Figure 1.
Identification of KIF1A cargo in neurons. Identification of KIF1A cargos using engineered motors and evaluation of KIF1A–cargo interaction in neuronal compartments. (A) Schematic depiction of the engineered motor assay in case of a negative (left) or positive (right) tail–cargo interaction. Upper panels show components of the assay, and lower panels show the readout in a neuron. (B) Schematic depiction of the structure of KIF5C and KIF1A motors. (C) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc-KIF1Atd, cargo-GFP, and FKBP-mRFP-KIF5Cmd without rapalog or with addition of 1 µM rapalog (N = 1, n = 15). (D) Representative images of hippocampal neurons coexpressing FRB-3myc-KIF1Atd, NPY-GFP, and FKBP-mRFP-KIF5Cmd without (upper panels) or with (lower panels) addition of 1 µM rapalog. Pink lines mark the cell soma. Blue dotted circles indicate examples of axonal tips. Zooms of the boxed regions are shown as a merge on the right. (E) Representative kymographs showing movement of NPY vesicles in the AIS before (left) and 15 min after (right) addition of 1 µM rapalog. (F) Quantification of the axonal entries of cargo before and 15 min after addition of 1 µM rapalog in neurons expressing FRB-3myc-KIF1Atd, cargo-GFP, and FKBP-mRFP-KIF5Cmd (N = 3, n = 17–27). (G) Quantification of the percentage of cargo vesicles colocalizing with KIF1Atd puncta (N = 3, n = 81–108). (H) Representative image of a neuron coexpressing KIF1Atd and GFP-NPY. White boxes on the merged image mark the axon (continuous line) and a dendrite (dotted line). TRIM46 staining in the axon is shown in the merged image. Merged zooms of the boxes are depicted on the right. Graphs show the NPY (green) and KIF1A (red) intensity along the line marked in the zooms. Quantification of the percentage of NPY colocalizing with KIF1A in the axon and dendrites is shown on the right (N = 3, n = 36 axons and 72 dendrites). Data are displayed as mean ± SEM. Mann–Whitney U test; ***, P < 0.001. Scale bars, 20 µm (D) and 10 µm (H).

Identification of KIF1A cargo in neurons. Identification of KIF1A cargos using engineered motors and evaluation of KIF1A–cargo interaction in neuronal compartments. (A) Schematic depiction of the engineered motor assay in case of a negative (left) or positive (right) tail–cargo interaction. Upper panels show components of the assay, and lower panels show the readout in a neuron. (B) Schematic depiction of the structure of KIF5C and KIF1A motors. (C) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc-KIF1Atd, cargo-GFP, and FKBP-mRFP-KIF5Cmd without rapalog or with addition of 1 µM rapalog (N = 1, n = 15). (D) Representative images of hippocampal neurons coexpressing FRB-3myc-KIF1Atd, NPY-GFP, and FKBP-mRFP-KIF5Cmd without (upper panels) or with (lower panels) addition of 1 µM rapalog. Pink lines mark the cell soma. Blue dotted circles indicate examples of axonal tips. Zooms of the boxed regions are shown as a merge on the right. (E) Representative kymographs showing movement of NPY vesicles in the AIS before (left) and 15 min after (right) addition of 1 µM rapalog. (F) Quantification of the axonal entries of cargo before and 15 min after addition of 1 µM rapalog in neurons expressing FRB-3myc-KIF1Atd, cargo-GFP, and FKBP-mRFP-KIF5Cmd (N = 3, n = 17–27). (G) Quantification of the percentage of cargo vesicles colocalizing with KIF1Atd puncta (N = 3, n = 81–108). (H) Representative image of a neuron coexpressing KIF1Atd and GFP-NPY. White boxes on the merged image mark the axon (continuous line) and a dendrite (dotted line). TRIM46 staining in the axon is shown in the merged image. Merged zooms of the boxes are depicted on the right. Graphs show the NPY (green) and KIF1A (red) intensity along the line marked in the zooms. Quantification of the percentage of NPY colocalizing with KIF1A in the axon and dendrites is shown on the right (N = 3, n = 36 axons and 72 dendrites). Data are displayed as mean ± SEM. Mann–Whitney U test; ***, P < 0.001. Scale bars, 20 µm (D) and 10 µm (H).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal