A C. elegans Zona Pellucida domain protein functions via its ZPc domain

Zona Pellucida domain (ZP) proteins are critical components of the body’s external-most protective layers, apical extracellular matrices (aECMs). Although their loss or dysfunction is associated with many diseases, it remains unclear how ZP proteins assemble in aECMs. Current models suggest that ZP proteins polymerize via their ZPn subdomains, while ZPc subdomains modulate ZPn behavior. Using the model organism C. elegans, we investigated the aECM assembly of one ZP protein, LET-653, which shapes several tubes. Contrary to prevailing models, we find that LET-653 localizes and functions via its ZPc domain. Furthermore, we show that ZPc domain function requires cleavage at the LET-653 C-terminus, likely in part to relieve inhibition of the ZPc by the ZPn domain, but also to promote some other aspect of ZPc domain function. In vitro, the ZPc, but not ZPn, domain bound crystalline aggregates. These data offer a new model for ZP function whereby the ZPc domain is primarily responsible for matrix incorporation and tissue shaping.

Introduction incorporation [45,46]. Structural data showed that the EHP is part of the ZPc fold [33], and suggested a model in which ZPn polymerization is inhibited prior to secretion via an intramolecular interaction with the ZPc domain and the C-terminus [35,44]. This interaction may be stabilized by binding between the EHP and an internal hydrophobic patch (IHP) in the linker between the ZPn and ZPc domains [44,47]. Under this model, cleavage at the CCS evicts the EHP and C-terminus, releases ZPc-mediated inhibition, and enables ZPn polymerization [44,48]. However, this model is based on in vitro studies of only a few proteins, most of which were modified to promote efficient secretion [30,49]. It remains unclear whether the ZPc and C-terminus have roles other than facilitating ZPn secretion and assembly, and if and how the ZPn and ZPc domains impact one another's function in vivo.
Here, we investigate the matrix assembly of the C. elegans ZP protein LET-653, which shapes multiple tube types. We identify the ZPc domain as the critical domain for LET-653 function and matrix localization and discover that cleavage is required for ZPc function. We also show that the isolated ZPc domain incorporates into aggregates in vitro. Together, these data offer a novel model for ZP protein function and matrix assembly.

