Figure 8. Effect of endocytic vesicle scission on phagophore formation.

A. Hela cells transfected with two rounds of control, clathrin-heavy chain or AP2 siRNA for 4 d were collected for immunoprecipitation with anti-Atg16L1 antibody (IP). Western blot for total lysate (bottom) and IP (top) were performed using anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1-Clathrin interaction with AP2 knockdown. Graph shows quantitation of the Atg16L1-clathrin or Atg16L1-AP2 with control, clathrin-heavy chain or AP2 knockdown from two independent experiments. B. HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (Control) or dominant-negative dynamin-II mutant (K44A Dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note co-localisation of Atg16L1 with PLC(PH) at the plasma membrane which is quantified in the graph. * - p<0.01. Scale bar – 5 μm. n = 20 cells. C. Hela cells treated with HBSS alone or HBSS with 80 μM dynasore for 5 h were collected for immunoprecipitation with anti-Atg16L1 antibody (IP). Western blot analyses for total lysate (TL) and IP were performed using anti-Atg16L1 and anti clathrin antibodies. Note the strong Atg16L1-Clathrin interaction with dynasore treatment. Graph shows quantitation from two independent experiments. All error bars in the graphs represent SEM. D. Representation of plasma membrane contribution to autophagosome precursor formation. Cholera toxin is internalized by clathrin-dependent and clathrin-independent endocytosis. Clathrin knockdown significantly decreases cholera toxin uptake ¹⁵, ²⁶. Clathrin-coated vesicles (CCV) budding immediately from the plasma membrane (EEA1-negative) are precursors to early endosomes (EE) ²⁷ (EEA1-positive). Previous studies showed that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes/multi vesicular bodies (LE/MVB) to form amphisomes, which are Atg16L1-negative, LC3-positive and positive for endosomal markers ¹¹, ¹² (red arrows). We show here that inhibition of clathrin-dependent internalization inhibits formation of early Atg16L1-positive precursors that mature to form phagophores and later autophagosomes (blue arrows). These Atg16L1-vesicles were positive for other early autophagosomal markers (Atg5 and Atg12), but not early endosomal markers (EEA1). AP2 is a clathrin adaptor at the plasma membrane while AP1 localises to TGN and endosomes.