A condensate dynamic instability orchestrates actomyosin cortex activation

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1–3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4–8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition [1][2][3] . Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex 4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion 9 , the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability 10 suppresses coarsening of the active micro-emulsion 11 , ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.
Morphogenesis involves forces that are generated within the actomyosin cortical layer of cells 1 . Improper cortical organization leads to an impairment of key cellular and developmental processes from as early as meiosis in oocytes to every subsequent cell division 12 . During meiotic maturation of oocytes, the actomyosin cortex transitions from inactive and non-contractile, to active and tension generating 2,3 . This transition can generate a spectrum of actomyosin cortical structures and dynamics, including an actin cap in the mouse oocyte 13 , actin spikes in starfish oocytes 14 and waves of Rho activation and F-actin polymerization in Xenopus 15 . Organizing the first active actomyosin cortex requires the recruitment and assembly of various cortical components as well as the polymerization of actin filaments 4 . These processes have to be coordinated across the entire cell surface in order to generate a uniform actomyosin cortical layer. Here we ask how the formation of an active and tension-generating actomyosin cortex during meiotic maturation in oocytes is orchestrated.

Actomyosin cortex activation
The hermaphrodite nematode Caenorhabditis elegans is a prime system for investigating actomyosin cortex formation during oocyte maturation [16][17][18] . In C. elegans, the onset of meiotic divisions and oocyte maturation coincides with ovulation and fertilization 16,18 . Oocytes are fertilized inside the hermaphrodite mother as they pass through the sperm-containing organ-the spermatheca 16 . To understand how the formation of the first actomyosin cortex during oocyte maturation is orchestrated, we visualized F-actin in C. elegans oocytes containing Lifeact::mKate2. We observed that, just before fertilization inside the mother, the oocyte cortical layer appears undeveloped with only sparse amounts of filamentous actin present (Fig. 1a, left). By contrast, shortly after fertilization a highly dynamic and dense actomyosin cortical layer is present below the plasma membrane (Fig. 1a, right and Supplementary Video 1). Importantly, we find that actomyosin cortex activation in the oocyte occurs through an intermediate stage that lasts approximately 10 min, results in a dynamic and contractile actomyosin cortical layer, and ends with the extrusion of the first polar body 19 (Supplementary Video 1). Strikingly, this intermediate stage is characterized by the transient appearance of thousands of F-actin-rich condensates at the cortical layer (Fig. 1a). Here we use the term condensate to refer to a dense assembly of specific molecular components maintained by collective molecular interactions. F-actin and its nucleators have previously been shown to form biomolecular condensates and evidence for liquid-like properties has been provided [5][6][7][8] . The F-actin-rich condensates we observe are highly dynamic and inherently unstable. They appear stochastically and each disappear after approximately 10 s. We next set out to investigate the nature of these transient F-actin-rich condensates. To better observe their dynamics, we took advantage of the fact that oocytes isolated from the mother can mature in the absence of fertilization 20 . This allowed us to develop a total internal reflection fluorescence (TIRF) assay 21 utilized under highly inclined and laminated optical sheet (HILO) conditions for imaging cellular structures near the cell membrane (Fig. 1b). This enabled a quantitative study of actomyosin cortex formation in isolated oocytes at high spatial and temporal resolution (Supplementary Video 2). F-actin polymerization is organized by nucleation pathway members such as N-WASP and the ARP2/3 complex, as well as the elongator Formin 22 . We first investigated the presence of these Article components in cortical condensates. We used three strains labelling F-actin (by expressing Lifeact::mKate2) together with either endogenously labelled N-WASP (WSP-1::GFP), capping protein (CAP-1::GFP) or Formin (CYK-1::GFP) 23 . In addition, we used a strain that endogenously labels both the ARP2/3 complex (ARX-2::mCherry) and N-WASP (WSP-1::GFP). As well as F-actin, we identified WSP-1, the  . j, Instantaneous concentrations of F-actin and WSP-1 within condensates from an ensemble of 36,930 condensates from 9 oocytes. Here 68% and 25% of instantaneous condensate concentrations fall within the outer and inner dark blue contour line, respectively. The light blue dot indicates the peak of preferentially maintained concentration pair of WSP-1 and F-actin in control oocytes. k, Normalized probability density functions of WSP-1::GFP (green lines) and Lifeact::mKate (magenta lines); the integrated condensate intensities are similar at 0, 4 and 8 min after the onset of oocyte maturation. ARP2/3 complex and the capping protein CAP-124 (Fig. 1c, Extended Data Fig. 1 and Supplementary Videos 3 and 4) as components of cortical condensates, but the Formin CYK-1 was absent 19,24 (Extended Data Fig. 1). This demonstrates that cortical condensates contain molecules that mediate branched F-actin nucleation, similar, for example, to CD44 nanoclusters, dendritic synapses and podosomes [25][26][27] . We also noted that during their approximately 10 s lifetime (Fig. 1f) cortical condensates were enriched first in WSP-1 and ARP2/3, and only then F-actin accumulated before first losing WSP-1 and ARP2/3 and then F-actin (Fig. 1d,e). Given the time at which they appear and the fact that they contain molecules that mediate branched F-actin nucleation, we speculate that dynamic cortical condensates play a role in the formation of the first oocyte cortex.
We next asked if cortical condensates constitute a phase that coexists with its surroundings. Such a phase is characterized by material properties (such as density) that are intensive, that is, independent of volume. We used the strain that simultaneously labels F-actin and WSP-1 to show that, throughout their brief lifetime (Fig. 1e,f), cortical condensates varied over two orders of magnitude in both Lifeact (A) and WSP-1 (W) integrated fluorescence intensities (Fig. 1g). We estimated the volume of cortical condensates from the cross-sectional area determined by segmentation 28 1i). This provides a relation between molecular content and volume, but does not imply that condensates are densely packed structures of only WSP-1 and F-actin. Whereas cortical condensates varied over two orders of magnitude in integrated fluorescence intensities (Fig. 1g), the respective concentrations of WSP-1   (yellow line) for the cortical condensate in a. The blue shaded region indicates the range of stoichiometry for which the volume dependence accounts for measured volumes (Extended Data Fig. 2). c, Mass flux phase portrait measured from 299,165 time points of 36,930 condensates from 9 oocytes (experiment, orange and grey arrows), and calculated from empirically determined growth laws (theory, yellow, green and blue arrows); see Extended Data Fig. 10 for separate representations. The colour scale denotes time rate change vector magnitudes. Thick lines indicate WSP-1 (green) and F-actin (magenta) nullclines from experiment; thin lines indicate theoretical nullclines. Absolute molecular amounts can be estimated for WSP-1 with 8 IU corresponding to approximately 100 WSP-1 molecules. d, Measured WSP-1 (green) and F-actin (magenta) growth rates as a function of stoichiometry display three regimes separated by the WSP-1 nullcline at stoichiometry approximately 0.85 and the F-actin nullcline at stoichiometry approximately 0.9. e,f, Linear dependence of relative WSP-1 (e) and F-actin (f) growth rates-in the unperturbed control (blue) and mild arx-2 RNAi (orange) and moderate (mod.) RNAi (red) cases-on effective F-actin volume fraction ϕ (Supplementary Information). Linearity holds within the blue shaded region (see b, Extended Data Fig. 2) and is indicated with lines, yielding the parameters k r , k l (e) and k b , k d (f). g, Reaction motif underlying the structure of c-f composed of WSP-1 self-recruitment, WSP-1 dependent F-actin polymerization, F-actin dependent WSP-1 loss and F-actin depolymerization.

