Annexin A2 promotes phagophore assembly by enhancing Atg16L⁺ vesicle biogenesis and homotypic fusion.

Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive.

Annexin A2 binds to endosomes following organelle destabilization by particulate wear debris.

Endosomal functions are contingent on the integrity of the organelle-limiting membrane, whose disruption induces inflammation and cell death. Here we show that phagocytosis of ultrahigh molecular weight polyethylene particles induces damage to the endosomal-limiting membrane and results in the leakage of cathepsins into the cytosol and NLRP3-inflammasome activation. Annexin A2 recruitment to damaged organelles is shown by two-dimensional DIGE protein profiling, endosomal fractionation, confocal analysis of endogenous and annexin A2-GFP transfected cells, and immunogold labelling.

Isolation of myelin.

The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane.