Figure 4
From: Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase
Characterization of dynamic BACE1 axonal transport and localization in functional synapses in cultured hippocampal neurons. (A-C) Axonal transport of BACE1-YFP. (A) Time-lapse images of transfected neurons (DIV12-13) were acquired at the rate of 1 frame/sec for 3–5 min. A representative frame and the kymograph of the image series corresponding to Additional file 2 are shown. (B) Maximum velocities of vesicles moving away (anterograde, n = 106) or toward (retrograde, n = 83) the cell body were quantified from kymographs of multiple axons imaged from five independent cultures and plotted. (C) Representative image montages of time-lapse sequences from a segment of the axon shown in Additional file 2 depict dynamic transport of BACE1-containing vesicles and tubulo-vesicular structures. Vesicles containing BACE1-YFP are transported in the anterograde (left panels) or the retrograde (right panels); several BACE1-positive structures also remain stationary (indicated by a green asterisks). Note the anterograde BACE1-containing vesicles emerging from the stationary larger vesicular structure. (D) Presynaptic localization of BACE1 in cultured hippocampal neurons. Live-cell images from a transfected neuron (DIV14) show colocalization of BACE1-YFP with synaptophysin-Cerulean. Sequential time-lapse images were acquired on the same axon (Additional file 3), and the dynamics of BACE1 and synaptophysin were analyzed by generating kymographs. Note that the large, stationary, BACE1-containing pleomorphic structures are positive for synaptophysin. (E) Localization of BACE1-Cerulean (BACE1-Cer) in functional synapses of DIV13 neurons labeled with FM1-43 (arrowheads). A kymograph of BACE1-Cer dynamics in the same axonal segment is shown in the bottom panel. Note that BACE1-Cer remains stationary (arrowheads) in structures that overlap with FM1-43 labeled synapses.