Background: LET-653 is a luminal ZP protein that shapes the duct and vulva tubes
LET-653 ( Fig 1A) is a component of a transient pre-cuticular luminal matrix that shapes multiple developing tubes [52,61]. LET-653 is essential to shape the tiny excretory duct tube, a unicellular tube that is less than a micron in diameter [52]. All let-653 mutants die as young larvae due to discontinuities in this tube lumen that prevent fluid excretion ( Fig 1B) [52,62]. let-653 mutants rescued in the duct cell survive into adulthood, but show more subtle defects in the shaping of other epithelial tissues, including the vulva, which is a large multicellular tube that, in adulthood, serves as the passageway through which embryos are laid [51,52,61] (Fig 1E). The excretory duct tube provides the most sensitive assay for LET-653 function, while both the duct (Fig 1C and 1D) and the vulva (Fig 1E-1G) allow rapid visual assessment of matrix incorporation via imaging of fluorescently-tagged LET-653 proteins in live animals.
Previously, we demonstrated that the LET-653 ZP domain is responsible for both tube shaping and incorporation into a membrane anchored aECM [52,61]. LET-653 is a secreted protein with two N-terminal PAN/Apple (plasminogen) domains and a C-terminal ZP domain [63] (Fig 1A). The PAN and ZP domains each confer different patterns of matrix localization, but only the ZP domain is capable of rescuing let-653 mutant lethality and both duct and vulva tube shape defects [52,61]. As observed for most other ZP proteins, the LET-653 C-terminus is cleaved, though LET-653 has no transmembrane or membrane association domain [52]. The cleaved C-terminus stays associated with the rest of the ZP domain in vivo, such that tagging LET-653(ZP) at either the N-or C-terminus yields the same localization pattern [52]. Fluorescence recovery after photobleaching (FRAP) analyses indicated that LET-653(ZP) has low mobility, consistent with matrix incorporation [52]. LET-653 has an N-terminal signal peptide (ss, pink) but lacks any other membrane-association domain. PAN; PAN/Apple domains, which are also found in the blood clotting factor plasminogen [81]. CCS (orange oval and scissors); consensus furin-type cleavage site (RRYR) located after the ZP domain and before the C-terminus. The linker is predicted to be N-glycosylated and heavily O-glycosylated. Pink box represents C-terminus. The LET-653 ZP domain aligns well with other ZP proteins, but has several unusual features. The ZPn domain lacks a key tyrosine residue thought to be important for ZPn polymerization [41,52], and the linker region between the ZP subdomains is unusually long, a feature whose impact is unclear [34]. B) Cartoons of the duct and pore cells of the excretory system and DIC images of let-653 mutants and WT C. elegans at L1 stage. Yellow; duct cell. Blue; pore cell. White; lumen. Black; cell junctions. Asterisk; luminal dilation. c; canal cell nucleus. B) Diagram of C. elegans 1.5 fold embryo. White; embryo. Gray; extraembryonic space. Arrowhead indicates duct/pore lumen. Red; canal cell. Yellow; duct cell. Blue; pore cell. D) Confocal image of a 1.5 fold embryo with CRISPR-tagged fulllength LET-653::SfGFP [61] with quantification of duct and extraembryonic fluorescence intensity. LET-653 is enriched in the duct lumen (arrowhead), buccal cavity and rectum. Fluorescence intensity was calculated within the indicated boxes to estimate matrix incorporation and overall expression levels, respectively. SfGFP alone shows minimal duct lumen fluorescence but strong extraembryonic fluorescence [52], while endogenously tagged full-length LET-653 shows strong duct lumen fluorescence but minimal extraembryonic fluorescence [61]. Quantification of average endogenous LET-653 fluorescence intensity per pixel in boxes drawn within the duct lumen and extraembryonic space. n = 10. AU; Arbitrary units. Error bars represent standard error. E) Cartoon of L4 larva. F-F') Confocal images of the vulva apical membranes, marked by PH::mCherry, and LET-653(ZP), which lines the dorsal vulE and vulF membranes. F') vulE and vulF membranes are faintly labelled by PH::mCherry (lines). G) Cartoon of vulva lumen and quantification of LET-653 fluorescence. The lumen is lined by 22 cells whose apical membranes define lumen shape. Cuticle lines the base of the vulva and a membranous hymen covers the top, generating a closed tube. Drawing a line across the dorsal region of the vulva allows quantification of LET-653 membrane vs. luminal localization. LET-653 fluorescence across the dashed line shows two peaks near the apical membranes and decreased fluorescence in the center of the lumen. Black; LET-653 fluorescence. Gray; membrane-associated and luminal regions whose fluorescence intensities were averaged. A line of 10 pixels thick (0.63 microns) was drawn across the vulva in the position indicated in panel H. Fluorescence intensity from 5 points proximal to each apical membrane (10 values total) were averaged and divided by the average fluorescence intensity across 10 points from the center of the vulva lumen. AU; arbitrary units. https://doi.org/10.1371/journal.pgen.1009188.g001

