Li R et al. (2016), Spindle function in Xenopus oocytes involves po...

XB-ART-52477

Mol Biol Cell 2016 Nov 01;2721:3273-3283. doi: 10.1091/mbc.E16-05-0338.

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Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling.

Li R , Leblanc J , He K , Liu XJ .

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Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results-most important, the different sensitivities of the spindle to BAPTA and EGTA-suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling.

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Species referenced: Xenopus
Genes referenced: btg3

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	FIGURE 1:. Lck-GCaMP3âdetected global calcium transients at egg activation. (A) Time line of experimental approach. All fluorescent probes (mostly mRNAs) were injected at GV stage. Frog oocytes (1.4 mm in diameter) exhibit a dark-pigmented animal hemisphere and a light vegetal hemisphere. GVBD is manifested with depigmentation at the center of the animal hemisphere (âmaturation spotâ), where all meiosis steps occur (and are imaged). Ana I, anaphase I; MI, metaphase I; MII, metaphase II; PB, polar body; Pg, progesterone; ProM, prometaphase; red lines, spindle microtubules; blue dots, chromosomes. (B) An MII oocyte injected with mRNAs for Lck-GCaMP3 and Lck-mCherry was imaged before (00:00) and at the indicated time after prick activation. The relative position shift, as marked by the first polar body (arrows), was due to the cortical contraction characteristic of egg activation. The graph shows relative fluorescence of the cortical region surrounding the first polar body (white dotted box), with values at 00:00 set as 1 (three oocytes, two experiments). **p < 0.001.
	FIGURE 2:. Polar body emission involves significant membrane addition. (A) Time series of an oocyte injected with mRNAs for Lck-GCaMP3 and RFE-H2B during first-polar-body emission. Arrow depicts the cortical spindle pole. (B) Time series of an oocyte injected with mRNAs for Lck-mCherry and GFP-H2B during first-polar-body emission. (C) The graph summarizes relative fluorescence at the cortical region around the spindle (white dotted box) at 00:00 and 00:10 of the same oocyte as in B, with values at 00:00 set as 1. Three oocytes, two experiments. NS, not significant. (D) Time series of an oocyte coexpressing Lck-GCaMP3 and Lck-Cherry imaged at similar time points as in A and B. Arrow, cortical spindle pole. (E) Time series (slightly tilted side view, with coordinates shown) of an oocyte injected with Lck-GCaMP3 and RFP-H2B imaged at metaphase II (00:00) and the indicated time after pricking. Arrow depicts the position of the cortical pole of the metaphase II spindle. Anaphase was noted at 00:12.
	FIGURE 3:. GCaMP3 failed to detect spindle-based calcium signals. (A) Time series of an oocyte injected with GCaMP3 and RFP-H2B imaged during first (1st) polar body emission. Arrow depicts metaphase I spindle (00:00 = 115 min after GVBD). Representative of three oocytes. (B) Time series of an oocyte injected with GCaMP3 and RFP-H2B imaged at metaphase II (00:00) and the indicated time after pricking. Arrow depicts metaphase II spindle. 2nd, second polar body. Representative of six oocytes.
	FIGURE 4:. BAPTA Ca2+ chelators, but not EGTA, cause spindle microtubule depolymerization in meiosis I. (A) Top, time series of an oocyte injected with 30 nl of 25 mM DB-BAPTA at 90 min after GVBD. The oocyte was subjected to time-lapse imaging immediately after DB-BAPTA injection (00:00). The schematics on the right depict side views of oocytes at 0 and 8 min after DB-BAPTA injection, respectively. Middle, time series (side view) of an oocyte injected (60 min after GVBD; 00:00) with 30 nl of 25 mM DB-BAPTA mixed with 5 mM CaCl2, depicting more gradual spindle collapse and chromosome clustering. Bottom, time series (side view) of an oocyte injected with 30 nl of 25 mM DB-BAPTA mixed with 25 mM CaCl2 60 min after GVBD, depicting no spindle collapse within a similar time frame. This series is composed of four image segments of the same oocyte, necessitated due to the cortical contraction. (B) Top, time series (side view) of an oocyte injected with 30 nl of 10 mM diazo-2 at 90 min after GVBD (00:00) followed by time-lapse imaging. Bottom, time series of an oocyte injected with 30 nl of 10 mM diazo-2 at 90 min after GVBD. Five minutes after diazo-2 injection, the oocyte was imaged (00:00), followed immediately by UV illumination for 3 min (00:00â€“00:03) and simultaneous time-lapse imaging. (C) Time series (side view) of an oocyte injected with 30 nl of 25 mM of EGTA at 90 min after GVBD. The oocyte was subjected to time-lapse imaging immediately after EGTA injection (00:00) and shows normal anaphase (00:20) and first-polar-body emission (00:28).
	FIGURE 5:. Both DB-BAPTA and EGTA buffers inhibited egg activation, but only DB-BAPTA caused collapse of metaphase II spindle. (A) Time series (side view) of a mature egg injected with 30 nl of water, followed immediately by time-lapse imaging (00:00), depicting anaphase (00:12) and emission of the second polar body (00:31). This series is composed of three image segments of the same oocyte, necessitated because significant cortical contraction required repositioning of the oocyte. (B) Time series of a mature egg injected with 30 nl of 25 mM DB-BAPTA (00:00), followed by immediate time-lapse imaging, depicting the lack of egg activation (no cortical contraction) but rapid collapse of metaphase II spindle. (C) Time series (side view) of a mature egg injected with 30 nl of 25 mM EGTA (00:00), followed by immediate time-lapse imaging, depicting no cortical contraction or changes of metaphase II spindle.
	FIGURE 6. DB-BAPTA inhibits Cdc42 activation but not RhoA activation. (A) Top, time series (side view) of a control oocyte showing the active Cdc42 (green) cap overlying the polar body chromosomes (00:10, arrow) and active Cdc42 at the polar body cortex (00:16, arrow). Bottom, time series of an oocyte injected with 20 nl of 30 mM DB-BAPTA immediately after anaphase initiation (upward arrow), depicting the lack of Cdc42 activation or polar body emission. (B) Top, time series (side view) of a control oocyte showing the active RhoA (green) contractile ring (00:11) and constriction âbelowâ the polar body chromosomes (00:13 and 00:16, arrow). Bottom, time series of an oocyte injected with 20 nl of DB-BAPTA immediately after anaphase initiation (upward arrow), showing active RhoA contractile ring (00:08) and constricting âaboveâ both sets of chromosomes (00:12, arrow).
	FIGURE 7:. Colocalization of calmodulin with spindle microtubules during polar body emission. Typical confocal images of oocytes fixed at the indicated stages, stained with anti-calmodulin and anti-tubulin, and counterstained with DAPI. The projection orientation is indicated for each oocyte, where the xy-plane is parallel to the oocyte surface and z is perpendicular, pointing toward the center of the oocyte. The approximate time (minutes from GVBD) of each stage is indicated at the bottom. E, early (prometaphase); red, tubulin; green, calmodulin; blue, DNA.

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