zar1 Antibody

What is ZAR1 and what is its primary function in cellular biology?

ZAR1 (Zygote arrest protein 1) is an mRNA-binding protein that plays a crucial role in embryonic development. It mediates the formation of MARDO (mitochondria-associated ribonucleoprotein domain), which is a membraneless compartment that stores maternal mRNAs in oocytes. The assembly of MARDO around mitochondria is directed by an increase in mitochondrial membrane potential during oocyte growth. ZAR1 promotes the formation of these phase-separated membraneless compartments by undergoing liquid-liquid phase separation upon binding to maternal mRNAs .

The protein is essential for female fertility and the oocyte-to-embryo transition, as it coordinates maternal mRNA storage, translation, and degradation. ZAR1 specifically binds to the 3'-UTR of maternal mRNAs, and these maternal mRNAs stored in the MARDO are translationally repressed until needed for embryonic development . This temporal control of maternal mRNA translation is critical for proper embryonic development following fertilization.

What are the molecular characteristics of ZAR1 protein that researchers should be aware of?

ZAR1 has several important molecular characteristics that researchers should understand when designing experiments. The protein contains a highly conserved C-terminal domain with a zinc finger motif that is critical for its RNA-binding function. This zinc finger is dependent on zinc ions (Zn²⁺) for proper functioning, as demonstrated by experiments showing that ZAR1 binds RNA in the presence of Zn²⁺ but not in its absence .

The predicted molecular weight of ZAR1 is approximately 46 kDa (specifically 45,873 Da according to calculations), which is important information when interpreting Western blot results . ZAR1 specifically binds to translation control sequences (TCSs) in the 3' UTR of target mRNAs, such as Wee1 mRNA, and this binding appears to have higher affinity compared to its paralog ZAR2 . Mutation studies have shown that conserved cysteines in the C-terminal domain are essential for RNA binding, further confirming the importance of the zinc finger structure .

How do ZAR1 and ZAR2 differ functionally and structurally?

While ZAR1 and ZAR2 are paralogs with similar domains, they exhibit distinct functional and expression characteristics. Both proteins have highly homologous C-terminal domains containing zinc fingers important for RNA binding, but ZAR1 demonstrates a significantly higher affinity for target RNAs such as the Wee1 3' UTR. Experimental data shows that the dissociation constant (Kd) for ZAR1 ranges from 3.5 nM to 40 nM, whereas ZAR2's Kd ranges from 60 nM to 440 nM, indicating that ZAR1 binds with approximately 10-fold higher affinity .

Expression patterns of ZAR1 and ZAR2 during oogenesis also differ. When protein levels per oocyte were compared in Xenopus, ZAR1 levels reached maximum in Stages I–III and then declined through Stages IV to VI, whereas ZAR2 levels peaked at Stages IV–V before declining in Stage VI. During oocyte maturation, ZAR1 levels remained constant, while ZAR2 levels decreased . These differences in expression profiles suggest that ZAR1 and ZAR2 may play complementary but distinct roles during early development, with ZAR1 potentially having greater importance during earlier developmental stages.

How can researchers effectively design experiments to study ZAR1's role in MARDO formation?

When designing experiments to study ZAR1's role in MARDO formation, researchers should consider a multifaceted approach that combines protein localization, RNA binding analysis, and functional studies. Since ZAR1 mediates the formation of mitochondria-associated ribonucleoprotein domains through liquid-liquid phase separation, microscopy techniques that can visualize membraneless organelles are essential. Immunofluorescence using specific ZAR1 antibodies (such as the rabbit polyclonal antibodies described in the literature) combined with mitochondrial markers can help visualize the association between ZAR1, maternal mRNAs, and mitochondria .

For RNA binding studies, researchers can employ electrophoretic mobility shift assays (EMSAs) as described in previous work with ZAR1. This technique was successfully used to demonstrate that the C-terminal domain of ZAR1 binds specifically to TCSs in the 3' UTR of mRNAs like Wee1 . When designing such experiments, it's important to include appropriate controls such as competition with unlabeled RNA and supershift assays with anti-ZAR1 antibodies to confirm specificity of binding. Additionally, researchers should consider the zinc-dependent nature of ZAR1's RNA binding activity when designing buffer systems for in vitro experiments .

