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

Basic Research Questions

• What is Tango7 and what are its main functions in Drosophila development?

Tango7 (Transport and Golgi organization 7) is a protein that functions as the eukaryotic translation initiation factor 3 subunit M (eIF3m). Initially described in a collection of targets linked to Golgi organization, Tango7 plays crucial roles in multiple developmental processes .

Key functions include:

• Collaboration with the Drosophila apoptosome to drive caspase-dependent cellular remodeling processes

• Regulation of initiator caspase Dronc activity through direct stimulation of the apoptosome

• Direction of caspase activity to specific subcellular locations during sperm individualization and salivary gland remodeling

Methodologically, studying Tango7 function typically involves genetic approaches (RNAi knockdown, mutant analysis), biochemical assays (protein interaction studies), and imaging techniques (immunofluorescence, fluorescent protein tagging).

• How does Tango7 interact with the Drosophila apoptosome?

Tango7 physically interacts with components of the apoptosome and modulates its activity through several mechanisms:

• Direct binding to both Dronc (initiator caspase) and Dark (Apaf-1 homolog) individually or together, as demonstrated by coimmunoprecipitation experiments and in vitro binding assays

• Localization to the active apoptosome compartment via its C-terminus

• Direct stimulation of apoptosome activity in a dose-dependent manner in reconstituted in vitro systems

Experimental evidence shows that Tango7 can increase the cleavage of pro-Drice C/A (a catalytically dead substrate) by the reconstituted apoptosome in a heat-sensitive manner . This enhancement is specific, as it is not observed when catalytically dead Dronc is used or when cytochrome c is added instead of Tango7 .

• What cellular processes require Tango7 activity?

Tango7 is involved in several critical developmental and cellular remodeling processes:

• Sperm individualization: Tango7 directs Dronc to the individualization complex (IC) to resolve individual sperm from a syncytium. In tango7 mutants, investment cones form properly but move asynchronously or stall at the nuclei .

• Dendrite pruning: Tango7 is required for Dronc-dependent pruning of dendritic arborizations during metamorphosis. Knockdown of tango7 blocks proper pruning despite fewer dendritic branches being formed .

• Salivary gland remodeling: Tango7 regulates cortical activity of caspases during reaper-induced programmed cell death, localizing to the cell cortex and directing Dronc to the plasma membrane .

• F-actin dismantling: Salivary gland-specific knockdown of tango7 disrupts cortical Dcp-1 activation and F-actin breakdown .

In each of these processes, Tango7 appears to direct caspase activity toward specific cellular remodeling functions rather than triggering complete cell death.

• What experimental approaches are used to study recombinant Tango7 function?

Multiple complementary approaches are employed to study recombinant Tango7:

Genetic approaches:

• TILLING (Targeting Induced Local Lesions in Genomes) to generate point mutations in the Tango7 locus

• RNAi-based silencing with multiple non-overlapping constructs to validate phenotypes

• Rescue experiments using genomic fragments spanning the Tango7 locus

Biochemical approaches:

• Production of recombinant Tango7 for in vitro activity assays

• Reconstitution of the apoptosome using recombinant Dronc and Dark with/without Tango7

• Coimmunoprecipitation to identify protein-protein interactions in cultured cells

Imaging approaches:

• Custom monoclonal antibodies against Tango7 (e.g., 1N13)

• Endogenous tagging with superfolder GFP (sGFP) to track dynamic localization

• Colocalization studies with markers of caspase activity (anti-CC3) and cytoskeletal structures (phalloidin)

Advanced Research Questions

• How does Tango7 regulate caspase activity during non-apoptotic cellular remodeling?

Tango7 employs multiple mechanisms to regulate caspase activity specifically for cellular remodeling rather than apoptosis:

• Spatial regulation: Tango7 localizes Dronc to specific subcellular compartments, including the individualization complex during spermatogenesis and the cell cortex in salivary glands .

• Independent of canonical adaptor: Unlike typical apoptotic scenarios, Tango7-mediated caspase activation at the salivary gland cell cortex and during dendrite pruning occurs independently of Dark (the canonical adaptor for Dronc) .

• Direct stimulation: Recombinant Tango7 directly enhances apoptosome activity in vitro, increasing substrate cleavage in a dose-dependent manner :

• Subcellular targeting: During sperm individualization, Tango7 discretely localizes to the leading edge of investment cones in the cystic bulge, directing caspase activity precisely where needed .

• What is known about the structural domains of Tango7 and how do they contribute to its function?

Tango7 contains several functional domains with specific roles:

• PCI domain: Tango7 contains a PCI (Proteasome, COP9, Initiation factor 3) domain that likely mediates protein-protein interactions .

• C-terminal region: The C-terminal 30 amino acids are critical for proper localization. The truncated Tango7^L protein (lacking these amino acids) fails to localize to the leading edge of investment cones in spermatids, resulting in male sterility despite otherwise normal development .

• Interaction interfaces: Biochemical studies demonstrate that Tango7 directly binds both Dronc and Dark . These interaction surfaces are essential for Tango7's ability to stimulate apoptosome activity.

• Modification sites: Coimmunoprecipitation experiments reveal that Dronc and Dark interact with a modified form of Tango7 , suggesting that post-translational modifications regulate Tango7 function.

Structural analysis reveals that Tango7 functions as both an eIF3 translation complex component and an apoptosome regulator, with the C-terminus specifically required for its role in cellular remodeling but dispensable for essential functions .

• How does Tango7 localization change during different developmental processes?

