Recombinant Drosophila sechellia Eukaryotic translation initiation factor 3 subunit M (Tango7)

What is Tango7 and what are its primary functions in Drosophila?

Tango7 (Transport and Golgi organization 7) in Drosophila functions as a dual-purpose protein with roles in both cellular remodeling and potentially in translation initiation. As an eukaryotic translation initiation factor 3 subunit M (eIF3m), it may participate in protein synthesis regulation. More prominently, Tango7 collaborates with the Drosophila apoptosome to drive caspase-dependent remodeling processes, particularly during spermatogenesis .

Research has demonstrated that Tango7 is essential for resolving individual sperm from a syncytium during spermatid individualization. It interacts directly with apoptosome components Dark and Dronc, localizing to the active apoptosome compartment via its C-terminus . Biochemical studies show that Tango7 directly stimulates apoptosome activity in vitro in a dose-dependent manner .

Studies of Tango7 orthologs in Caenorhabditis elegans and yeast suggest it may also function as a noncore factor associated with the COP9 signalosome complex and/or the eIF3 translation complex, indicating its potential involvement in multiple cellular pathways .

How does Tango7 differ from typical apoptosis-regulating proteins?

Unlike classic apoptosis regulators that primarily control cell death pathways, Tango7 appears to specify the Drosophila apoptosome for cellular remodeling rather than cell demolition . This represents a fascinating example of how apoptotic machinery can be repurposed for non-lethal cellular processes.

While viable tango7 mutants exhibit wing phenotypes characteristic of apoptotic mutants, they don't show the global cell death defects typically seen in core apoptotic pathway mutations . Furthermore, unlike proteins that simply inhibit or activate apoptosis, Tango7 appears to modulate the specificity of the apoptosome, directing it toward remodeling functions .

The protein's ability to stimulate apoptosome activity while preventing cell death suggests it may regulate substrate specificity, subcellular localization, or intensity of caspase activation. This contrasts with binary regulators of apoptosis that simply turn the process on or off .

What experimental approaches are used to study Tango7 function in Drosophila?

Multiple complementary approaches have been employed to investigate Tango7 function:

• Genetic analysis: Creation of various tango7 mutant alleles, including the tango7L allele that truncates 30 amino acids from the C-terminus. These mutants show male sterility and characteristic wing phenotypes .

• Rescue experiments: Generation of a genomic rescue construct (Tango7 VK006) using site-specific integration of a 20-kb bacterial artificial chromosome spanning the Tango7 locus, which effectively rescues mutant phenotypes .

• Immunolocalization: Anti-Tango7 antibody staining reveals localization to the cystic bulge and waste bag during spermatid individualization, co-localizing with active caspases (anti-CC3) specifically at these structures .

• Biochemical assays: In vitro reconstitution of the apoptosome using recombinant Dronc and Dark with or without recombinant Tango7, assaying for caspase activity using catalytically dead pro-Drice C/A as a substrate .

• Protein-protein interaction studies: Co-immunoprecipitation experiments in cultured S2R+ cells demonstrating physical interactions between Tango7 and apoptosome components Dark and Dronc .

These multidisciplinary approaches collectively establish Tango7's role in specifying the apoptosome for cellular remodeling rather than cell death.

What methods are used to express and purify recombinant Tango7?

The expression and purification of recombinant Tango7 typically follows these methodological steps:

• Gene cloning and vector construction: The Tango7 coding sequence is PCR-amplified and cloned into expression vectors with appropriate tags (His, GST, or FLAG) to facilitate purification .

• Expression system selection: For functional studies of Tango7, both prokaryotic (E. coli) and eukaryotic expression systems (insect cells) have been employed. The choice depends on the specific requirements for protein folding, post-translational modifications, and yield .

• Protein expression: Optimization of expression conditions including temperature, induction time, and inducer concentration to maximize protein yield and solubility.

