, 2010 and Wu et al., 2011). Tubulovesicular structures carrying APP and BACE-1 moved bidirectionally selleck chemical within the dendritic shaft and the movement was microtubule dependent, resembling the movement of motor-driven vesicular cargoes (Figures S1E and S1F; Movie S1; Table S1; also see Tang et al., 2012). As shown in the kymographs (Figure 1B), localization of both stationary and mobile APP/BACE-1 particles were largely nonoverlapping (Figure 1B, bar graph). Subtle differences were also seen in the transport kinetics of APP and BACE-1 vesicles (Figure 1C), further suggesting that these two cargoes were largely conveyed in distinct
organelles. Next, to examine the biogenesis of APP/BACE-1 cargoes, we focused on the distribution of these proteins within the neuronal soma. As both
APP and BACE-1 are transmembrane proteins, they would be expected to traffic via the ER→Golgi (biosynthetic) pathway. In accordance with this, we found significant colocalization of APP and BACE-1 in the perinuclear region (Figure 1D)—a distribution reminiscent of the ER-Golgi network in these neurons (Dresbach et al., 2006; also see overlap with Golgi marker galactosyl-transferase [GalT] below). However, we also saw BACE-1 particles Paclitaxel chemical structure (Figure 1D, arrowheads) that did not colocalize with APP, suggesting that BACE-1 may be sorted into a distinct compartment after trafficking via the ER→Golgi pathway. If the latter was true, conditions inhibiting the emergence of Golgi-derived vesicles would be expected to “trap” APP/BACE-1 within the Golgi network.
To test this idea, we incubated cultured neurons at 20°C for 2 hr—conditions expected to block the exit of Golgi-derived proteins (Dresbach et al., 2006). Indeed, we found that the perinuclear colocalization of APP/BACE-1 was significantly increased under these conditions (Figure 1D, inset), further suggesting that the subset of somatic BACE-1 that failed to colocalize with APP at 37°C Bay 11-7085 was a consequence of post-Golgi processing. Increased colocalization of BACE-1 with GalT at 20°C also supports this overall model (Figure 1D). Moreover, APP/BACE-1 colocalization decreased over 6–36 hr posttransfection (Figure 1D, bottom), further suggesting a differential sorting after biogenesis. The spatial segregation of APP and BACE-1 was also evident in sucrose density gradients of P100 (“vesicle pellet”; DeBoer et al., 2008; see fractionation strategy in Figure S1G) mouse brain fractions in vivo, in which endogenous holo-APP/BACE-1 were largely localized to distinct fractions (Figure 1E). Next, we sought to determine the specific organelles carrying BACE-1 and APP in neurons.