Article
and F-actin within the cortical condensates were significantly more restricted in their variation (Fig. 1h). This is also reflected in the emergence of a preferred pair of F-actin and WSP-1 concentrations maintained on average by the ensemble of cortical condensates (Fig. 1j). We conclude that, on the one hand, cortical condensates are maintained far from equilibrium: they are highly dynamic and each disassemble after approximately 10 s. On the other hand, cortical condensates display signatures of a multicomponent condensed phase: they occupy a volume determined by their molecular content and show an enrichment of WSP-1 and F-actin at concentrations distinct from their external environment 29,30 . Hence, the ensemble of stochastically appearing, growing and subsequently dissolving cortical condensates effectively forms a chemically active micro-emulsion, which, despite continuous turnover, maintains a steady size distribution that does not coarsen 11 (Fig. 1k). Both the properties of a condensed phase and the mechanisms underlying its formation and dissolution can be revealed by a study of growth kinetics.

Cortical condensate growth laws
To study the growth kinetics of these cortical condensates, we quantified their compositions and volumes over time ( Fig. 2 and Supplementary Methods). For a single representative cortical condensate, Fig. 2a,b shows the time evolutions of (1) WSP-1 and F-actin total condensate intensity, (2) stoichiometry and (3) volume (Supplementary Notes 1 and 2). For the example shown, WSP-1 precedes F-actin in both growth and loss, stoichiometry grows monotonically with time, and volume first increases and then decreases, and is well captured by summing volume contributions from F-actin and WSP-1. We noted that neighbouring condensates followed similar trajectories in composition and volume despite forming stochastically and at different times (Fig. 1c-e and Extended Data Fig. 9). Thus, at a given time, neighbouring cortical condensates that share their external environment can be at different stages of their internal life cycle. We conclude that the growth kinetics postnucleation are governed by condensate internal composition.  How does the internal composition of a cortical condensate influence its growth and shrinkage? To answer this question we developed a general method to quantitatively study compositional dynamics in an ensemble of multicomponent condensates based on an analysis of the mass flux into the condensates (mass balance imaging 31 ). For this, we quantified the time rate change of protein amounts within cortical condensates as a function of their internal F-actin and WSP-1 amounts. This time rate change of amounts is represented by a vector field, which defines average trajectories in the space of WSP-1 and F-actin amounts (Fig. 2c). Consistent with the representative example (Fig. 2a), average trajectories form loops that pass through three subsequent regimes: an early growth regime in which condensates first grow in WSP-1 and subsequently in F-actin amounts; a transition regime in which WSP-1 is lost while F-actin amounts still increase; and a disassembly regime with loss of both WSP-1 and F-actin. The nullcline of WSP-1 dynamics (the green line in Fig. 2c), that is, the WSP-1 amounts above which condensates grow and below which they shrink in WSP-1 content, reflects an F-actin-dependent critical WSP-1 amount for WSP-1 growth. Stoichiometry is constant on lines that pass through the origin, and hence the WSP-1 nullcline corresponds to a threshold stoichiometry of approximately 0.85. F-actin growth dynamics change from growth to shrinkage at a similar but slightly higher stoichiometry of approximately 0.9 (the magenta line in Fig. 2c shows the F-actin nullcline). We conclude that cortical condensates become unstable and change from growth to disassembly in the transition regime between the two nullclines.
The three regimes (growth, above the WSP-1 nullcline; transition, between the two nullclines; disassembly, below the F-actin nullcline in Fig. 2c) are also visible when plotting WSP-1 and F-actin growth rates as a function of stoichiometry (Fig. 2d). Because the stereotypical compositional trajectories (Fig. 2a,b) involve a monotonic increase in stoichiometry with time, the x axis of Fig. 2d also represents a progression through time. The dependence of growth rates on stoichiometry reveals the mutual regulation of WSP-1 and F-actin, and can be depicted by the reaction motif shown in Fig. 2g. Processes I and II are mediated by WSP-1, whereas processes III and IV are mediated by F-actin. Process I corresponds to WSP-1 self-recruitment, evidenced by the fact that at low stoichiometry, corresponding to condensates consisting of mainly WSP-1, the WSP-1 growth rate is largest (Fig. 2d). Process II denotes WSP-1 dependent F-actin growth, reflected by a decrease of the F-actin growth rate as stoichiometry increases. This is most evident in the transition regime of Fig. 2d. Process III denotes F-actin-dependent loss of WSP-1, reflected by the fact that WSP-1 growth rates decrease with increasing stoichiometry. This suggests that F-actin counteracts the ability of WSP-1 to self-recruit, similar to previously reported negative feedback of F-actin on its nucleation via Rho 15,32 . Finally, process IV denotes F-actin depolymerization, reflected by the fact that F-actin is lost fastest at the highest stoichiometry (Fig. 2d). Further support for this reaction motif is provided by an analysis of WSP-1 and F-actin growth rates at constant WSP-1 and F-actin amounts (Extended Data Fig. 9), and an analysis of the impact of RNA interference (RNAi) of proteins involved in regulating F-actin (Extended Data Fig. 4).
The shape of the measured phase portrait (Fig. 2c) and the shape of the growth rates Ẇ for WSP-1 and Ȧ for F-actin as a function of stoichiometry (dots denote time derivatives; Fig. 2d) suggest the following empirical growth laws that define a non-linear dynamical system 33 (Fig. 2c, Extended Data Fig. 3