LET-653 functions via the ZPc domain
Transgenic structure/function experiments were performed to dissect ZP domain aECM assembly and tube shaping function. ZP domain fragments were expressed under a 2.2 kb let-653 promoter fragment and tagged with Superfolder GFP (SfGFP) [52,64]. These transgenes were expressed at levels comparable to endogenous, SfGFP-tagged LET-653 as assessed by fluorescence levels (Fig 1D, S1 Fig). For each transgene, multiple independent lines were tested in three assays. To determine function, we tested the ability of transgenes to restore viability to let-653(cs178) null mutants (Fig 1B). To assay localization, we imaged the developing duct tube in 1.5 fold embryos (Fig 1C and 1D), and the developing vulva tube in L4 larvae (Fig 1F  and 1G). In such assays, the positive control LET-653(ZP) localized transiently to the excretory duct lumen (Fig 2A, S2 Fig) and along specific apical membranes within the vulva tube (Figs 1F and 1G and 2A).
To determine which part of the LET-653 ZP domain is functionally important, we first expressed constructs containing either the ZPn domain and linker or the ZPc domain and Cterminal tail (referred to henceforth as simply ZPn or ZPc). Both proteins were efficiently secreted, but LET-653(ZPn) was expressed at slightly lower levels than LET-653(ZPc) in embryos (Fig 2B-2E). The ZPn domain transgene did not rescue let-653 mutant lethality ( Fig  2F). This protein localized weakly to the duct lumen and had no specific localization in the vulva (Fig 2B, 2D and 2E). In contrast, LET-653(ZPc) rescued let-653 mutants ( Fig 2F). This protein robustly lined the duct and vulva lumens (Fig 2C-2E). These data indicate that LET-653(ZPc) is sufficient for both function and proper localization, and suggest that this domain binds to a partner that helps recruit it to the membrane-anchored matrix.
To test whether the observed function and localization were due to activity of the main part of the ZPc domain or to the C-terminal tail that follows the CCS, we attempted to express each segment independently. The LET-653 C-terminus contains a hydrophobic region that may correspond to an EHP ( Fig 3A). To generate a truncated ZPc construct, we removed most (42/ 78 amino acids) of the C-terminus, leaving only this short, hydrophobic region (Fig 3A and  3B). LET-653(ZPc-½Cterm) was not secreted and did not rescue let-653 mutant duct defects (Fig 3B and 3D). This is consistent with prior results showing that the C-terminus is required for secretion of LET-653(full ZP)-containing proteins, though not for secretion of LET-653 (ZPn) ( [52]; Fig 2B). We conclude that the full C-terminus is required for proper trafficking and secretion of the ZPc domain.
Next, we tested if the LET-653 C-terminus was sufficient for function or localization. Our attempts to make transgenic animals expressing only the LET-653 C-terminal tail failed, suggesting that the C-terminal tail is toxic, perhaps due to its hydrophobicity ( Fig 3A). Instead, to test C-terminus activity, we attached it to the ZPn domain ( Fig 3C). This resulted in a secreted protein that did not rescue let-653 mutant duct defects and did not localize to the duct lumen (Fig 3C and 3D). In the vulva, most LET-653(ZPn+Cterm) protein formed large, irregular clumps, but some of the protein localized along the vulva apical membrane (Fig 3C). These observations suggest that the C-terminus does have some capacity for aggregation and/or matrix binding. However, the full ZPc domain is required for tube shaping and appropriate apical matrix localization.

LET-653 cleavage promotes ZPc function
Because ZP cleavage is thought to promote ZPn polymerization, we examined how it impacts LET-653(ZP) function. To test the role of cleavage in LET-653, we mutated the best candidate furin cleavage site (CCS) from RRYR to AAYA in a transgene encoding the full ZP domain (Fig 4A and 4B). Western blotting confirmed that, unlike wild-type LET-653, the mutated  (Fig 4F). Although this uncleavable protein still localized to the duct lumen, it localized weakly, or not at all, to the apical membrane in the vulva lumen ( Fig 4B-4D), and was unable to rescue let-653 mutant lethality ( Fig 4E). LET-653(ZP, AYAA) was expressed at similar levels and cleared from the duct lumen at the same time as the wild-type ZP domain ( Fig 4C, S2 Fig), indicating that differences in stability or clearance do not account for these results. We conclude that cleavage promotes LET-653(ZP) function and localization.
To determine how cleavage enables LET-653(ZP) function, we mutated the cleavage site in a transgene expressing LET-653(ZPc). If, as previously proposed [35], cleavage relieves an inhibitory ZPn-ZPc interaction, then cleavage should no longer be required when the ZPn is absent. In contrast, if cleavage changes ZPc conformation to enable ZPc interactions, then cleavage should still be required in the absence of ZPn. LET-653(ZPc, AYAA) localized efficiently to the apical membrane of the vulva and to the duct lumen of embryos, and partially of 1, where membrane fluorescence is equal to luminal fluorescence. Error bars; standard error. F) Both integrated (Is) and extrachromosomal (Ex) transgenes containing LET-653(ZP) rescued let-653 mutant lethality. Non-transgenic animals are pooled siblings from experiments in each group; nontransgenic animals never survive. 0/3 independent transgenic lines containing LET-653(ZPn) were functional, but 3/3 lines containing LET-653(ZPc) (either with or without an SfGFP tag) were functional. ��� p<0.0001, two-tailed Fisher's Exact test.

