York-Andersen AH et al. (2020), A calcium-mediated actin redistribution at egg ...

ECB-ART-49884

Mol Reprod Dev 2020 Feb 01;872:293-304. doi: 10.1002/mrd.23311.

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A calcium-mediated actin redistribution at egg activation in Drosophila.

York-Andersen AH , Hu Q , Wood BW , Wolfner MF , Weil TT .

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Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.

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	Figure 1. Actin is reorganized at egg activation. Time series of an ex vivo‐activated mature oocyte (a–c; n = 6) and in vivo‐activated egg (d; n = 4) expressing F‐Tractin.tdTomato. Maximum Z‐projection (a–d), three‐dimensional (3D) projection at a 45° angle (a′–d′) and a single Z‐plane at 20 µm depth (a″–d″) are shown for each time point. Images were acquired using an Olympus FV3000 confocal microscope. (a–a″) Before the addition of activation buffer, actin is in a smooth cortical distribution around ridges in the cortex (as shown in Movie 1). (b–c″) Following egg activation (3–12 min, as shown in Movie 2) and (27–35 min, as shown in Movie 3), actin is reorganized in larger, more widely distributed foci. 3D projection shows that the cortex has expanded and that actin is no longer associated with the ridge. Single plane images show that a cortical enrichment preactivation is lost and that actin is not as enriched at the cortex after activation. (d–d″) In vivo‐activated eggs collected from the oviduct have a similar actin distribution as mature oocytes activated ex vivo (compare c–c″ [Movie 3] to d–d″ [as shown in Movie 4]). Scale bar = 10 µm (a–d′)
	Figure 2. A dynamic actin cytoskeleton is required for a calcium wave at egg activation. (a–b′,g,g′) Egg chamber expressing GFP‐moesin. Images were acquired using an Olympus FV3000 confocal microscope. (a,b) Fluorescence recovery after photobleaching (FRAP) of GFP‐moesin before and after ex vivo egg activation (n = 5). GFP‐moesin labeling actin at the lateral cortex is reduced to 25% of original fluorescent intensity following a bleaching step. In both Stage 14 mature egg chambers before activation (a) and after activation (b) recovery can be detected. In the unactivated oocyte, full recovery is complete at 12 min post‐bleaching. However, in the activated oocyte recovery is substantially faster post‐activation (14 vs. 45 s recovery halftime, n = 5). Note that movement caused by the addition of activation buffer (AB) meant that recovery could only be recorded for 1 min and 15 s postbleach. The dashed white box denotes the bleached region. (c) Graph showing calcium wave phenotypes when mature oocytes are treated with AB and the actin perturbing drugs latrunculin A, cytochalasin D, or phalloidin. A “full wave” indicates the calcium wave traversed the whole oocyte, a “partial wave” indicates that the calcium wave initiated but did not traverse the whole oocyte, and a “no wave” phenotype indicates that a calcium wave never initiated. When only AB is added, 82% of oocytes displayed full waves. The addition of latrunculin A shows a nonsignificant decrease in full waves to 72% (n = 20; p = .5). Mature oocytes treated with cytochalasin D or phalloidin show a significant decrease in full waves observed to 28% and 32%, respectively (n = 35 for each, Fisher's exact statistical analysis, p = .0001 [***]). (d) Graph showing calcium wave phenotypes when mature oocytes are treated with AB in GCaMP3 controls, overexpression of Spire B and knockdown of scar. When Spire B is overexpressed there is a significant decrease, to 40%, in the number of activated mature oocytes that show a full calcium wave, and an increase in the number of “no wave” phenotypes to 58% (n = 12, Fisher's exact statistical analysis, p < .05). When scar is knocked down using RNAi, there were no significant changes in the calcium wave phenotype. (e) Boxplot showing the speeds of calcium entry into the mature oocyte in control (GCaMP3) and scar RNAi mature oocytes (n = 15 and n = 18). Speed of calcium entry was determined as the time taken from the addition of AB to the time of first GCaMP fluorescence detection in the oocyte. Initial calcium entry was observed by eye and confirmed through quantification of the fluorescence signal, using a threshold of a 10% increase from the oocyte background. Images were taken every 3 s. There was a significant decrease in the time taken for calcium to enter the oocyte in the scar RNAi background (p < .05). (f,f′) Stage 14 egg chambers fixed and labeled for actin with phalloidin (n = 10). More actin is detected at the cortex in a wild‐type egg chamber (f) compared with a scar RNAi expressing egg chamber (f′). White arrowheads mark the lateral cortex of the egg chamber. (g,g′) In the Stage 14 egg chamber, before egg activation, GFP‐moesin is less enriched at the posterior pole (g) of the oocyte (white arrowheads) as compared with elsewhere of the oocyte cortex (paired t test p < .05, n = 20). Observation of GFP‐moesin at the anterior pole was challenging due to the presence of the dorsal appendages. However, in some (~40%; n = 25) oocytes Moesin is depleted compared with the lateral cortex (g′) and in these oocytes the depletion is significant (paired t test p < .05, n = 10). Single plane image (a,b,f,g). Scale bar = 10 µm (a,b), 20 µm (f,g)
