To clarify the intracellular sites where ALS2 functions, we analyzed the subcellular localization of ALS2 in developing cultured hippocampal neurons. After several hours of plating, neurites begin to sprout from the
Amyloid beta-peptide (42-1) (Stage 2), and then a single axon and shorter processes are elongated within 48 h (Stage 3) [19]. At Stage 2–3, ALS2 was colocalized with F-actin, particularly in membrane ruffles at the edge of the growth cones (Fig. 1A–C). ALS2 was also localized onto F-actin-coated vesicles in the growth cones (Fig. 1D–F). Due to the lower sensitivity of the anti-ALS2 Ab used, we were unable to define the detailed subcellular localization of endogenous ALS2 molecules. To overcome this difficulty, we used hippocampal neurons from ALS2-tg mice. The ALS2-tg hippocampal neurons normally developed in vitro and were morphologically indistinguishable from wild-type (WT) neurons. Exogenously expressed ALS2 was predominantly present in the growth cones as well as in the cell bodies ( Fig. 1G–I), consistent with the distribution of endogenous ALS2 in WT neurons. Notably, ALS2 was localized to membrane ruffles and the F-actin-coated large vesicles in the growth cones ( Fig. 1J–L). These vesicles appear to share the features with those of macropinosomes [20], which are F-actin-coated, large (diameter; >0.2 μm) and phase-bright organelles [21] formed via a mode of endocytosis called macropinocytosis at a
base of membrane ruffles [22] and [23].