Functional studies of MARDO formation might employ mutational analysis of ZAR1's key domains. Given that conserved cysteines in the C-terminal domain are critical for RNA binding, site-directed mutagenesis of these residues could help elucidate structure-function relationships. RNA immunoprecipitation followed by sequencing (RIP-seq) could be employed to identify the complete repertoire of maternal mRNAs bound by ZAR1 in vivo, providing insights into the global role of ZAR1 in maternal mRNA regulation.

What are the optimal experimental conditions for studying ZAR1's interaction with maternal mRNAs?

Studying ZAR1's interaction with maternal mRNAs requires careful consideration of experimental conditions that preserve the integrity of both the protein and its target RNAs. Based on published research, several critical parameters emerge for optimal results. First, buffer conditions should include zinc ions (Zn²⁺), as ZAR1's RNA binding activity is zinc-dependent through its zinc finger domain . Experiments have shown that omitting zinc from binding buffers abolishes ZAR1's RNA binding capacity.

For in vitro binding assays such as EMSAs, using the conserved C-terminal domain of ZAR1 fused to a tag like GST has proven effective. The full-length ZAR1 does not bind RNA effectively in vitro, similar to observations with ZAR2 . This suggests that the N-terminal domains may have regulatory effects on RNA binding that are overcome in the cellular context, potentially through interactions with other proteins or post-translational modifications.

When designing RNA probes for binding studies, researchers should focus on the 3' UTR regions of potential target mRNAs, particularly those containing TCSs. Experiments with Wee1 mRNA demonstrated that ZAR1 binds specifically to TCSs, and mutating these sequences significantly reduces binding . Using fluorescently labeled RNA probes (such as Cy5-labeled probes) allows for sensitive detection of binding interactions. Competition assays with unlabeled RNAs can confirm binding specificity, as demonstrated by experiments where unlabeled wild-type Wee1 RNA competed for binding but unrelated RNAs or those with mutated TCSs did not .

How can researchers interpret conflicting ZAR1 antibody results across different developmental stages?

Interpreting conflicting ZAR1 antibody results across developmental stages requires careful consideration of several factors including protein isoforms, post-translational modifications, and methodological variations. Research has shown that ZAR1 antibodies may recognize multiple bands in earlier stages of oogenesis but only a single band in Stage VI oocytes and during oocyte maturation . This variability could stem from several sources that researchers should systematically investigate.

First, consider the possibility of post-translational modifications that change during development. The multiple bands observed in early oogenesis could represent differentially phosphorylated or otherwise modified forms of ZAR1 that converge to a single form in later stages. To test this hypothesis, researchers could employ phosphatase treatments of protein samples before Western blotting or use phospho-specific antibodies if available.

Alternative splicing may also contribute to conflicting results. Published research has identified both Zar1a and Zar1b sequences in Xenopus, suggesting the existence of splice variants . Researchers should design PCR primers to detect potential splice variants and correlate their expression with the protein bands observed. Using antibodies raised against different regions of ZAR1 (N-terminal versus C-terminal domains) might help distinguish between splice variants.

Methodological variations in sample preparation can also lead to conflicting results. Protein degradation during sample handling could produce artificial bands. This possibility can be addressed by including protease inhibitors in lysis buffers and comparing fresh versus stored samples. Cross-reactivity with related proteins (like ZAR2) should be ruled out by using highly specific antibodies. The published research demonstrates that carefully validated ZAR1 antibodies do not cross-react with ZAR2, despite sequence similarities .

What are the best practices for Western blot analysis using ZAR1 antibodies?

Successful Western blot analysis using ZAR1 antibodies requires attention to several critical factors. Based on published protocols and commercial antibody recommendations, the following best practices emerge. For sample preparation, researchers should use appropriate lysis buffers containing protease inhibitors to prevent degradation of ZAR1 protein. Given the predicted molecular weight of ZAR1 at approximately 46 kDa , standard SDS-PAGE conditions with 10-12% acrylamide gels should provide adequate separation.

Validation of antibody specificity is crucial for interpreting results correctly. This can be accomplished through several approaches: (1) peptide competition, where pre-incubation of the antibody with the immunizing peptide should abolish specific binding, as demonstrated in published work ; (2) use of positive and negative control samples with known ZAR1 expression; and (3) correlation with mRNA expression data. When analyzing developmental samples, researchers should consider normalizing to appropriate loading controls, as protein content changes dramatically during oogenesis and early development. Published work has used tubulin as a normalization control for ZAR1 expression studies .