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rescued let-653 mutant duct defects (Fig 5B, 5C-5E). Western blots confirmed that LET-653 (ZPc, AYAA) was rarely cleaved (Fig 5F). We conclude that cleavage is not required for localization of the isolated ZPc domain but does promote its function. These results suggest that LET-653(ZPc) can bind to a relevant matrix partner without being cleaved, but that cleavage  1). p>0.01, Mann Whitney two-tailed U test. Error bars; standard error. D) Ratio of apical membrane-associated to lumen fluorescence in the L4 vulva (see Fig 1). ��� p<0.0001, Mann Whitney two-tailed U test. Dashed line indicates a ratio of 1, where membrane fluorescence is equal to luminal fluorescence. Error bars; standard error. E) LET-653(ZP)::SfGFP was partly functional in a let-653 mutant background, though rescue was not as strong as that seen with N-terminally tagged transgenes (compare to Fig 2F). Three independent lines of LET-653(ZP, AYAA):: SfGFP had little activity. Although the comparisons to non-transgenic siblings are not statistically significant, the few survivors likely represent some biological function for LET-653(ZP, AYAA), as let-653 mutants never survive [52]. ��� p<0.001, two-tailed Fisher's Exact test. F) Western blot demonstrating abrogation of cleavage. SfGFP::LET-653(ZP) runs at~120 kD while LET-653(ZP)::SfGFP runs at~40 kD, indicating that LET-653 is cleaved at its C-terminus as previously reported [52]. LET-653(ZP, AYAA)::SfGFP runs at nearly the same size as N-terminally tagged LET-653, indicating it is not cleaved. Loading control is RFP, which is co-expressed from transgenes containing LET-653 and whose expression is proportional to the amount of LET-653 in transgenic C. elegans. Blot is representative of n = 4 biological replicates.  Fig 4A) resulted in a protein that ran at~60kD, close to the estimated size of the entire protein fragment based on amino acid sequence. Loading control is RFP (see Fig 4). Blot is representative of n = 3 biological replicates. https://doi.org/10.1371/journal.pgen.1009188.g005

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may release inhibition of this binding by the ZPn domain. Cleavage also changes other functional properties of the LET-653(ZPc) module and/or releases the C-terminus for function.

LET-653(ZPc) incorporates stably into the aECM
To test if LET-653(ZPc) incorporates stably into the aECM, we performed FRAP analysis. In line with previous results [52], roughly 50% of LET-653(ZP) fluorescence was bleached and this fluorescence showed incomplete recovery, indicating that at least some of LET-653(ZP) is immobile in its aECM layer (Fig 6A, S3 Fig). In contrast, bleaching of secreted SfGFP was difficult to detect, likely due to swift recovery and replacement of SfGFP molecules from other parts of the vulva lumen (S3 Fig). A limited amount of LET-653(ZPc) fluorescence could be effectively bleached, though similar to LET-653(ZP), the bleached portion showed limited recovery (Fig 6B-6E, S3 Fig). Resistance to bleaching could indicate that 1) a portion of LET-653(ZPc) is highly mobile, similar to SfGFP alone, and recovers before bleaching can be detected; or 2) LET-653(ZPc) integrates into an aECM structure that shields the fluorescent protein from bleaching. In any case, incomplete recovery of the bleached portion of LET-653 (ZPc) is consistent with a portion of the protein stably incorporating into an aECM layer.

