Design on What Was Actually Done

 Quick Analysis Plan Copied

 Long Analysis Plan

1. Sourced quantitative foundation

The ATG16L1 FIP200-interacting region (FIR) binds FIP200 Claw with Kd 1.33 μM (FP) and several ATG8 proteins (GABARAPL1 4.59, LC3C 6.27, LC3A 7.28, GABARAP 8.11, LC3B 18.10, GABARAPL2 179 μM via ITC); these interactions are mutually exclusive, and engineered mutants DRIF (D239R/I240F) selectively disrupt FIP200 binding while IQIQ (I240Q/I243Q) abolishes both ATG16L1 FIR binding affinities.

FIP200 Claw point mutants confirm the interface: R1573E weakens binding ~258-fold (343.59 μM) versus WT FIP200 Claw mutant affinities.

2. Experimental design to test the recruitment model

Experiment A — Requirement of PI3P–WIPI2 axis. In ATG16L1-knockout HeLa rescued with WT ATG16L1, compare starvation-induced LC3B lipidation and p62 degradation under (i) VPS34 inhibition, (ii) WIPI2 knockdown, (iii) WIPI2 rescue. Prediction: lipidation drops when PI3P/WIPI2 recruitment is blocked WIPI2 recruitment model.

Experiment B — Decouple FIP200 vs ATG8 binding. Express DRIF and IQIQ mutants in ATG16L1-null cells. DRIF preserves ATG8 binding but disrupts FIP200; IQIQ abolishes both. Measure autophagic flux (LC3B-II, p62) and CASM (monensin-induced lipidation). The reported pattern — FIR–FIP200 essential for flux, FIR–ATG8 promoting CASM — provides the falsification criterion DRIF/IQIQ functional separation.

Experiment C — Alternative branch control. Because ATG16L2 heterodimerizes with ATG16L1 and diverts it from WIPI2-positive membranes toward VAIL lysosomes (β-propeller residues K484/R522 essential), co-express ATG16L2 as a competitive test: canonical WIPI2-dependent lipidation should decrease while LAMP1/VAIL lipidation increases ATG16L2 VAIL branch.

Experiment D — In vitro reconstitution. Use PI3P-containing liposomes, purified WIPI2, ATG16L1 FIR peptides, ATG12-ATG5-ATG16L1, ATG3 and ATG7 to test whether WIPI2–ATG16L1 binding stimulates ATG8-PE conjugation specifically on PI3P(+) membranes; include PI3P(–) liposomes and R1573E FIP200 mutant as negative controls. Analogous PI3P-specific liposome flotation/BLI logic has been validated for other PI3P effectors PI3P liposome assay precedent.

3. Analysis plan (bioinformatics/coding)

Python3 pipeline: quantify LC3B-II/tubulin ratios from western blots (densitometry), fit binding curves with scipy for Kd validation, and perform 3-4 biological replicate t-tests/ANOVA with statsmodels. Readouts: (i) ATG16L1-WIPI2 colocalization (Pearson coefficient), (ii) LC3B-II/total LC3B lipidation index, (iii) p62 degradation rate. Predicted outcome hierarchy: WT > DRIF (partial flux defect) > IQIQ (severe) > WIPI2 knockdown (near-abolished) Graded mutant phenotype.

Blind spots and limitations: structural studies used truncated FIR fragments, overexpression may create non-physiological stoichiometries, and in vivo relevance of the ATG16L2 branch remains untested; ATG8 lipidation is not strictly essential for autophagosome formation in mammals, so readouts must combine flux markers rather than relying on LC3B-II alone Lipidation non-essential caveat. Disconfirming result: if a DRIF mutant preserves normal starvation flux, the FIR–FIP200 essentiality claim is falsified Falsification criterion.