How should researchers design RNA binding experiments to study ZAR1's interaction with target mRNAs?

Designing effective RNA binding experiments for ZAR1 requires careful consideration of protein constructs, RNA probes, and assay conditions. Based on published research, electrophoretic mobility shift assays (EMSAs) provide a robust method for studying ZAR1-RNA interactions in vitro. For protein expression, researchers should consider using the conserved C-terminal domain of ZAR1 (containing the zinc finger) fused to a tag such as GST for purification and detection. Full-length ZAR1 has been reported not to bind RNA effectively in vitro, similar to observations with ZAR2 .

RNA probe design is critical for successful binding studies. Researchers should focus on the 3' UTR regions of potential target mRNAs, particularly those containing translation control sequences (TCSs). Fluorescently labeled probes (such as Cy5-labeled RNA) allow for sensitive detection of binding. When studying novel potential targets, researchers should include positive controls such as the Wee1 3' UTR, which has been confirmed to bind ZAR1 .

Binding buffer composition significantly impacts ZAR1-RNA interactions. Critically, buffers must contain zinc ions (Zn²⁺) as ZAR1's RNA binding is zinc-dependent. Experiments have shown that omitting zinc from binding buffers abolishes ZAR1's RNA binding capacity . To establish binding specificity, researchers should perform competition assays with unlabeled RNAs (both specific target RNAs and non-specific control RNAs) and supershift assays using antibodies against ZAR1 or its fusion tag. Mutational analysis of putative binding sites in target RNAs can further confirm binding specificity and identify critical nucleotides for interaction.

What technical considerations are important when selecting or validating ZAR1 antibodies for specific experimental applications?

Selecting and validating ZAR1 antibodies for specific experimental applications requires consideration of several technical factors. First, researchers should identify the epitope recognized by the antibody and assess its conservation across species of interest. Commercial ZAR1 antibodies are available targeting different regions of the protein - for example, antibodies raised against synthetic peptides within the N-terminus (amino acids 33-49) or C-terminus (amino acids 267-286) of ZAR1, or against amino acids 220-270 of human ZAR1 . Epitope location can affect antibody performance in different applications; for instance, some N-terminal ZAR1 antibodies work well for Western blot but not for immunoprecipitation .

Species reactivity is another critical consideration. Commercial antibodies list reactivity with specific species, such as human, mouse, and rat samples . When working with other species, researchers should assess sequence homology in the epitope region to predict potential cross-reactivity. Antibody validation should include multiple methods to confirm specificity. Peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific binding, provide strong evidence for specificity . Comparison of results across multiple antibodies targeting different epitopes of ZAR1 can further increase confidence in specificity.

For each experimental application, optimization of conditions is essential. For Western blotting, researchers should determine optimal antibody dilution, blocking conditions, and detection methods. For immunoprecipitation, buffer conditions that preserve native protein structure while minimizing non-specific binding are critical. For immunofluorescence, fixation and permeabilization methods should be optimized to preserve epitope accessibility. Researchers should also be aware that post-translational modifications might affect epitope recognition, potentially explaining why multiple bands are observed in Western blots of early oogenesis samples but not in later developmental stages .

How can researchers address inconsistent Western blot results when detecting ZAR1 in different tissue types?

Inconsistent Western blot results when detecting ZAR1 across different tissue types can stem from multiple factors that researchers should systematically address. First, consider the natural variation in ZAR1 expression levels. ZAR1 is primarily expressed in oocytes and early embryos, with expression profiles changing during development. As documented in Xenopus studies, ZAR1 levels are highest in early oocyte stages (Stages I-III) and decline through later stages . Therefore, inconsistent results between reproductive and non-reproductive tissues may simply reflect biological reality rather than technical issues.

Sample preparation techniques should be optimized for each tissue type. Different tissues may require different lysis buffers to effectively extract ZAR1. Include appropriate protease inhibitor cocktails to prevent degradation, especially important when comparing tissues with different protease compositions. Flash-freezing samples immediately after collection can help preserve protein integrity. For reproductive tissues, consider developmental timing carefully, as ZAR1 expression changes dramatically across oogenesis and early embryonic development .