Article
ere WSP-1 self-recruitment depends linearly on W through the recruitment rate k r , consistent with the ability of WSP-1 molecules to dimerize 34,35 (process I). Interactions between F-actin and WSP-1 result in ARP2/3 mediated branched nucleation and a subsequent increase in the amounts of F-actin 36,37 . This behaviour is captured by the term k where k b is a kinetic coefficient describing branching and condensate volume V v A v W = + A W depends on molecular amounts (see above and also Supplementary Information; process II). Branched nucleation coincides with a loss in WSP-1; this loss is captured by k AW V l with the kinetic coefficient k l describing the branching-dependent loss of WSP-1 (ref. 38 ; process III). Finally, F-actin is lost with rate k d , consistent with severing and depolymerization 39 (process IV) (see also the simplified depiction in Extended Data Fig. 6). Note that these four coefficients together capture all the relevant molecular processes inside the condensates. This may include processes not discussed above. The mathematical form of all four terms is determined by the observation that the relative growth rates W W / and A A / are linear functions of the effective F-actin volume fraction ϕ =  Figure 2e,f also allow us to estimate k r , k l , k b and k d . With these estimates, the simple growth laws describe the experimental data well, and capture the entire mass flux phase portrait together with the composition-dependent critical sizes as reflected by nullclines (Fig. 2c).
specifies a critical amount of WSP-1 above which WSP-1 amounts grow and below which WSP-1 amounts shrink. Notably, this critical amount is similar to a critical droplet size for nucleation and growth, but here it stems from biochemical reactions and not from condensation physics. The resulting growth laws exhibit a fixed point at A W ( , ) = (0, 0) with a stable (F-actin) and an unstable (WSP-1) direction. After nucleation, condensate dynamics follow a homoclinic orbit, initially growing rapidly in the unstable direction before turning and eventually undergoing disassembly while moving along the stable direction back towards the fixed point. Together, this represents a dynamic instability of condensates that shares similarities with the dynamic instability of microtubules 10 : cortical condensates transition from unstable growth to shrinkage, which limits their size, and can display stochastic rescue events (Extended Data Fig. 7).

