Recombinant Macaca fascicularis Atlastin-2 (ATL2)

Basic Research Questions

What experimental systems are optimal for studying ATL2-mediated endoplasmic reticulum (ER) fusion?

• Methodological approach:

  • Use liposome fusion assays with ER-mimetic lipid compositions (e.g., phosphatidylcholine, phosphatidylethanolamine, cholesterol) to reconstitute ATL2 activity in vitro .

  • For in vivo studies, employ HEK293T cells or Drosophila melanogaster models, as these systems allow visualization of ER morphology via fluorescent markers (e.g., BiP-sfGFP-HDEL) and assessment of GTPase-dependent fusion .

  • Prioritize isoform-specific analysis: ATL2-1 (auto-inhibited) requires co-factors like ATL3 for fusion, while ATL2-2 (lacking the C-terminal inhibitory helix) is constitutively active .

How do researchers validate ATL2 function in ER network maintenance?

• Key techniques:

  • High-resolution confocal microscopy to quantify ER tubular networks vs. sheet structures .

  • GTPase activity assays (e.g., malachite green phosphate detection) to measure catalytic efficiency .

  • Genetic complementation: Express ATL2 in ATL-deficient cells and assess ER morphology rescue .

Advanced Research Challenges

How to resolve contradictions in ATL2 isoform activity across studies?

• Case example: ATL2-1 shows no fusogenic activity in vitro but supports ER fusion in cellular assays .

  • Strategy:

    • Identify co-factors (e.g., M1-spastin for ATL1) that relieve autoinhibition in specific isoforms .

    • Compare lipid compositions of experimental systems; ER-mimetic liposomes (with cholesterol) enhance ATL2-2 activity by 40% vs. synthetic lipids .

  • Validation: Perform proteomics on ER microsomes to identify interacting partners that modulate ATL2-1 in vivo .

What methodologies address ATL2’s role in disease contexts (e.g., cancer)?

• Experimental design:

  • Clinical correlation: Analyze ATL2-2 mRNA/protein levels in tumor vs. normal tissues using TCGA or METABRIC datasets .

  • Pathway modulation: Knock down ATL2 in luminal breast cancer models and assess proliferation via RNA-seq (focus on MYC/E2F/G2M pathways) .

  • Survival analysis: Use Cox regression to link high ATL2-2 expression with patient outcomes (HR = 1.334 for BC-specific mortality) .

Data-Driven Insights

Table 1: Fusogenic Activity of Human Atlastin Paralogs

Table 2: Impact of Pathogenic Mutations on ATL2 Function

Methodological Recommendations

How to optimize ATL2 purification for functional assays?

• Protocol:

  • Express ATL2 in E. coli with a His-tag and purify via nickel-affinity chromatography .

  • Include 2% CHAPS in buffers to maintain solubility of transmembrane domains .

  • Validate activity using single-turnover GTPase assays to avoid non-enzymatic hydrolysis artifacts .

What controls are critical when analyzing ATL2 knockout phenotypes?

• Essential controls:

  • Rescue experiments with wild-type ATL2 and GTPase-dead mutants (e.g., K80A) .

  • Co-staining with ER markers (e.g., calnexin) to distinguish ER defects from off-target effects .

  • Isoform-specific siRNA to avoid cross-targeting ATL1/ATL3 .