Protein loading and transfer efficiency can vary between tissue types due to differences in sample composition. Use appropriate loading controls that are consistently expressed across the tissues being compared. For reproductive tissues, traditional housekeeping genes like GAPDH or β-actin may not be ideal as their expression can change during development. Consider normalization to total protein using stains like Ponceau S as an alternative approach. Optimize transfer conditions for the molecular weight of ZAR1 (approximately 46 kDa) .

Finally, consider antibody-specific factors. Different antibodies may recognize different isoforms or post-translationally modified versions of ZAR1. Some commercial antibodies are raised against specific regions of human ZAR1 , which may have varying degrees of conservation across species. Always include positive controls (tissues known to express ZAR1) and negative controls (tissues or cell lines with ZAR1 knocked down) when possible.

What methodological approaches can resolve contradictory data regarding ZAR1's RNA binding specificity?

Resolving contradictory data regarding ZAR1's RNA binding specificity requires a comprehensive, multi-method approach that addresses potential technical and biological variables. First, researchers should standardize protein preparation methods. The C-terminal domain of ZAR1 containing the zinc finger has been successfully used for in vitro RNA binding studies, while full-length ZAR1 does not bind RNA effectively in vitro . Protein expression systems (bacterial, reticulocyte lysate, etc.) can affect protein folding and post-translational modifications, potentially impacting binding properties.

RNA probe design and preparation are critical variables. Inconsistencies may arise from differences in RNA secondary structure, which can be influenced by buffer conditions and temperature. Researchers should perform RNA folding predictions and structure mapping to ensure consistent RNA conformations across experiments. The sequence context around core binding motifs can also influence binding; studies with Wee1 mRNA demonstrated that ZAR1 binds to translation control sequences (TCSs), but mutating these sequences only partially abolished binding, suggesting additional binding determinants .

Buffer conditions significantly impact RNA-protein interactions. ZAR1's RNA binding is zinc-dependent, so zinc concentration in binding buffers is a critical parameter . Salt concentration, pH, and the presence of competing ions can also affect binding specificity. Researchers should systematically vary these parameters to determine optimal conditions for specific binding. Temperature can influence both RNA structure and protein binding kinetics, so consistent temperature control across experiments is essential.

To definitively resolve contradictions, researchers should employ multiple complementary techniques. In addition to EMSAs, consider methods such as filter binding assays, RNA immunoprecipitation followed by sequencing (RIP-seq), or CLIP-seq (cross-linking immunoprecipitation followed by sequencing) to identify binding sites in vivo. Surface plasmon resonance or isothermal titration calorimetry can provide quantitative binding parameters. Functional validation through reporter assays or in vivo mutational analysis of putative binding sites can connect biochemical binding data to biological function.

How should researchers interpret ZAR1 expression data across different model organisms and developmental stages?

Interpreting ZAR1 expression data across different model organisms and developmental stages requires careful consideration of evolutionary conservation, technical variables, and biological context. ZAR1 is conserved across vertebrates but with varying degrees of sequence similarity. When comparing expression patterns, researchers should first establish orthology relationships through phylogenetic analysis. For example, Xenopus laevis has been shown to have both Zar1a and Zar1b sequences, and proper classification of Zar sequences should be based on characteristic amino acids and chromosomal context .

Different detection methods may yield apparently contradictory results. Western blotting detects protein levels but may miss isoforms or modified forms if antibodies lack appropriate specificity. RT-PCR or RNA-seq measure transcript levels, which may not directly correlate with protein abundance due to translational regulation. Immunohistochemistry or immunofluorescence provide spatial information but can be affected by epitope accessibility. When possible, researchers should employ multiple complementary methods and validate commercial antibodies for species-specific applications.

Developmental timing is crucial for proper interpretation. In Xenopus, ZAR1 levels per oocyte reach maximum in Stages I–III and then decline through Stages IV to VI, while during oocyte maturation, ZAR1 levels remain constant . When comparing across species, consider equivalent developmental stages rather than absolute timing. The observation of multiple ZAR1 bands in early oogenesis but a single band in late oogenesis suggests developmental regulation of post-translational modifications or isoform expression, which may vary between species.

Functional context should guide interpretation of expression differences. ZAR1's role in maternal mRNA storage and regulation may be conserved across species, but the specific target mRNAs and regulatory mechanisms might vary. ZAR1 and ZAR2 show different expression profiles during oogenesis , suggesting complementary but distinct roles. Researchers should consider the entire regulatory network in which ZAR1 functions, including potential interacting proteins and target mRNAs, which may vary across species and developmental contexts.

What are the emerging techniques that could advance our understanding of ZAR1's role in early embryonic development?

Several cutting-edge techniques show promise for unraveling ZAR1's complex functions in early embryonic development. CRISPR-Cas9 gene editing offers unprecedented precision for creating model organisms with specific ZAR1 mutations. By introducing mutations in functional domains such as the zinc finger or creating conditional knockouts, researchers can dissect ZAR1's role at specific developmental stages while avoiding the complete fertility failure associated with conventional knockouts. Domain-specific mutations could distinguish ZAR1's roles in RNA binding versus MARDO formation.

Advanced imaging techniques like super-resolution microscopy and live-cell imaging can reveal the dynamics of ZAR1-containing ribonucleoprotein complexes during development. These techniques could visualize the formation and dissolution of MARDO structures in real-time, providing insights into how ZAR1 mediates the storage and regulated release of maternal mRNAs. Combining these imaging approaches with techniques like fluorescent in situ hybridization (FISH) could simultaneously track ZAR1 protein and its target mRNAs .

Transcriptome-wide approaches for mapping RNA-protein interactions in vivo, such as enhanced CLIP (eCLIP) or individualized nucleotide resolution CLIP (iCLIP), could comprehensively identify ZAR1's RNA targets during different developmental stages. These techniques provide single-nucleotide resolution of binding sites, allowing precise characterization of ZAR1's binding motifs across different target mRNAs. Complementary techniques like ribosome profiling could correlate ZAR1 binding with translational regulation of maternal mRNAs.

Single-cell approaches represent particularly promising avenues for future research. Single-cell RNA-seq and single-cell proteomics could reveal heterogeneity in ZAR1 expression and function among individual oocytes or blastomeres, potentially explaining variable developmental competence. Spatial transcriptomics techniques could map the distribution of ZAR1 and its target mRNAs within the complex cytoplasm of oocytes and early embryos, providing insights into how spatial regulation contributes to developmental timing and cell fate decisions.

How might the study of ZAR1 contribute to advances in assisted reproductive technologies?

ZAR1's critical role in oocyte-to-embryo transition positions it as a potentially valuable marker and therapeutic target for improving assisted reproductive technologies (ARTs). Research has established that ZAR1 is essential for female fertility through its coordination of maternal mRNA storage, translation, and degradation . This function is fundamental to early embryonic development before the embryonic genome is activated. Understanding ZAR1 expression and activity could therefore provide new diagnostic tools for assessing oocyte quality and developmental potential.

Non-invasive methods for evaluating ZAR1 activity could be developed by identifying secreted factors or accessible biomarkers that correlate with proper ZAR1 function. Since ZAR1 regulates specific maternal mRNAs, the translation products of these mRNAs might be detectable in follicular fluid or culture media. Alternatively, small amounts of cumulus cells, which are connected to oocytes via gap junctions, might reflect aspects of oocyte ZAR1 activity that could be measured without compromising oocyte integrity.

For in vitro maturation (IVM) of oocytes, a procedure increasingly used in fertility treatments, optimization of culture conditions to support proper ZAR1 function could improve developmental outcomes. Research has shown that ZAR1 activity depends on zinc ions , suggesting that careful attention to trace element composition in culture media might be important. Additionally, since ZAR1 mediates the formation of mitochondria-associated RNP domains that are directed by increases in mitochondrial membrane potential , culture conditions that support optimal mitochondrial function might enhance ZAR1-dependent processes.

In more advanced applications, transient modulation of ZAR1 activity might help rescue developmental potential in compromised oocytes. This could involve techniques to deliver exogenous ZAR1 protein, stabilize endogenous ZAR1, or provide key ZAR1-regulated factors to bypass defects in the ZAR1 pathway. Such approaches would require detailed understanding of ZAR1's partners and targets in different species, including humans, and careful validation in model systems before clinical application.

What technological developments are needed to better study ZAR1's interactions with the maternal transcriptome?

