from the cell periphery), and also around the nucleus in cells where nuclear fluorescence was observed. Peripheral fluorescence was calculated by subtraction of the cytoplasmic fluorescence from the outer region of interest. In cases where nuclear fluorescence was observed, cytosolic fluorescence was calculated by subtraction of nuclear

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fluorescence. Ratios of peripheral to cytosolic fluorescence were calculated on the basis of the mean fluorescence per pixel in the respective regions rather than total fluorescence to eliminate any variations in the data arising from the size of the selected regions of interest. In cells expressing GFP-FAPPI, regions of interest around the fluorescence observed at the Golgi were drawn and quantified as average fluorescence intensity per pixel.

2.5.7 Quantification of STIM1 puncta

STIM1 puncta, induced by store depletion, were counted under various conditions where phosphatidyl inositide species were depleted. Cells were cotransfected with STIM1-EYFP and PM-FRB-RFP/RFP-5-ptase-domain or with STIM1-EYFP, PM-FRB- RFP/RFP-5-ptase-domain and Cerulean-Orai1, and preincubated with various combinations of lipid kinase inhibitors as described in section 2.5.5. Cells were then treated with 2 µM thapsigargin for 10 minutes. Control cells overexpressing STIM1- EYFP alone were treated with thapsigargin alone for 10 minutes. For quantification of STIM1-EYFP puncta formation, the puncta were selected as spots of high fluorescence intensity ranging from approximately 0.5 – 1.0 μm in diameter and counted blindly. Accuracy of puncta quantification was verified by independent blind counting.

2.5.8 Image analysis

All Images were processed using the CorelDraw software package (Corel Corporation, Ottawa, Canada) or ImageJ Java-based imaging software (NIH, USA).

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To measure changes in intracellular Ca2+ ([Ca2+]i), HeLa cells were loaded with 5 μM

of the cytosolic Ca2+ indicator, Fluo-4 AM (Molecular Probes, Paisley UK) for 30 minutes at room temperature. Cells were was three times in Calcium free Sodium Hepes buffer and mounted in a perfusion chamber on the stage of an inverted Leica AOBS SP2 confocal microscope. During experiments a gravity-fed perfusion system was used to exchange extracellular solutions. Fluo-4 was excited using a 488 nm laser and emission fluorescence was collected between 495-545 nm. To allow for comparison between different cells from different experiments, Fluo-4 fluorescence measurements for each cell were calculated as the ratio of the fluorescence at the first time point and to eliminate variability between experiments in the resting fluorescence level these values are shown as F/F0 normalised to the peak ratio for the control cells in each experiment.

2.5.10 Bimolecular fluorescence complementation (BiFC) Assays

HeLa cells were transfected with Golli-YC, STIM1-CT-YN, full length STIM1-YN and Orai1-YC or Orai1-YN BiFC constructs either alone or in complementary combinations. For further control experiments, each of these constructs was transfected in HeLa cells along with the complementary empty BiFC vector (YFP-YC and YFP-YN), or with both YFP-YC and YFP-YN alone. Cells were treated with thapsigargin (2 µM) or histamine (100 µM) in the presence of 2 mM external Ca2+ for 60-90 minutes at room temperature before imaging. The EYFP emission spectra profiles were generated by performing Lambda scans in transfected cells between 510-580 nm, and each scan collected emitted light from within a 10 nm range.

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Emission fluorescence values were calculated as the ratio of the fluorescence to the peak fluorescence value in each experiment.