The LET-653 ZPc domain binds aggregates in vitro
To test whether the LET-653 ZPc domain polymerizes or aggregates, we expressed tagged LET-653 fragments in Drosophila S2R+ cells. LET-653(ZP) or LET-653(ZPc) were efficiently secreted and properly cleaved by S2R+ cells, as assessed by Western blotting (S4 Fig). As previously observed, another C. elegans ZP protein, DYF-7, formed large fibrils on the surface of S2R+ cells [54], but LET-653 fragments formed no such structures and instead appeared diffuse (S4 Fig). However, using higher magnification, we observed large crystalline aggregates throughout the cell media (Fig 7). Aggregates were present but not fluorescent in media from S2R+ cells transfected with empty vector (EV), SfGFP, or the LET-653(ZPn) domain (Fig 7A,  7B, 7D and 7H). In contrast, aggregates were fluorescent in media containing LET-653(ZP) or LET-653(ZPc) (Fig 7C, 7E, 7F and 7H). Media containing LET-653(ZP, AYAA) had crystalline aggregates that were similar in brightness to those of media containing LET-653(ZP) (Fig 7F-7H). The number of aggregates varied between technical replicates and did not correlate with genotype ( S5 Fig). We conclude that the aggregates consist primarily of components derived from S2R+ cells or media, and that LET-653 binds these aggregates but does not stimulate their formation. Furthermore, efficient aggregate binding in vitro correlates well with efficient matrix incorporation and function in vivo.
To test the properties of the observed aggregates, we examined whether they stained with amyloid dyes or were heat sensitive. The dye Nile Red stained aggregates whether secreted LET-653(ZPc) was present or not (Fig 8). This dye binds amyloid structures, as well as hydrophobic materials and some salt crystals [65][66][67]. To test heat sensitivity, we heated freshly thawed media to 50˚C for fifteen minutes. Imaging revealed that fluorescent aggregates remained after heating (Fig 8C). The fact that these aggregates can be labeled with dyes and are non-heat-soluble indicates that they may have some biochemical characteristics of amyloid. Future studies are needed to determine the composition of these aggregates and any relationship they might have to the endogenous matrix partners of LET-653 in vivo.

Discussion
Although ZP proteins are critical components of aECMs in metazoans, how ZP proteins assemble in aECMs remains poorly understood. In this study, we investigated how one C. elegans ZP protein, LET-653, assembles into aECMs. We found that, counter to prevailing models for ZP assembly, LET-653 functions and localizes via its ZPc domain (Fig 9A and 9B). Cleavage of the C-terminus may release inhibition of the ZPc by the ZPn domain, but also promotes ZPc function via some other mechanism. In vitro, LET-653(ZPc) spontaneously incorporates into large aggregates. These data demonstrate that ZPc domains can function independently to assemble in aECMs, functions that were previously attributed only to ZPn domains.

ZPc domains can function independently of ZPn domains
ZPc domains have been generally assumed to support ZPn domains and not to function independently [30]. Initial surveys of ZP proteins across phyla suggested that some contain ZPn domains without ZPc domains, and yet are still capable of polymerization [36], while proteins with only ZPc domains were not thought to exist [30]. However, we and others found that C. elegans does have five predicted ZPc-only proteins [34,68]. In proteins like uromodulin that have polymerizing ZPn domains, ZPc domains are thought to inhibit the ZPn domain [30]. While our study was under review, two groups reported cryo-EM structures for full-length uromodulin that suggest filament assembly involves interactions of both ZPn and ZPc domains with linker regions of other monomers, which would suggest that both domains are functionally important [69,70]. In the TGFβ receptors Betaglycan and Endoglin, ZPc domains dimerize and can bind ligands, but have not been reported to form higher-order structures [28,29,71]. Our results indicate that ZPc domains can function independently of ZPn domains for both matrix incorporation and tissue-shaping.
In fact, in the case of LET-653, the ZPn domain inhibits ZPc activity, perhaps to confine ZPc activity to precise times and places within the aECM. We did not detect any independent role for the ZPn domain in shaping the duct lumen, and this domain is not able to localize specifically in the vulva aECM. This result is surprising given the centrality of ZPn domains to most models of ZP function [30]. The LET-653 ZPn domain lacks a key tyrosine residue present in most polymerizing ZP proteins [41,52], which could explain functional differences between the LET-653 ZPn domain and others. Intriguingly, this tyrosine residue is also absent in a number of other C. elegans ZP proteins [61], the Drosophila protein Zye [50] and some mammalian proteins including Endoglin [41] and Oit3 (S6 Fig). Researching additional ZP proteins may reveal that both ZPn and ZPc domains can have diverse functions, and that either domain, or both domains simultaneously, can direct matrix incorporation.
The LET-653 ZPc domain has additional cysteines compared to some ZP proteins. ZPc domains fall into several families, with 4, 6, or 8 cysteines [30,34]. The LET-653 ZPc domain has 8, as does the Zona Pellucida ZP protein ZP3 [30,33]. Like LET-653, ZP3 has a cleaved Cterminus that can bind the ZPc domain [33]. It is possible that all ZPc domains with 8 cysteines have analogous assembly mechanisms. Testing the assembly of additional proteins in this class may expose patterns in ZP structure and function.