Advancing our understanding of ZAR1's interactions with the maternal transcriptome requires several technological developments. First, improved methods for isolating intact ribonucleoprotein complexes from oocytes and early embryos are needed. The large size and complex cytoplasm of oocytes present challenges for traditional biochemical approaches. Development of gentle fractionation techniques that preserve native ZAR1-RNA interactions while allowing separation from other cellular components would enable more precise characterization of these complexes. Combining such fractionation with mass spectrometry could identify proteins that cooperate with ZAR1 in regulating maternal mRNAs.

Methods to visualize ZAR1-RNA interactions in living oocytes and embryos would provide unprecedented insights into the dynamics of maternal mRNA regulation. This might involve adaptation of techniques like MS2 tagging of target mRNAs combined with fluorescently tagged ZAR1, allowing real-time visualization of interactions. Development of biosensors that report on ZAR1 activity or the translational status of its target mRNAs could provide functional readouts in living cells.

Finally, computational tools specifically designed for analyzing the unique features of maternal transcriptomes are needed. The maternal transcriptome includes many specialized transcripts and regulatory elements that may not be well-annotated in standard genome databases. Improved algorithms for identifying regulatory motifs in 3' UTRs, predicting RNA secondary structures that might influence ZAR1 binding, and integrating various omics data types would enhance our ability to interpret experimental results and generate testable hypotheses about ZAR1's regulatory networks.

How does ZAR1 antibody performance compare across different commercial sources and applications?

Comparing ZAR1 antibody performance across commercial sources reveals important differences in specificity, sensitivity, and application versatility that researchers should consider when selecting reagents. Based on available data, several commercial antibodies target different epitopes of ZAR1, which influences their performance characteristics. The Abcam antibody (ab129875) is a rabbit polyclonal antibody generated against a synthetic peptide within human ZAR1, suitable for Western blot applications, and has been validated for human samples . The Boster Bio antibody (A12936-1) is also a rabbit polyclonal antibody but targets amino acids 220-270 of human ZAR1 and is reported to react with human, mouse, and rat samples in Western blot applications .

Specificity validation methods vary between commercial sources, which can impact confidence in antibody performance. The Abcam antibody has been validated using peptide competition assays, where pre-incubation with the immunizing peptide blocks antibody binding in Western blots . Additionally, this antibody shows a band at the predicted molecular weight of 46 kDa in Western blots of 293 cell lysates. Research-grade antibodies described in the literature include those raised against N-terminal (amino acids 33-49) or C-terminal (amino acids 267-286) peptides of ZAR1 . Interestingly, the antibodies raised against the N-terminal domain performed well in Western blot but not in immunoprecipitation, while those against the C-terminal domain did not produce antibodies specific to ZAR1 .

For application versatility, most commercially available antibodies have been primarily validated for Western blot applications . More extensive application testing and validation would benefit the research community, particularly for techniques like immunoprecipitation, immunofluorescence, and chromatin immunoprecipitation that might reveal new aspects of ZAR1 biology. When selecting antibodies for novel applications, researchers should conduct their own validation studies using appropriate positive and negative controls.

What are the key differences in ZAR1 function and expression between mammalian and non-mammalian vertebrate models?

Comparative analysis of ZAR1 across vertebrate species reveals both conserved and divergent aspects of its function and expression. ZAR1 appears to be conserved across vertebrates, reflecting its fundamental role in early development, but with notable species-specific adaptations. In both mammalian and non-mammalian models, ZAR1 functions in maternal mRNA regulation and is essential for female fertility, but the specific mechanisms and expression patterns show interesting variations.

Expression timing differs between mammalian and non-mammalian species. In Xenopus, ZAR1 protein levels per oocyte reach maximum in early oogenesis (Stages I–III) and then decline through later stages, while remaining constant during oocyte maturation . This contrasts with mammalian models where ZAR1 expression persists through maturation and early embryonic divisions until maternal-to-zygotic transition. These differences likely reflect the varying developmental strategies and timing of maternal-to-zygotic transition between species.

Gene duplication events have created species-specific differences in the ZAR family. Xenopus laevis has been shown to have both Zar1a and Zar1b sequences, as well as Zar2a and Zar2b . This gene duplication and subfunctionalization may allow for more specialized roles of different ZAR family members in amphibian development. Mammals appear to have fewer ZAR family members, potentially requiring individual ZAR proteins to fulfill multiple functions that are distributed among paralogs in non-mammalian vertebrates.