Tango7 exhibits dynamic localization patterns that correlate with its function:

• During spermatogenesis:

  • Conspicuously localizes to the cystic bulge and waste bag

  • Colocalizes with cleaved Caspase-3 (CC3) specifically at these structures

  • Discretely localizes to the leading edge of investment cones

• In salivary glands:

  • Displays robust cortical localization at -8h PF (hours post-formation)

  • This cortical localization is lost by 0h PF

  • Shows subcellular dynamics similar to Dronc

The localization of Tango7 depends on its C-terminus, as the truncated Tango7^L protein fails to properly localize to the individualization complex . This dynamic localization pattern suggests that Tango7 is recruited to specific subcellular compartments precisely when and where caspase activity is required for cellular remodeling.

• What are the challenges in analyzing Tango7 function across different genetic backgrounds?

Analyzing Tango7 function presents several methodological challenges:

• Maternal contribution effects: Like other apoptosome components, zygotic expression of Tango7 might be dispensable for embryonic programmed cell death due to maternally loaded transcript . Abundant Tango7 transcript is detected even in embryos genetically lacking the Tango7 locus .

• Allele-specific effects: Different Tango7 alleles reveal distinct aspects of its function:

  • Tango7^E (Q135* nonsense mutation) is lethal at first instar larval stage

  • Tango7^L (W358* nonsense mutation) is viable but male-sterile

  • This allelic series helps distinguish essential functions from specific roles in cellular remodeling

• Context-dependent phenotypes: In D. virilis, hybrid dysgenesis can result in germline transposition of diverse transposable elements and mitotic recombination within the female germline , potentially affecting the expression and function of genes including Tango7.

• Species-specific differences: D. virilis has significantly higher recombination rates than D. melanogaster , which may affect genetic mapping and analysis of Tango7 function across species.

• How can contradictions in experimental data about Tango7 function be resolved?

Resolving contradictions in Tango7 research requires systematic approaches:

• Considering context-dependent effects: Tango7 has different functions in different tissues. For example, knockdown of tango7 reduces Dronc protein levels in some contexts but not in salivary glands .

• Distinguishing direct vs. indirect effects: Tango7 has dual roles as both a component of the eIF3 translation complex and a regulator of the apoptosome . Distinguishing which role is responsible for specific phenotypes requires careful experimental design.

• Using multiple loss-of-function approaches:

  • RNAi with non-overlapping constructs (Tango7 #1 and Tango7 #2) to validate specificity

  • Point mutations that affect specific domains (e.g., Tango7^L affecting the C-terminus)

  • Tissue-specific knockdown to avoid systemic effects

• Rigorous rescue experiments: Testing whether wild-type Tango7 can rescue mutant phenotypes confirms specificity . The 20-kb bacterial artificial chromosome spanning the Tango7 locus (Tango7^VK006) effectively rescues sterility and caspase activation defects in tango7^L/L males .

• What methods are most effective for studying recombinant Tango7 protein interactions?

For studying Tango7 protein interactions, multiple complementary approaches are recommended:

• In vitro reconstitution assays: Testing the ability of recombinant Tango7 to stimulate apoptosome activity using purified components (Dronc, Dark) and substrates (pro-Drice C/A) .

• Coimmunoprecipitation studies:

  • In cultured cells: Cotransfection of tagged constructs (Tango7-V5, Flag-Dronc, Dark-Myc) followed by immunoprecipitation

  • In tissues: Using endogenously tagged proteins to capture physiological interactions

• Direct binding assays: Testing whether recombinant Tango7 interacts with irrelevant His-tagged proteins (negative control) versus Dronc or Dark individually or together .

• Localization studies: Colocalization analysis of Tango7 with apoptosome components and caspase activity markers in tissues undergoing remodeling .

These techniques have successfully demonstrated that Tango7 physically interacts with both Dronc and Dark, stimulates apoptosome activity, and localizes to active caspase compartments during cellular remodeling processes .

• How does Tango7 function differ in apoptotic versus non-apoptotic contexts?

Tango7 functions differently depending on cellular context:

In non-apoptotic remodeling:

• Acts independently of the canonical apoptosome adaptor Dark

• Localizes to specific subcellular structures (individualization complex, cell cortex)

• Directs caspase activity toward specific substrates involved in cellular remodeling

• Restricts caspase activity to prevent complete cell destruction

In apoptotic contexts:

• Required for cell death in certain scenarios

• Cells depleted for Tango7 resist apoptosis prior to effector caspase activation

• Regulates Dronc protein levels post-transcriptionally

• Does not affect stimulus-dependent loss of DIAP1, unlike canonical apoptosis regulators

This context-dependent function allows Tango7 to direct caspase activity either toward controlled cellular remodeling or full apoptotic execution, depending on developmental requirements.

• What are the best approaches for visualizing Tango7 activity in vivo?

Optimal approaches for visualizing Tango7 activity include:

• Direct visualization:

  • Custom monoclonal antibodies against Tango7 (e.g., 1N13 raised against epitope ELLGTYTADN)

  • Endogenous tagging with superfolder GFP (sGFP)

• Caspase activity reporters (as proxies for Tango7 function):

  • Drice-based sensors (DBS) detect initiator caspase activation

  • Rapid (RDBS) and delayed (DDBS) reporter sensors distinguish between immediate and delayed caspase activation

  • These sensors consist of a Dronc recognition element fused to either Histone-GFP (RDBS) or QF transcription factor (DDBS)

• Colocalization studies:

  • Anti-cleaved Caspase-3 (anti-CC3) to mark regions of caspase activation

  • Phalloidin staining to visualize F-actin structures being remodeled

  • AXO49 staining as a marker for advanced spermatogenesis

• Lineage tracing systems:

  • G-TRACE (combines UAS-nRFP, UAS-FLP, and FRT-stop-FRT-nEGFP) for tracking cell lineages undergoing Tango7-dependent remodeling

These approaches provide complementary information about Tango7 localization, activity, and functional outcomes in developing tissues.