Courjaret R and Machaca K (2016), Xenopus Oocyte As a Model System to Study Store...

XB-ART-52502

Front Cell Dev Biol 2016 Jun 24;4:66. doi: 10.3389/fcell.2016.00066.

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Xenopus Oocyte As a Model System to Study Store-Operated Ca(2+) Entry (SOCE).

Courjaret R , Machaca K .

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Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) influx pathway at the cell membrane that is regulated by Ca(2+) content in intracellular stores. SOCE is important for a multitude of physiological processes, including muscle development, T-cell activation, and fertilization. Therefore, understanding the molecular regulation of SOCE is imperative. SOCE activation requires conformational and spatial changes in proteins located in both the endoplasmic reticulum and plasma membrane. This leads to the generation of an ionic current of very small amplitude. Both biochemical and electrophysiological parameters of SOCE can be difficult to record in small mammalian cells. In this protocol we present the different methodologies that enable the study of SOCE in a unique model system, the frog oocyte, which provides several advantages and have contributed significantly to our understanding of SOCE regulation.

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Species referenced: Xenopus
Genes referenced: clca1.3 dtl orai1 pmch stim1

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	Figure 1. Triple voltage jump protocol used to monitor the intracellular Ca2+ concentration ([Ca2+]i) in the Xenopus oocyte. The first depolarizing jump (1) measures the Cl− current through CaCC at rest or during Ca2+ release from intracellular stores, mirroring [Ca2+]i. The hyperpolarizing voltage jump (2) drives Ca2+ through SOCE channels that in turn induce Cl-efflux. The final depolarizing pulse (3) reports the activation of the CaCC by the Ca2+ influx produced during the previous hyperpolarizing pulse (blue trace). The difference between current obtained in 1 and 3 is termed IClT and reports Ca2+ influx.
	Figure 2. CaCC as an endogenous sensor for Ca2+ release and SOCE in the oocyte. (A) Simultaneous reading of ICl1 and IClT show the transient increase in ICl1 (b) during store depletion by ionomycin (10 μM, 20 s) and the following increase in IClT due to SOCE activation (c). (B) Currents traces corresponding to the different time points in (A). (C) Bar chart illustrating the increase in the ionomycin-induced SOCE (IClT) in cells expressing STIM1 and Orai1 (S/O) compared to uninjected oocytes (Naïve).
	Figure 3. Recording of SOCE current in Xenopus oocytes. (A) Intracellular injection of BAPTA (final concentration 2-5 mM) inhibits CaCC activation. (B) The SOCE current is activated by store depletion using bath applied ionomycin (10 μM, 20 s) and is totally inhibited by La3+ (100 μM). The current is measured at a membrane potential of −120 mV. (C) Current traces obtained using a voltage ramp from −120 mV to +40 mV at time points indicated in (B). (D) Current-voltage relationship of the SOCE current (La3+-sensitive) obtained after subtraction of the La3+ resistant fraction (c) from the global current (b). The current is dramatically increased in oocytes overexpressing STIM1 and Orai1. Recordings in (A–C) are made on a cell overexpressing STIM1 and Orai1.
	Figure 4. Confocal imaging of STIM1 and Orai1 in Xenopus oocytes. (A) Confocal z-stacks are obtained from oocytes overexpressing mCherry tagged STIM1 (Ch-STIM1) and GFP-Orai1. The pinhole aperture is set to 1 Airy unit and the images are taken every 0.5 μm. Two selected planes are illustrated, the Plasma Membrane (defined as the maximum expression level of GFP-Orai1) and the intracellular/ER compartment (Deep) approximately 10 μm deep in the cell and at the maximum of Ch-STIM1 expression in control conditions (Ctr). Treatment of the cells with TPEN (5 mM, 10 min), IP3 (2.5 μM final) induces the translocation of Ch-STIM1 to the plasma membrane and the co-clustering of the proteins, visible as dense yellow dots in the merged images. Below each panel (Ctr and TPEN) is an orthogonal reconstitution (Ortho) of a plane perpendicular to the membrane illustrating the distribution of both proteins across the z-axis. (B) Timeline of the formation of Ch-STIM1 clusters following application of ionomycin (10 μM). The images are captures using an open pinhole aperture on the confocal to compensate for the vertical movement of STIM1.
	Figure 5. Orai1 internalization during meiosis. Confocal stacks are performed on oocytes (A) and eggs (B) expressing both STIM1-Ch and Orai1-GFP. Oocyte maturation is associated with internalization of Orai1. This is visible as the removal of Orai1 from the plasma membrane in the oocyte and its enrichment in an endosomal compartment in confocal sections (deep), on the orthogonal reconstruction (ortho) and on the fluorescence curves as a shift of the Orai1 intensity curve toward the ER as marked by STIM1.
	Figure 6. Analysis of the movement of STIM1 and Orai1 after store depletion. (A) The fluorescence intensity of each channel is plotted vs. the position on the z-axis and normalized to its maximum. The separation between the Ch-STIM1 and GFP-Orai1 peaks disappears after store depletion induced by TPEN due to co-localization of both proteins at the membrane focal plane as illustrated in Figure 4. The translocation of the STIM1 proteins can also be highlighted using the area under the curve with a reference point set at the peak of the Orai1 signal. The intracellular fraction is labeled in pink and the “membrane” one (above the Orai1 peak) in blue. (B) Co-localization plots of mCh-STIM1 and GFP-Orai1 fluorescence obtained at the membrane plane (defined as the maximum of the GFP-Orai1 signal) before and after store depletion by TPEN. The Pearson's correlation coefficient (Pcc) is indicated on each plot.
	Figure 7. Measuring SOCE clustering parameters. A thresholded image of the Orai1 clusters (from Figure 4) is processed with the “analyze particle” plugin of ImageJ. The distribution of the areas of individual clusters is analyzed and the SD of the data is used to define a cutoff value that allows limiting the size of the analyzed particles. The nearest neighbor distance gives a measure of the cluster density (NND).

References [+] :

Arredouani, Regulation of store-operated Ca2+ entry during the cell cycle. 2010, Pubmed