Figure 7. Coordinate movement of TrkB and cytoplasmic dynein complexes containing IC-1B in neurons. (A) Comparison of the colocalization of TrkB with IC-2C and IC-1B dynein complexes. Hippocampal neurons were cotransfected with TrkB-GFP and either mRFP–IC-2C or mRFP–IC-1B and imaged in living cells; the number of dynein, Trk, and overlapping puncta was then determined in the transfected neurons. The percentages of dynein puncta that colocalized with TrkB were determined for each neuron, averaged, and graphed with the standard error. Dynein containing IC-1B (green) was significantly more likely (P < 0.005, Student's t test) to be associated with the TrkB carrier vesicles than dynein containing IC-2C (orange). 15 IC-1B–transfected neurons were analyzed; a total of 211 dynein puncta and 270 TrkB puncta were counted, and 41 of the puncta were colocalized. 10 IC-2C–transfected neurons were analyzed; a total of 302 dynein puncta and 282 TrkB puncta were counted, and 10 of the puncta were colocalized. (B) Live cell imaging. Hippocampal neurons were cotransfected with TrkB-GFP and mRFP–IC-1B. TrkB-GFP (green) and mRFP-dynein image sets were collected simultaneously as described in the Materials and methods. The panels are individual frames derived from Video 5 (available). The coordinately moving spots are indicated with white arrows. Panels from left to right: (Dynein) movement of the dynein puncta (red); (TrkB) movement of TrkB puncta (green); (Overlay) overlay of the dynein (red) and TrkB (green) puncta, the moving yellow puncta is indicated with an arrow; (Offset) vertical offset of the dynein puncta (red, bottom) and TrkB puncta (green, top) to more clearly demonstrate the coordinate movement of the two proteins. The time interval for each frame from the start of the video is indicated on the right. Bar, 10 μm. (C) IC-1 dynein is preferentially associated with Trk containing vesicles from brain cortex. Membrane-bounded organelles were isolated from fresh rat brain cortex, and the fractions were analyzed by SDS-PAGE and Western blotting; the postnuclear supernatant (S1) and pellet (P1), high-speed supernatant (S2), and high-speed membrane fraction (P2) are indicated. The presence of Trks, ICs, and synaptophysin (Sy) in the fractions was determined by probing the blots with antibodies to Trk and synaptophysin and the pan IC antibody, 74.1. Trk containing organelles were purified by immunoaffinity purification on magnetic beads from the membrane fraction (P2) with antibodies to Trk (αTrk) or control IgG (IgG) and analyzed by SDS-PAGE and Western blotting. The IC isoforms present in the α-Trk immunoprecipitate were identified by probing the blot with an antibody specific to the IC-2 isoforms (IC-2) followed by the pan IC antibody (74.1). As a positive control for the presence of both IC-1 and IC-2 on the blot, dynein immunoprecipitated from the testis was loaded on a neighboring lane (Dy).
Figure 7.

Coordinate movement of TrkB and cytoplasmic dynein complexes containing IC-1B in neurons. (A) Comparison of the colocalization of TrkB with IC-2C and IC-1B dynein complexes. Hippocampal neurons were cotransfected with TrkB-GFP and either mRFP–IC-2C or mRFP–IC-1B and imaged in living cells; the number of dynein, Trk, and overlapping puncta was then determined in the transfected neurons. The percentages of dynein puncta that colocalized with TrkB were determined for each neuron, averaged, and graphed with the standard error. Dynein containing IC-1B (green) was significantly more likely (P < 0.005, Student's t test) to be associated with the TrkB carrier vesicles than dynein containing IC-2C (orange). 15 IC-1B–transfected neurons were analyzed; a total of 211 dynein puncta and 270 TrkB puncta were counted, and 41 of the puncta were colocalized. 10 IC-2C–transfected neurons were analyzed; a total of 302 dynein puncta and 282 TrkB puncta were counted, and 10 of the puncta were colocalized. (B) Live cell imaging. Hippocampal neurons were cotransfected with TrkB-GFP and mRFP–IC-1B. TrkB-GFP (green) and mRFP-dynein image sets were collected simultaneously as described in the Materials and methods. The panels are individual frames derived from Video 5 . The coordinately moving spots are indicated with white arrows. Panels from left to right: (Dynein) movement of the dynein puncta (red); (TrkB) movement of TrkB puncta (green); (Overlay) overlay of the dynein (red) and TrkB (green) puncta, the moving yellow puncta is indicated with an arrow; (Offset) vertical offset of the dynein puncta (red, bottom) and TrkB puncta (green, top) to more clearly demonstrate the coordinate movement of the two proteins. The time interval for each frame from the start of the video is indicated on the right. Bar, 10 μm. (C) IC-1 dynein is preferentially associated with Trk containing vesicles from brain cortex. Membrane-bounded organelles were isolated from fresh rat brain cortex, and the fractions were analyzed by SDS-PAGE and Western blotting; the postnuclear supernatant (S1) and pellet (P1), high-speed supernatant (S2), and high-speed membrane fraction (P2) are indicated. The presence of Trks, ICs, and synaptophysin (Sy) in the fractions was determined by probing the blots with antibodies to Trk and synaptophysin and the pan IC antibody, 74.1. Trk containing organelles were purified by immunoaffinity purification on magnetic beads from the membrane fraction (P2) with antibodies to Trk (αTrk) or control IgG (IgG) and analyzed by SDS-PAGE and Western blotting. The IC isoforms present in the α-Trk immunoprecipitate were identified by probing the blot with an antibody specific to the IC-2 isoforms (IC-2) followed by the pan IC antibody (74.1). As a positive control for the presence of both IC-1 and IC-2 on the blot, dynein immunoprecipitated from the testis was loaded on a neighboring lane (Dy).

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