Sun L, Yu F, Ullah A, Hubrack S, Daalis A, Jung P, Machaca K.

	Figure 1. ER remodeling and IP3 receptor clustering during meiosis.A. Functional clustering of elementary Ca2+ release events during oocyte maturation. Xenopus oocytes were injected with 10 µM caged IP3 and 40 µM Oregon-green. Oocyte maturation was induced with progesterone and both immature oocytes and fully mature eggs were imaged in linescan mode at 488 nm with the 405 nm laser at low intensity (0.2%) to continuously uncage cIP3. The same region in the cell was scanned continuously in linescan mode with the x-axis representing time and the y-axis space. The single isolated Ca2+ puffs observed in the oocyte coalesce into larger release events referred to as single release events (SRE). B. The ER remodels during oocyte maturation to form large patches in the egg. Oocytes were injected with GFP-IP3 receptor (IP3R) (50 ng/cell) and mCherry-KDEL (10 ng/cell). Images show the formation of ER patches in both animal and vegetal hemisphere to which IP3 receptors localize (Scale bar, 2 µm). C. Frequency of ER patches on the animal and vegetable poles (n = 166; 14 frogs). D. ER patch density and area (n = 38). E. Width of elementary Ca2+ release events in the egg as compared to the width of ER patches.
	Figure 2. A. ER patches remodel continuously.Examples of images from a time series from an egg expressing mCherry-KDEL (Scale bar, 2 µm). B. Elementary Ca2+ release events in the egg localize to ER patches. mCherry-KDEL expressing eggs injected with cIP3 and Oregon-green were line-scanned with the 405 nm laser on (0.2%). Left image show Ca2+ release events and the right ER distribution. The xy image with the linescan area is shown on top of the ER linescan. Histograms of Ca2+ release (red trace) and ER distribution (black trace) are shown for four selected areas as indicated by the numbers.
	Figure 3. Sensitization of Ca2+ release in ER patches.Eggs expressing mCherry-KDEL were injected with cIP3, and Oregon-green and imaged ∼1 hr after GVBD. A–B. IP3 receptors in ER patches respond to IP3 concentrations not detected by receptors in the reticular ER. ER distribution and Ca2+ release were imaged over time, while continuously uncaging cIP3 only within two ROIs as indicated by the boxes using the 405 nm laser. One ROI was placed over an ER patch (P) and the other over a reticular ER domain (Ret). Ca2+ release events over the entire time course within the two ROIs are shown on the right. B. Same experimental conditions as in A, except that uncaging with the 405 nm laser was performed over the entire field continuously. The white box shows the localization of the first release event to an ER patch. C–D. A single sub-threshold uncaging pulse results in preferential Ca2+ release from ER patches as compared to the reticular ER. In this case a single uncaging pulse was applied (405 nm laser 50% power) only within the circular ROIs (white circles) as indicated by ‘Uncage’ on the traces in the lower panel. One ROI is over an ER patch and the other over the reticular ER. D. Same imaging conditions over a small area (inset, 9.15×3 µm) where a single uncaging pulse was applied over the entire imaged area. Ca2+ release was then analyzed from two region as indicated by the boxes representing an ER patch and the neighboring reticular ER. The lower traces show an expanded time scale of Ca2+ release.
	Figure 4. A. IP3 receptors lateral mobility in ER patches and reticular ER.Fluorescence recovery after photobleaching (FRAP) in oocytes and eggs expressing GFP-IP3 receptor (n = 7–8; mean±SE). The bleaching ROIs in eggs were positioned over ER patches or reticular ER. Recovery kinetics were fitted with a monoexponential decay function. Recovery kinetics show that the lateral mobility of IP3 receptors is comparable within ER patches and the reticular ER. B–D. Mathematical modeling. Modeling the time delay in Ca2+ release as a function of IP3 receptor cluster size. As IP3 receptor cluster size increases the delay in response to an IP3 pulse decreases. C. Modeling Ca2+ release spatial and amplitude distribution from two IP3 receptor clusters of varying size (20 and 1000) in response to a sub-threshold IP3 pulse. D. Simulation of the dependence of Ca2+ release within an IP3 receptor cluster as a function of IP3 concentration for varying Ca2+ diffusion coefficients. Lowering the Ca2+ diffusion coefficient results in less Ca2+-dependent cooperativity between neighboring receptors. Decreasing Ca2+ diffusion coefficient leads to decreased sensitivity of IP3 receptors as illustrated by the threshold IP3 concentration required to initiate Ca2+ release.
	Figure 5. Role of ER remodeling in Ca2+ wave propagation.Cells expressing mCherry-KDEL to visualize ER distribution were injected with caged-IP3 and Oregon-Green to image Ca2+ dynamics. Top panel shows an enlarged region from two cells, one with ER patches and one without. Ca2+ waves were induced by uncaging IP3 within an ROI (red circle) and wave propagation speed was measured. Example images of wave propagation are shown. These experiments were performed on the animal hemisphere (n = 12–13).
	Figure 6. MPF is required for ER remodeling during meiosis.A. Simplified kinase cascade activated during oocyte maturation. Indicated in red is the action of different molecular and pharmacological modulators. The arrow indicates activation and the bar inhibition. B. Confocal fields from individual cells expressing mCherry-KDEL and treated as indicated showing ER structure. After imaging cells were lysed and subjected to Western blotting analysis for phosphor-MAPK (p-M), phosphor-Cdc2 (p-C) and tubullin (Tub) as the loading control (Scale bar, 2 µm). Mos indicates cells injected with Mos RNA, CyR: Cyclin B1 RNA; MOS+Wee: inject Wee1 RNA and incubate cells overnight before Mos RNA injection; CyR+U0126: pre-treat with U0126 (50 µM) for 1 h before Cyclin B1 RNA injection. The effectiveness of these treatments is illustrated in Western blots performed on individual oocytes after confocal imaging. The top band (Tub) represents the tubulin loading control, the middle band phosphorylated MAPK (p-M) and the lower band phosphorylated cdc2 at Tyr-15 (p-C). MAPK phosphorylation indicates its activation, whereas the phosphorylation of Cdc2, the kinase subunit of MPF, indicates its inhibition. C–D. Sensitivity of IP3-dependent Ca2+ release measured as indicated in the text.

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