• Purification strategy:

  • Initial capture by affinity chromatography using the fusion tag

  • Further purification through ion exchange chromatography

  • Final polishing by size exclusion chromatography

  • Removal of tags using specific proteases when necessary

• Quality control:

  • SDS-PAGE and Western blotting to verify purity and identity

  • Activity assays to confirm functional integrity (e.g., ability to stimulate apoptosome activity)

In vitro studies have successfully used recombinant Tango7 to demonstrate its direct stimulation of apoptosome activity, indicating that the purified protein retains its functional properties .

How is Tango7 function assessed in cellular and biochemical contexts?

Assessment of Tango7 function utilizes both cellular and biochemical approaches:

Cellular assays:

• Spermatid individualization analysis: Phalloidin staining of testes to visualize actin-rich investment cones (ICs), which form properly but move asynchronously in tango7 mutants .

• Caspase activation detection: Anti-cleaved caspase-3 (CC3) antibody staining to assess caspase activity in wild-type versus tango7 mutant tissues .

• Localization studies: Immunofluorescence of Tango7 in relation to active caspases and cellular structures during spermatid individualization .

• Fertility assessment: Male fertility tests to quantify functional consequences of Tango7 mutations .

Biochemical assays:

• Apoptosome reconstitution: In vitro assembly of the apoptosome using recombinant Dronc and Dark with or without Tango7 .

• Caspase activity measurements: Using fluorogenic substrates or detecting cleavage of pro-Drice C/A to assess apoptosome activity .

• Protein interaction studies: Pull-down assays and co-immunoprecipitation to demonstrate physical interactions between Tango7 and apoptosome components .

• Domain mapping: Using truncated proteins to identify functional domains, such as the finding that the C-terminal 30 amino acids are critical for Tango7 localization and function .

These complementary approaches provide a comprehensive assessment of Tango7's roles in both cellular and biochemical contexts.

How does the C-terminus of Tango7 influence its localization and function?

The C-terminus of Tango7 plays a crucial role in both its subcellular localization and function during spermatid individualization. Research has revealed several key aspects of its importance:

• Localization determinant: Wild-type Tango7 discretely localizes to the leading edge of investment cones in the cystic bulge during spermatid individualization. In contrast, the truncated Tango7L protein, which lacks the C-terminal 30 amino acids, is notably absent from this location in tango7L/L spermatids .

• Functional requirement: Despite normal expression levels, Tango7L is unable to support proper spermatid individualization, indicating that the C-terminus is essential for the protein's function in this process .

• Apoptosome recruitment: The data suggests the C-terminus may serve as a specialized adapter domain that directs Tango7 and possibly the associated apoptosome to specific subcellular locations where controlled caspase activity is required .

• Specific vs. general functions: Interestingly, the C-terminus appears dispensable for viability (Tango7L/L flies are viable) but essential for fertility, indicating a tissue-specific requirement for this domain in reproductive tissues .

• Protein interactions: The C-terminus likely mediates specific protein-protein interactions required for proper localization and function at the investment cones .

These findings highlight how protein localization signals can direct the activity of caspase-regulating proteins to specific subcellular compartments, enabling non-lethal, remodeling functions of the apoptotic machinery.

What is the role of Tango7 in non-apoptotic caspase activation during spermatogenesis?

Tango7 plays a pivotal role in directing caspase activity toward cellular remodeling rather than cell death during spermatogenesis, particularly during the process of spermatid individualization:

• Compartmentalized caspase activation: Tango7 conspicuously localizes to the cystic bulge and waste bag during spermatid individualization, co-localizing with active caspases specifically at these structures . This restricted localization helps ensure caspase activity remains confined to appropriate cellular compartments.

• Apoptosome stimulation: Biochemical studies demonstrate that Tango7 directly stimulates apoptosome activity in a dose-dependent manner in vitro . This stimulation is heat-sensitive and requires catalytically active Dronc, indicating a specific regulatory mechanism.

