El-Jouni W et al. (2008), Internalization of plasma membrane Ca2+-ATPase ...

XB-ART-38408

Dev Biol 2008 Dec 01;3241:99-107. doi: 10.1016/j.ydbio.2008.09.007.

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Internalization of plasma membrane Ca2+-ATPase during Xenopus oocyte maturation.

El-Jouni W , Haun S , Machaca K .

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A transient increase in intracellular Ca(2+) is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca(2+) signal during oocyte maturation. The first Ca(2+) transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca(2+) signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation.

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Species referenced: Xenopus laevis
Genes referenced: atp2b1 edil3 mapk1 mos pcyt1b tbx2 tf wee1

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	Fig. 2. GFP-PMCA traffics in a similar fashion to endogenous PMCA. (A) Subcellular localization of endogenous PMCA and GFP-PMCA. Oocytes and eggs were fixed, sectioned, and stained with a pan PMCA antibody (red signal). Differential interference contrast (DIC), GFP (green), and wheat germ agglutinin (WGA, pink) images of the same slice are also shown. WGA-Alexa633 (pink) was used to stain the plasma membrane. The scale bar is 10 μm. (B) Similar staining as in panel A, except that an anti-Flag antibody was used to detect injected GFP-PMCA (Flag). For panels A and B the oocyte examples are representative of at least 30 similar cells and the egg examples are representative of 5–15 similar cells. All cells examined showed robust PMCA enrichment at the cell membrane in oocytes and undetectable levels in eggs. (C) Left panel: Time course of PMCA expression using the pan PMCA antibody at 1 (1D) and 4 days (4D) after injection, as compared to uninjected cells (Uninj) (left panel). An equivalent of one cell was loaded in each lane. Right panel: Expression of GFP-PMCA as compared to endogenous PMCA using a PMCA1 isoform specific antibody (right panel). Oocytes refer to cells before progesterone treatment (Ooc) and eggs are mature cells (> 3 h after GVBD). An equivalent of 1/2 cell was loaded per lane.
	Fig. 3. Kinase-dependent modulation on PMCA internalization. (A) Signaling cascade regulating Xenopus oocyte meiosis entry. The steps at which the molecular and pharmacological manipulations used act are shown in green. (B) Live cell imaging of PMCA internalization. Oocytes expressing GFP-PMCA were subjected to one of the following treatments: injected with Cyclin B1 RNA (Cyclin); pre-treated with U0126 (50 μM) for 1 h before cyclin RNA injection (U-Cyclin); injected with Mos RNA (Mos); or injected with Wee1 RNA and incubated overnight before Mos RNA injection (Wee-Mos). The cell membrane was stained with WGA-Alexa633. The inset shows that in the Wee-Mos treatment, GFP-PMCA internalization is delayed but not completely inhibited. The examples shown are representative of 76 and 29 oocytes and eggs respectively, and 11–15 cells each for treatments used to manipulate the kinase cascades. All cells analyzed showed a similar pattern of PMCA localization to the examples shown, except for the Wee-Mos treatment where in 3/11 cells PMCA was internalized to similar levels as Mos. Western blot analysis for phospho-MAPK (P-MAPK) and phospho-MPF (P-MPF) of each of the scanned cells is shown on the right. The equivalent of 1/2 a cell was loaded. The scale bar is 20 μm.
	Fig. 4. PMCA internalization. (A) To interfere with raft-dependent endocytosis oocytes were treated 2% methyl-β-Cyclodextrin (βMCD) 9 h before progesterone stimulation. GFP-PMCA internalization is inhibited in cells pretreated with βMCD during oocyte maturation (Egg — βMCD) as compared to control cells (Egg). For the C3 Exoenzyme treatment, oocytes were injected with 1.7 ng C3 exoenzyme 1 h before progesterone stimulation (Schmalzing et al., 1995). The C3 exoenzyme treatment increases membrane invaginations leading to a diffuse membrane appearance in the oocyte (Ooc). These invaginations appear as large circular structures at the cell membrane focal plane (inset). A cross section through these circular regions (red line) shows the invagination at the membrane (inset top). Even though membrane area is increased in C3 exoenzyme treated cells PMCA internalization is unaffected (Egg). The scale bar is 10 μm. (B) GFP PMCA expressing cells were labelled with Alexa555-labeled B subunit of the cholera toxin (CTB) and imaged around GVBD to visualize PMCA internalization. In a cross-section the GFP-PMCA and CTB signals co-localize (Cross). In a focal plane that is 10–20 μm below the cell membrane (Sub-memb) the CTB and GFP-PMCA signals also co-localize. A close up view and line scan of fluorescence intensity through one of the PMCA and CTB positive vesicles shows CTB in the luminal core of the vesicle, while PMCA localizes to the vesicle membrane. The scale bar is 5 μm. (C) GFP-PMCA expressing cells at GVBD were labeled Alexa633-labeled transferrin and chased for different time points before imaging. No co-localization is observed between GFP-PMCA and transferrin. The scale bar is 10 μm. The examples shown are representative of 5–8 cells for each treatment.
	Fig. 5. Deletion analyses. (A) Oocytes were injected with RNA (5–7 ng) of each of the GFP-PMCA deletions (DEL1, DEL2, and DEL5), then activated with progesterone 4–5 days after injection. Internalization was assayed by imaging cells stained with WGA-Alexa633. For DEL3 and DEL4 no signal was observed at the cell membrane following injection of different RNA concentrations up to 12 days for DEL3 and 16 days for DEL4. Rather the products of these deletions localize intracellularly to elongated vesicle-like structures (Del3, Del4). The examples shown are representative of 5–10 cells for each deletion. Scale bar is 20 μm. (B) Expression of the full length and deletions. Anti-PMCA blot from uninjected oocytes (Uninj), oocytes injected with full-length GFP-PMCA and the 5 deletions. For the full-length, DEL1, DEL2, and DEL5 lysates were collected 5–7 days after injection. DEL3 and DEL4 lysates were collected 9 days and 16 days post-injection respectively. The bracket marks the electrophoretic mobility of full-length and different deletion mutants (GFP-PMCA-DEL). Endogenous PMCA is also indicated (PMCA).

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