Cleavage promotes two separable aspects of ZPc function
Intramolecular interactions between ZPn and ZPc domains during cellular trafficking are thought to hold ZP proteins in an inactive conformation to prevent premature

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oligomerization [30]. C-terminal cleavage, perhaps by proteases at the plasma membrane, relieves this auto-inhibition to allow matrix incorporation [43,45,48]. Our data are consistent with this model, although the relative roles of the ZPn vs ZPc domains are reversed. Our data further suggest that cleavage promotes two distinct roles of the ZPc domain: 1) the ability to bind to a matrix partner that determines spatial specificity; and 2) some other change that is essential for tube-shaping function. Cleavage may relieve ZPn-dependent inhibition of the first role, but the second role still requires cleavage even in the absence of the ZPn domain.
The relevant matrix partner(s) of LET-653(ZPc) are not known, but they appear to be located on the apical surfaces of the excretory duct tube and vulE and vulF vulva cells. Although let-653 is expressed by all vulva cells [52], genetic perturbations to increase or eliminate vulE or vulF cell types showed that these cells have a specific ability to recruit LET-653 (ZP) [61]. Furthermore, vulF cells contain large matrix-secreting vesicles whose contents appear disorganized in let-653 mutants, suggesting that LET-653 may traffic through the same compartment with its partner(s) [61]. Identifying the relevant partner(s) may require a combination of genetic and biochemical approaches and would likely offer crucial insight into how LET-653 incorporates into matrix and shapes tubes.
The second ZPc function enabled by cleavage might be oligomerization, binding to a second partner, and/or release of the hydrophobic C-terminus for function. A portion of LET-653(ZPc) appears immobile in the aECM in vivo (Fig 6) and incorporates into crystalline aggregates in vitro (Fig 7), consistent with the possibility that LET-653(ZPc) may oligomerize to form its matrix layer in vivo. Our data further indicate that the cleaved C-terminus stays associated with the ZPc domain, since LET-653(ZPc) tagged at either end results in identical localization patterns. Models for ZP organization generally show the C-terminus binding to the linker [35], but the crystal structure of the protein ZP3 indicates the C-terminus can bind the ZPc domain [41]. Furthermore, the C-terminus alone can confer aggregation properties when attached to the ZPn domain (Fig 3). We propose that a cleavage-dependent change in the relative conformations of the main ZPc and C-terminus may facilitate oligomerization and interactions with other partners (Fig 9B).
LET-653 incorporates into roughly spherical aggregates that resemble salt crystals. These aggregates may consist largely of protein or could represent protein-salt complexes. Intriguingly, salt appears to stimulate uromodulin to form filaments [35]. Furthermore, the ZP protein Oit3 (also known as LZP), a proposed partner of uromodulin, can form spherical aggregates of unknown organization in vitro [31]. However, how these Oit3 aggregates assemble, if they include other co-factors, such as uromodulin or salts, and whether they form in vivo is not clear. Previous in vitro studies of ZP proteins have been challenging, as many cell types fail to cleave or secrete intact ZP proteins appropriately [33,35]. S2R+ cells fortuitously express a compatible protease and offer a straightforward model to assess ZP assembly and to characterize ZP-containing aggregates.

Conclusion
ZP proteins are a diverse family of aECM components that are found across the animal kingdom. ZP proteins shape many apical surfaces [14], changes in ZP protein expression or structure are implicated in the evolution of morphological features [72] and speciation [73], and ZP protein loss contributes to multiple human diseases. This work harnessed C. elegans to demonstrate a new mode of ZP protein assembly in which the ZPc domain is the primary functional unit. Future work should take advantage of additional tractable models to characterize the true diversity of ZP protein function and assembly mechanisms.