• Phenotypic specificity: Like animals compromised for apoptosome components dronc and dark, tango7 mutant males are sterile due to defects in spermatid individualization . Investment cones form properly but move asynchronously through the syncytia, indicating a specific requirement for Tango7 in coordinating this process.

• Molecular interactions: Tango7 physically interacts with both key components of the Drosophila apoptosome - Dronc and Dark - both in vitro and in cultured cells . These interactions likely enable Tango7 to regulate apoptosome activity in specific contexts.

• Cell death vs. remodeling: Unlike typical cell death contexts where caspase activation leads to cellular destruction, Tango7-regulated caspase activity during spermatogenesis promotes cellular remodeling by controlling the resolution of individual sperm from a syncytium .

This non-apoptotic function of caspases represents an elegant example of how the apoptotic machinery can be repurposed for developmental processes requiring precise cellular remodeling rather than cell elimination.

How do mutations in Tango7 affect male fertility in Drosophila?

Mutations in Tango7 cause complete male sterility in Drosophila through defects in spermatid individualization, a process essential for producing functional sperm:

• Complete sterility: All viable tango7 mutants (including tango7L/L) are male-sterile, indicating an absolute requirement for Tango7 function in male reproduction .

• Spermatogenesis defects: The sterility is specifically caused by failure in spermatid individualization, the process that resolves individual sperm from a syncytium .

• Investment cone abnormalities: In tango7 mutants, investment cones form properly but either stall at the nuclei or move asynchronously through the syncytia, similar to defects seen in dronc and dark mutants .

• Caspase activation defects: Tango7 is required for caspase activation during spermatid individualization, as demonstrated by reduced anti-CC3 staining in mutant testes .

• Localization failure: The truncated Tango7L protein fails to localize to the leading edge of investment cones, suggesting that proper subcellular localization is essential for Tango7's function in spermatogenesis .

• Genetic rescue: The sterility phenotype can be completely rescued by a genomic Tango7 construct (Tango7 VK006), confirming that these defects are specifically due to loss of Tango7 function .

These findings establish Tango7 as an essential factor for male fertility, functioning through regulation of non-apoptotic caspase activation during the critical process of spermatid individualization.

What experimental approaches can be used to study the interaction between Tango7 and apoptosome components?

Several sophisticated experimental approaches can be employed to study the interactions between Tango7 and apoptosome components (Dark and Dronc):

• In vitro biochemical assays:

  • Reconstitution experiments: Using recombinant proteins to assemble the apoptosome with or without Tango7, as demonstrated in studies showing dose-dependent stimulation of caspase activity .

  • Pull-down assays: Direct binding can be assessed using tagged recombinant proteins, as shown in experiments where Tango7 was found to bind to Dronc or Dark individually or together .

  • Activity measurements: Caspase activity assays using pro-Drice C/A as a substrate to quantify the stimulatory effect of Tango7 on apoptosome function .

• Cellular interaction studies:

  • Co-immunoprecipitation: Experiments in cultured S2R+ cells demonstrated that Tango7 interacts with both Dronc and Dark in a cellular context .

  • Bimolecular fluorescence complementation (BiFC): To visualize protein interactions in live cells.

  • Förster resonance energy transfer (FRET): To measure proximity between fluorescently tagged proteins in cells.

• Structural approaches:

  • Hydrogen-deuterium exchange mass spectrometry: To map interaction interfaces.

  • Cryo-electron microscopy: To visualize the apoptosome structure with and without Tango7.

  • X-ray crystallography: Of co-crystals or individual domains to determine atomic-level interactions.

• Domain mapping experiments:

  • Deletion constructs: To identify specific regions required for interaction, as suggested by the importance of Tango7's C-terminus .

  • Site-directed mutagenesis: Of predicted interface residues to disrupt specific interactions.

  • Peptide competition assays: Using synthetic peptides corresponding to putative interaction regions.

These approaches, used in combination, can provide comprehensive insights into how Tango7 interacts with and regulates the apoptosome.