Transition to unbounded growth
To understand how the transition from condensate growth to condensate disassembly is orchestrated, we used RNAi to perturb the interplay between WSP-1 and F-actin. RNAi of upstream signalling molecules that regulate F-actin assembly, such as RHO-1 (Rho GTPase), CYK-1, CDC-42 and CHIN-1 (CDC-42 GAP), as well as multivalent adaptors VAB-1 (Ephrin receptor) and NCK-1 (Nck) did not affect condensate dynamics [40][41][42] (Extended Data Fig. 4 and Supplementary Notes 4 and 7). This suggests that cortical condensate dynamics are governed by feedback structures independent of the major signalling pathways that regulate the actomyosin cortex 43 . WSP-1 mediates branched F-actin nucleation through the ARP2/3 complex 4,35 , and we thus performed RNAi against ARX-2 (ARP2 in the ARP2/3 complex in C. elegans 44 ). Oocytes showed reduced numbers of cortical condensates for less than 20 h of arx-2 RNAi, whereas cortical condensates were absent for more than 20 h of RNAi (Fig. 3a,b and Extended Data Fig. 7A), consistent with a general reduction of F-actin branched nucleation 45 .
We used the reduction in the number of cortical condensates as a measure of the strength of the perturbation, and distinguished between mild (30 to 70 condensates per oocyte), moderate (70 to 120 condensates per oocyte) and strong arx-2 RNAi (no dynamic cortical condensates). Mass balance imaging of cortical condensates in the mild and moderate conditions revealed that, in comparison with the unperturbed case, compositional trajectories are progressively tilted towards the WSP-1 axis (Fig. 3c). The mathematical form of the growth laws is maintained, but the associated coefficients are changed (Fig. 2e,f). Mild and moderate arx-2 RNAi reduced the rate of WSP-1 self-recruitment k r by 17 ± 4% and 15 ± 4%, respectively, and increased the coefficient k l for moderate arx-2 RNAi by 20 ± 6% (1 ± 5%). The branching coefficient k b remained essentially unchanged, indicating that ARP2/3 amounts are not rate limiting for ARP2/3 mediated branching inside cortical condensates. The dominant effect of mild and moderate arx-2 RNAi is an approximately 1.7-and 2.7-fold increase, respectively, of the F-actin loss rate k d . This is consistent with previous findings that the ARP2/3 complex protects F-actin from depolymerization in vitro 46 . In addition to the changes of coefficients, mild and moderate arx-2 RNAi both reduced the average F-actin concentration by a factor of approximately 1.4 and 1.5, respectively, and increased the average WSP-1 concentration by a factor of approximately 1.5 and 1.8, respectively (Fig. 3d). In all three cases, no RNAi control, and mild and moderate arx-2 RNAi, the pairs of average concentrations fall on the line of constant total density of WSP-1 and F-actin together (Fig. 3d, yellow dashed line; see Supplementary Information). We conclude that the ARP2/3 complex, largely through its impact on F-actin disassembly, governs the transition from condensate growth to condensate disassembly and determines the ensemble-averaged pair of internal concentration along the line of constant total density. Strong depletion of ARX-2 by RNAi (more than 20 h of RNAi feeding at 20 °C) resulted in a loss of dynamic cortical condensates, and a considerably altered cortical architecture with large persistent patches of F-actin and WSP-1 (Fig. 3b, right, Supplementary Video 5 and Extended Data Fig. 8). We asked if this phenotype can be understood given the condensate growth laws above. It is not possible to determine the four growth law coefficients for strong arx-2 RNAi using mass balance imaging, because there are no dynamic condensates. However, the systematic change of both the F-actin loss rate k d and the condensate number per oocyte for increasing strength of arx-2 RNAi enabled us to provide a lower-bound estimate of k d for the strong RNAi condition (Extended Data Fig. 7). We find that at and above this estimated value of k d the system crosses a critical point at which the two nullclines switch their position, with the F-actin nullcline now above the WSP-1 nullcline (Fig. 3c, right). This causes a notable change in the growth dynamics of cortical condensates, with a complete loss of the homoclinic orbits that transition from growth to shrinkage. Instead, condensates exhibit unbounded growth consistent with the emergence of large persistent patches of F-actin and WSP-1 (Fig. 3b, right; note that we expect unbounded growth to ultimately become limited by effects we have not considered in our description, such as the depletion of the monomer pool). In conclusion, our analysis suggests that a switching of nullcline positions in strong arx-2 RNAi leads to uncontrolled F-actin growth and impaired cortical activation in the oocyte, and therefore impaired later development 22,47 (Supplementary Notes 5 and 6).