Staging and microscopy
Embryos were selected at the 1.5-fold stage based on morphology. Larvae were staged by size and vulva lumen morphology. Fluorescent, brightfield, Differential Interference Contrast (DIC), and Dodt (an imaging technique that simulates DIC) [75] images were captured on a Zeiss Axioskop compound microscope fitted with a Leica DFC360 FX camera or with a Leica TCS SP8 confocal microscope (Leica, Wetzlar Germany). Images were processed and merged using ImageJ.

Quantification of LET-653 fluorescence
To confirm that LET-653 transgenes were adequately expressed, their fluorescence intensity was measured in embryos within boxes drawn on single confocal slices. Boxes were drawn either to fit the small duct lumen using the ImageJ "Rotated rectangle" tool or were drawn to roughly 3x3 microns within the extraembryonic space (Fig 1D). The average fluorescence intensity per pixel within those regions was measured using the ImageJ "Mean gray value" tool.
Measurements of LET-653 enrichment at the vulva vulE and vulF cell apical membranes were performed as in Bidaud-Meynard et al. [76]. 10 pixel-thick-lines were drawn across the dorsal region of the vulva, including both apical membranes. 5 pixel values representing LET-653 fluorescence intensity at each apical membrane (totaling 10 values) were averaged and divided by 10 pixel values from the luminal region directly between the vulE and vulF apical membranes ( Fig 1G). All measurements are taken from single confocal Z-slices. Final values reported are ratios.

let-653 transgenes
All let-653 transgenes were generated by inserting LET-653 fragments into the plasmid pJAF5, which contains a 2.2 kb fragment containing the let-653 promoter and upstream regulatory region [52]. All plasmids were generated via restriction enzyme cloning and were verified by Sanger sequencing. let-653 encodes three isoforms of a protein with two N-terminal PAN/ Apple domains and a ZP domain. As the shortest isoform, LET-653b, was previously shown to be sufficient for function, all structure-function constructs are based on LET-653b (GenBank accession no: X91045.1) [52]. Plasmids were co-injected at concentration 17 ng/μL with marker pHS4 (lin-48pro::mRFP) at concentration 50 ng/μL. Integrated transgenes were generated using TMP/UV as described [77] and include: In most cases, two or three independent lines of each transgene were assayed for rescue activity and gave similar results. Only a single line per transgene was used for the image quantifications shown. A complete list of transgenes, primers, and plasmids generated for this study can be found in S2 Table. FRAP Specimens were mounted on 10% agarose pads containing 20 mM sodium azide and 10 mM levamisole in M9 after Gill et al., 2016 [52]. FRAP was performed using Leica Application Suite X software FRAP module on a Leica TCS SP8 MP confocal microscope. Three regions of interest of 3 × 3 μm were defined within the wizard: a bleached region of interest (bleached ROI), an unbleached ROI of similar fluorescence intensity, and a background ROI drawn in a region that did not include an animal. Maximum laser intensity was used for bleaching experiments and experiments were carried out over the course of a single imaging session. Mean fluorescence intensities within the ROIs were measured at specified intervals. Background ROI values were subtracted from both the bleached and unbleached ROI values. Corrected bleach ROI values then were divided by corrected unbleached ROI values (bleached-background / unbleached-background), and the resulting ratios were normalized such that the maximum intensity was set to 1. The following experimental time-course was used: 20 prebleach frames every 0.4 sec, 10 bleach frames every 0.4 sec, and 90 postbleach frames every 2.0 sec. Pre-and postbleach laser intensity was set to 1% and bleach laser intensity was set to 100%. FRAP plots were created using Prism (Graphpad, San Diego California). Mobile fraction = (recovery fluorescence-postbleach fluorescence) / (prebleach fluorescence-postbleach fluorescence) [78].