How does recombinant Tango7 stimulate apoptosome activity in vitro?

Recombinant Tango7 has been shown to directly stimulate apoptosome activity in vitro through several mechanisms:

• Dose-dependent stimulation: Biochemical studies demonstrate that increasing concentrations of Tango7 progressively enhance the ability of the Dark-Dronc apoptosome to cleave pro-Drice C/A . This dose-response relationship indicates a direct stimulatory effect rather than an all-or-none switching mechanism.

• Direct physical interaction: Tango7 directly binds to both Dark and Dronc, the core components of the Drosophila apoptosome . This physical interaction likely forms the basis for its stimulatory effect.

• Protein structure dependence: The stimulatory effect is abolished when Tango7 is heat-inactivated, indicating that the native protein structure is essential for this function . This suggests that specific structural elements or domains of Tango7 are required for apoptosome stimulation.

• Catalytic activity requirement: Stimulation is not observed when catalytically dead Dronc mutants are used, confirming that Tango7 enhances the enzymatic activity of the complex rather than serving as an alternate proteolytic enzyme .

• Specificity of effect: Unlike cytochrome c, which activates the mammalian apoptosome but not the Drosophila equivalent, Tango7 specifically stimulates the Drosophila apoptosome . This highlights the evolutionary divergence in apoptosome regulation between species.

These findings establish Tango7 as a direct stimulator of apoptosome activity, potentially functioning as a specificity factor that directs this complex toward cellular remodeling functions.

What are the implications of Tango7's dual role in translation initiation and apoptosis regulation?

Tango7's dual functionality as both a component of the eIF3 translation complex and a regulator of apoptosome activity has several profound implications:

• Integrated cellular regulation: Tango7 may serve as a molecular link between protein synthesis and cellular remodeling pathways, potentially allowing cells to coordinate these processes during development or stress responses. This represents a form of integrated regulation where a single protein influences multiple cellular processes.

• Evolutionary repurposing: Studies of Tango7 orthologs suggest an evolutionary trajectory where a protein initially involved in basic cellular processes (translation) acquired specialized roles in apoptosome regulation in more complex organisms . This exemplifies how evolution can repurpose existing proteins for new functions.

• Context-dependent regulation: The cell must have mechanisms to direct Tango7 to different functions in different contexts. These could include post-translational modifications, alternative splicing, or interactions with different protein partners in specific cellular compartments.

• Specialized non-lethal caspase activation: Tango7's role in spermatid individualization demonstrates how apoptotic machinery can be repurposed for non-lethal cellular remodeling . This challenges the traditional view of caspases as exclusively cell death effectors.

• Developmental timing control: The connection to translation machinery suggests potential mechanisms for coordinating protein synthesis with developmental remodeling events, ensuring proper timing and execution of complex cellular processes.

This multifunctionality highlights the sophisticated ways cells achieve regulatory complexity from a limited proteome, with individual proteins serving different roles depending on cellular context.

How can comparative studies between D. sechellia and other Drosophila species inform our understanding of Tango7 evolution?

Comparative studies between D. sechellia and other Drosophila species can provide valuable insights into Tango7 evolution:

• Adaptive evolution analysis: D. sechellia has adapted to its toxic host Morinda citrifolia through molecular changes affecting dopamine metabolism . Similar comparative analyses of Tango7 sequences across Drosophila species could reveal whether this protein has undergone adaptive evolution in D. sechellia.

• Functional conservation testing: Cross-species genetic rescue experiments could determine whether Tango7 from D. sechellia can substitute for its counterpart in D. melanogaster and vice versa, highlighting functional conservation or divergence.

• Host plant adaptation links: D. sechellia shows massive apoptosis in oogenesis when fed standard diet, which is dramatically reduced when flies are raised on morinda diet . This suggests connections between apoptotic regulation and host plant adaptation that might involve Tango7.