	Figure 3. Actin wavefront dynamics follow calcium changes at egg activation. Time series of a mature egg chamber coexpressing GCaMP3 and F‐Tractin.tdTomato (a), or only expressing F‐Tractin.tdTomato (b) or Lifeact::mCherry (c). (a) At activation with activation buffer (AB) (n = 9), a calcium wave (a) initiates from the posterior pole (t = 0′) and propagates across the entire egg. A corresponding wavefront of F‐tractin (a′) initiates from the posterior pole and traverses the egg. Analysis of the fluorescence intensity shows that the calcium wave initiates on average 106 s after the addition of AB (SEM = 11.5 s), traverses the whole oocyte by 267 s (SEM = 42 s) and has recovered back to its initial level by 737 s (SEM = 148.37 s). A wave of filamentous actin (F‐actin) follows behind this calcium wave, initiating on average 220 s after the addition of AB (SEM = 60 s). The actin wave traverses the whole oocyte by 503 s (SEM = 72 s) and has recovered to its initial state by 829 s (SEM = 222 s). On average, the F‐actin wave lags behind the calcium wave by 103 s. As shown in Movie 6. Images were acquired using an inverted Leica SP5 confocal microscope sequentially, with a scan time of approximately 30 s per Z‐stack. (b) F‐actin, labeled by F‐tractin (n = 15), shows a posterior to anterior wavefront following the addition of AB (t = 0′). The wave initiates (t = 4′), propagates across the oocyte to the anterior pole (t = 10′) and recovers (t = 16′). As shown in Movie 7. Images were acquired using an inverted Leica SP5 confocal microscope. (c) F‐actin, labeled by Lifeact (n = 10), shows a similar actin wavefront as F‐tractin (b) in its initiation (t = 3′), propagation (t = 9′30″) and recovery (t = 15′). The bright fluorescence outside of the egg chamber is ovarian tissue associated with the dissection, as shown in movie 6, 7, and 9. Images were acquired using an inverted Leica SP5 confocal microscope. Image a projection of 40 µm (a,b,c). Scale bar = 60 µm (a–c). AB, activation buffer; SEM, standard error of the mean
	Figure 4. Actin wavefront requires the calcium wave at egg activation. Time series of mature egg chambers expressing F‐Tractin.tdTomato (a) or mature egg chambers coexpressing (b,c) GCaMP3 and F‐Tractin.tdTomato (b′,c′). (a) Time series of mature egg chambers expressing F‐Tractin in a sarah mutant background (sra A108/sra A426) (n = 7). The addition of activation buffer to mature egg chambers causes an initial dispersion of cortical actin (t = 4′), but does not initiate a wavefront of filamentous actin (F‐actin) (t = 15′). Images were acquired using an inverted Leica SP5 confocal microscope. (b–b′) Addition of 2‐aminoethoxydiphenyl borate (a TRP and IP3 pathway inhibitor) to modified Robb's Buffer prevents initiation of the calcium wave and an F‐actin wavefront is not observed (t = 13′55″; n = 14). Images were acquired using a Zeiss LSM880 confocal microscope. (c–c′) Application of pressure to the lateral cortex of the mature egg chamber induces a local increase calcium (c) (white arrowhead, t = 0′) (n = 12). This local increase recovers over time (t = 9′30″). Application of pressure does not result in a local F‐actin increase (c′) (white arrowhead, t = 0′), nor does it trigger an F‐actin wavefront. Images were acquired using a Zeiss LSM880 confocal microscope. Image a projection of 40 µm (a) or 90 µm (b,c). Scale bar = 60 µm (a–c)
	Figure 5. The pattern of actin reorganization follows calcium changes. Time series of mature egg chambers coexpressing GCaMP3 and F‐Tractin.tdTomato (a,b,d) or coexpressing GCaMP3 and Lifeact::mCherry (c). (a–a′) The addition of activation buffer (AB) with sodium orthovanadate initiates a calcium wave that is sustained (t = 40′; n = 10). An increase in filamentous actin (F‐actin) is similarly seen and is also maintained (t = 40′). Images were acquired using an inverted Leica SP5 confocal microscope. (b–b′) Following the addition of distilled water (DW; n = 20), the mature egg chamber undergoes a global increase in intracellular calcium (t = 0′) which slowly recovers to basal levels (t = 15′). F‐actin displays an initial dispersion (t = 3′) followed by a global increase (t = 9′30″), as shown in Movie 10. Images were acquired using an inverted Leica SP5 confocal microscope. (c) Following the addition of AB (n = 5), the calcium wave initiates from the anterior pole (t = 0′) and is followed by the wavefront of F‐actin from the same pole (t = 5′). Images were acquired using an inverted Leica SP5 confocal microscope. (d) Following addition of modified Robb's Buffer (RB) (n = 4), the calcium wave initiates from both anterior and posterior poles (t = 0′) and traversing the oocyte (t = 4′10″). The F‐actin wavefront initiates at the same sites as the calcium wave (t = 4′10″) and propagates with similar characteristics. Images were acquired using a Zeiss LSM880 confocal microscope. (e) Diagram showing the interaction between calcium and actin during Drosophila egg activation. Cortical actin is relatively rigid before egg activation. It is less dense near the oocyte poles, where calcium waves initiate ex vivo. The calcium wave that progresses through the oocyte precedes a wavefront of actin reorganization. The actin wave and the calcium wave display similar speeds and are interdependent for propagation and completion. After completion of the calcium wave, cortical actin appears more dynamic in distribution. Actin is represented in pink and calcium in blue. The diagram was created with BioRender.com. Image a projection of 40 µm (a–c) or 90 µm (d). Scale bar = 60 µm (a–d) [Color figure can be viewed at wileyonlinelibrary.com]

References [+] :

Becker, Reorganization of filamentous actin and myosin-II in zebrafish eggs correlates temporally and spatially with cortical granule exocytosis. 1999, Pubmed