Drosophila cell culture and media experiments
S2R+ cells were cultured under standard conditions [79]. Cells were transfected using Effectene (Qiagen, 301427, Venlo Netherlands). Plasmids for transfection were generated by inserting cDNAs containing let-653b into pAC5.1 as XbaI-EcoRI fragments alongside the unc-54 3' UTR as an EcoRI-ApaI fragment. Three sets of S2R+ cells were independently transfected with 1 μg DNA of the indicated constructs. Cells were grown under standard conditions for 72 hours before imaging and media collection. Cells were imaged under brightfield and fluorescence microscopy using a Leica SP5 confocal microscope, and media and cells were then collected for further analysis. Media was collected by pipetting and was then centrifuged for 1 min at 10,000. Cells were washed in PBS then lysed via incubation for 20 minutes at 4˚C in 1 ml lysis buffer (50 mM Tris-HCl pH 7.4, 500 mM NaCl, 1% NP40, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 1× complete protease inhibitor cocktail (Roche, Basel Switzerland) and 1 mM PMSF). Cell lysates were centrifuged for 20 minutes at 4 C at 10,000 rpm to remove debris. Media and cell lysates were stored at -80˚C until ready for use. All three sets of transfections showed evidence of LET-653 secretion and cleavage as assessed by Western blotting. Two out of two tested sets of transfections showed evidence of ZPc binding to aggregates.
To image aggregates, 3 μL freshly thawed media was pipetted onto glass slides. DIC images for counting aggregates were obtained with a Zeiss Axioskop. 5 evenly spaced 92 mm x 92 mm fields of view were imaged per slide. To quantify aggregate number, DIC images were first subjected to a grayscale threshold filter in ImageJ (default threshold; limits: 85-255) and then aggregates were counted using the 3D Particle Counter plugin. The number of aggregates per field of view was adjusted to represent the number of aggregates per μL. To measure fluorescence intensity of aggregates, a box of 5 x 5 microns was drawn within aggregates and the average intensity per pixel (Mean gray value) was recorded from a single confocal Z-slice. A minimum of 5 aggregates per sample were measured.
To measure aggregate stability at different temperatures, 12 μL of media was thawed and aliquoted. 3 μL of this media was immediately pipetted onto glass slides for imaging. The remaining 12 μL of media was heated to 50˚C in a thermocycler for 15 minutes and then a portion was immediately imaged. Another portion was reserved and cooled to 4˚C in a thermocycler for 15 minutes before imaging. Aggregate number from DIC images was quantified as described above, except that before thresholding, images were processed using the ImageJ plugin "Background Correction". To stain aggregates with Nile Red, a solution of 100 mM in acetone was mixed with media for a final concentration of 1 mM and the sample was then imaged immediately.
Western blotting was performed on 5 μL of media or cell lysate as described below.

Western blotting
Early embryos were collected via the alkaline bleach method [80] from two 60 mm plates of nearly confluent worms and embryos were allowed to develop in M9 for 4-5 hours until they reached 1. Loading controls were carried out as described above using 1:1000 anti-RFP antibody (Abcam, ab62341) and 1:10,000 anti-rabbit-HRP antibody (GE Healthcare, NA934V). RFP corresponds to the lin-48p::mRFP coinjection marker present in all transgenes, and therefore reflects the number of transgenic animals present in each sample.  [52]. Grey shading corresponds to known beta-strands in ZP3, blue indicates conserved cysteines and yellow indicates conserved aromatic residues. Like LET-653, Oit3 lacks the conserved aromatic residue in the F strand between Cys3 and Cys4, and DMBT1 lacks the second conserved aromatic residue after Cys4. These two residues interact in the ZP3-N crystal structure [41] and are thought to be critical for ZPn polymerization. Genbank accession numbers: Ce_LET-653b: NP_001021337; Ce_DYF-7: NP_509630; Dm_Dpy: NP_001245875; Hs_UMOD: NP_003352; Hs_TectA: NP_005413; Hs_ZP3: NP_001103824; Hs_DMBT1: NP_004397; Hs_Oit3: NP_689848; Hs_Eng: NP_001108225. (DOCX) S1 Table. Strains generated in this study. All strains used or generated in this study are listed with full genotypes. (DOCX) S2 Table. Plasmids generated in this study. All plasmids generated in this study are listed with their contents and mechanism of assembly.