Ensemble properties
How do the growth kinetics lead to a specific pair of average internal concentrations and therefore a specific stoichiometry? To address this question, we change variables from F-actin amount A and WSP-1 amount W to effective F-actin volume fraction ϕ v A V = / A and condensate volume V. Figure 4a shows that the calculated phase portrait in the ϕ-V plane obtained by a change of variables of the empirically determined growth laws is consisted with the experimental one determined from measured WSP-1 and F-actin amounts and measured condensate volumes. Figure 4b shows that the transition from condensate growth (red) to shrinkage (blue) occurs at an effective F-actin volume fraction of approximately 0.8, corresponding to a stoichiometry of approximately 0.86. Notably, at this stoichiometry the rate of change of condensate volumes and stoichiometry is slowest (orange dotted lines in Fig. 4a,b,d), implying that the ensemble of dynamic condensates is governed by this slowly varying, thus dominant, stoichiometry. Hence, the peak of the concentration histograms (Figs. 1j and 3d) occurs at the point at which the line of dominant stoichiometry intersects with the line of constant total density (Fig. 4d).

Intensive chemical reaction dynamics
We also recognized that the time evolutions of the effective F-actin volume fraction ϕ̇ and the WSP-1 and F-actin concentrations are independent of condensate volume (Supplementary Notes 8-10). Thus, condensate dynamics are intensive, which is consistent with mass action kinetics in well-mixed systems. However, conventional mass action kinetics change reactant concentrations at constant volume, but usually do not involve assembly and disassembly as is the case here. Note that intensive condensate dynamics are not consistent with the conventional kinetics of nucleation and growth of liquid-like condensates, in which assembly rates depend on condensate size 48,49 . This reveals that cortical condensates exhibit an unconventional chemical kinetics in which mass action governs assembly and disassembly, and therefore the effect of mass action dynamics on concentrations in condensates is modified (Supplementary Notes 8-10). Note, however, that even though cortical condensates do not assemble via classical nucleation and growth, the intensive condensate dynamics show that the condensate material behaves as a well-mixed phase with size-independent properties. Intensive reaction dynamics are expected to arise in situations in which the time for diffusion across the condensate is shorter than the typical time associated with a chemical reaction. The condensate dynamic instability limits cortical condensate size. Therefore, reaction dynamics remain intensive and the resultant chemically active micro-emulsion maintains a steady-state size distribution (Fig. 1k) 11 .

Discussion
To conclude, cortical condensates represent a new type of nonequilibrium biomolecular condensate that assembles and disassembles via a non-linear dynamic process governed by mass action chemical kinetics. They recruit molecules that drive branched nucleation of F-actin and support the activation of the actomyosin cortex. The dynamics of the growth and shrinkage of cortical condensates are similar to the dynamic instability of growing and shrinking microtubules 10 , but arises in a bulk assembly that forms a phase. We suggest that the formation and subsequent dissolution of cortical condensates via a condensate dynamic instability serves to control autocatalytic F-actin nucleation and prevents runaway growth during the activation of the first cortical actin meshwork in the C. elegans oocyte.

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Fig. 10 | Theory and experimental phase portraits from Fig. 2c presented separately. A, Non-RNAi experimental phase portrait from Fig. 2c. B, Non-RNAi theory phase portrait from Fig. 2c. Solid lines indicate WSP-1 (green) and F-actin (Magenta) nullclines, dashed lines indicate WSP-1 (green) and F-actin (magenta) mild arx-2 RNAi nullclines, and dotted lines indicate WSP-1 (green) and F-actin (magenta) moderate arx-2 RNAi nullclines.

Corresponding author(s): Stephan Grill
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No sample size pre-calculation was performed. Each measured oocyte, contained sufficient condensate events (always >5000) to conduct the phase portrait analysis described in our manuscript and reach all the main conclusions. For comparison between the control and two RNAi datasets, data was combined from multiple oocytes in each condition. These sample sizes were chosen to ensure >100 condensates in each phase portrait bin in all three datasets -the number 100 was chosen to be well above that suggested by an analysis of the variance in phase portrait vectors..

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All data and analysis results were replicable for multiple imaged C. elegans oocytes. Data from n oocytes in any given condition were combined for further analysis with n as stated across the manuscript and supplement.
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