• Reproductive biology differences: D. sechellia has unique reproductive adaptations linked to dopamine metabolism . Investigating whether Tango7's role in reproduction differs between species could reveal connections to these specialized adaptations.

• Molecular signature identification: Sequence analysis focused on specific domains (particularly the C-terminus that is crucial for localization and function) could identify signatures of selection that might explain species-specific differences in Tango7 function.

• Expression pattern comparison: Analysis of Tango7 expression patterns in reproductive tissues across Drosophila species could reveal regulatory changes that contribute to reproductive adaptations.

Such comparative approaches would illuminate how proteins involved in basic cellular processes can evolve to support species-specific adaptations while maintaining core functions.

What are the potential roles of post-translational modifications in regulating Tango7 function?

Post-translational modifications (PTMs) likely play important roles in regulating Tango7's diverse functions:

• Evidence for modified forms: Coimmunoprecipitation experiments revealed that Dronc and Dark interact with a modified form of Tango7 , suggesting that PTMs might regulate these interactions.

• Context-specific regulation: PTMs could serve as molecular switches that direct Tango7 toward either its translation-related functions or its apoptosome-regulatory role in specific cellular contexts.

• Localization control: The critical importance of Tango7's C-terminus for its localization to investment cones suggests this region might be subject to modifications that control subcellular targeting.

• Activation/deactivation mechanisms: Phosphorylation, ubiquitination, or other modifications could regulate Tango7's ability to stimulate apoptosome activity in a context-dependent manner.

• Interaction surface modulation: PTMs could alter the surfaces that mediate Tango7's interactions with Dark, Dronc, or components of translation machinery, thereby controlling its functional partnerships.

• Developmental timing: Temporally regulated modifications could ensure that Tango7's apoptosome-stimulating activity is activated only at appropriate developmental stages, such as during spermatid individualization.

While specific PTMs on Tango7 have not been thoroughly characterized, the evidence for modified forms and the protein's context-specific functions strongly suggest that such modifications play important regulatory roles.

How might CRISPR/Cas9 genome editing be used to study Tango7 function in D. sechellia?

CRISPR/Cas9 genome editing offers powerful approaches for studying Tango7 function in D. sechellia:

• Generation of precision mutations:

  • Creating domain-specific mutations (particularly in the C-terminus) to dissect functional regions

  • Engineering separation-of-function mutations that affect either translation or apoptosome regulation

  • Introducing specific amino acid changes to test evolutionary hypotheses

• Endogenous tagging strategies:

  • Inserting fluorescent tags (GFP, mCherry) for live imaging of Tango7 localization

  • Adding epitope tags (FLAG, HA) for improved biochemical studies

  • Creating split-GFP systems to visualize protein interactions in vivo

• Regulatory element analysis:

  • Modifying promoter or enhancer regions to study expression regulation

  • Creating reporter constructs to monitor Tango7 expression patterns

  • Engineering inducible systems for temporal control of expression

• Cross-species domain swapping:

  • Replacing D. sechellia Tango7 domains with those from D. melanogaster to identify regions responsible for species-specific functions

  • Creating chimeric proteins to map interaction domains with apoptosome components

• Functional genomics approaches:

  • Conducting CRISPR screens to identify genetic interactors with Tango7

  • Performing parallel mutations across multiple Drosophila species for comparative functional analysis

  • Creating allelic series to assess the phenotypic consequences of progressive protein modification

The application of these CRISPR-based approaches to D. sechellia, which has unique adaptations to its toxic host plant , could reveal how Tango7 function has evolved in this specialist species compared to its generalist relatives.

What methodological approaches can address the challenges in studying Tango7's structure-function relationship?

Several methodological approaches can address the challenges in elucidating Tango7's structure-function relationship:

These complementary approaches would provide a comprehensive understanding of how Tango7's structure relates to its dual functionality in translation and apoptosome regulation, potentially revealing how proteins can acquire new functions while maintaining ancestral roles.