Figure 5. Identification of KIF1A... | Rockefeller University Press

Figure 5.

$Identification of KIF1A adaptors for specific cargo transport. Identification of a phosphorylation mechanism in KIF1A-cargo binding and Arl8A and MADD as adaptors regulating KIF1A specificity for DCVs and lysosomes or SVs, respectively. (A) Schematic depiction of the structure of KIF1A_1106–1698 mutated constructs and KIF1A_5*Ala. (B and C) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc–fused mutated KIF1A_1106–1698 (B) or KIF1A_5*Ala (C), marker-GFP, and FKBP-mRFP-KIF5Cmd, without or with addition of 1 µM rapalog. Dotted gray line marks the normalized intensity for KIF1A_1106–1698 (B) or KIF1A_657–1698 (C) with rapalog treatment (N= 1, n = 15). (D) Quantification of the axonal entries of NPY vesicles before and 15 min after addition of 1 µM rapalog in neurons expressing FRB-3myc-KIF1Atd, NPY-GFP, and FKBP-mRFP-KIF5Cmd in control or after treatment with 1 µM KN-93 or 1 µM KN-92 for 20 h (N = 3, n = 30). (E) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc–fused adaptors, cargo-GFP, and FKBP-mRFP-KIF5Cmd, without or with addition of 1 µM rapalog (N = 1, n = 10–15). (F and H) Representative kymographs showing movement of NPY (F) or LAMP1 (H) vesicles in the AIS in control or when transfected with Arl8A/B shRNA. (G and I) Quantification of the fraction of static, anterograde, and retrograde NPY (G) or LAMP1 (I) traces in control or when transfected with Arl8A/B shRNA (N = 3, n = 29–30). (J) Representative images of hippocampal neurons expressing GFP-Rab3 in control or when cotransfected with MADD shRNA. (K) Quantification of the polarity index of GFP-Rab3 in control or MADD KD (N = 3, n = 29–30). (L) Representative images of the soma of hippocampal neurons immunostained for Rab3 in control or when cotransfected with MADD shRNA. Dotted red lines mark the cell soma. (M) Quantification of the ratio of endogenous Rab3 intensity in a transfected cell to that of a surrounding cell in control or MADD KD (N = 3, n = 29–30). (N) Representative images of the soma of hippocampal neurons immunostained for Arl8A in control, when cotransfected with KIF1A shRNA, or with KIF1A shRNA and KIF1A deletion constructs. Dotted red lines mark the cell soma. (O) Quantification of the ratio of endogenous Arl8A intensity in a transfected cell to that of a surrounding cell in control, KIF1A KD, and rescue with KIF1A deletion constructs (N = 3, n = 26–30). Data are displayed as mean ± SEM. Unpaired t test (G, I, and K) and Mann–Whitney U test (B–E, M, and O), *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 10 µm (L and N) and 20 µm (J).$

Identification of KIF1A adaptors for specific cargo transport. Identification of a phosphorylation mechanism in KIF1A-cargo binding and Arl8A and MADD as adaptors regulating KIF1A specificity for DCVs and lysosomes or SVs, respectively. (A) Schematic depiction of the structure of KIF1A_1106–1698 mutated constructs and KIF1A_5*Ala. (B and C) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc–fused mutated KIF1A_1106–1698 (B) or KIF1A_5*Ala (C), marker-GFP, and FKBP-mRFP-KIF5Cmd, without or with addition of 1 µM rapalog. Dotted gray line marks the normalized intensity for KIF1A_1106–1698 (B) or KIF1A_657–1698 (C) with rapalog treatment (N= 1, n = 15). (D) Quantification of the axonal entries of NPY vesicles before and 15 min after addition of 1 µM rapalog in neurons expressing FRB-3myc-KIF1Atd, NPY-GFP, and FKBP-mRFP-KIF5Cmd in control or after treatment with 1 µM KN-93 or 1 µM KN-92 for 20 h (N = 3, n = 30). (E) Quantification of the normalized ratio of cargo intensity in axonal tips to that of the soma of hippocampal neurons coexpressing FRB-3myc–fused adaptors, cargo-GFP, and FKBP-mRFP-KIF5Cmd, without or with addition of 1 µM rapalog (N = 1, n = 10–15). (F and H) Representative kymographs showing movement of NPY (F) or LAMP1 (H) vesicles in the AIS in control or when transfected with Arl8A/B shRNA. (G and I) Quantification of the fraction of static, anterograde, and retrograde NPY (G) or LAMP1 (I) traces in control or when transfected with Arl8A/B shRNA (N = 3, n = 29–30). (J) Representative images of hippocampal neurons expressing GFP-Rab3 in control or when cotransfected with MADD shRNA. (K) Quantification of the polarity index of GFP-Rab3 in control or MADD KD (N = 3, n = 29–30). (L) Representative images of the soma of hippocampal neurons immunostained for Rab3 in control or when cotransfected with MADD shRNA. Dotted red lines mark the cell soma. (M) Quantification of the ratio of endogenous Rab3 intensity in a transfected cell to that of a surrounding cell in control or MADD KD (N = 3, n = 29–30). (N) Representative images of the soma of hippocampal neurons immunostained for Arl8A in control, when cotransfected with KIF1A shRNA, or with KIF1A shRNA and KIF1A deletion constructs. Dotted red lines mark the cell soma. (O) Quantification of the ratio of endogenous Arl8A intensity in a transfected cell to that of a surrounding cell in control, KIF1A KD, and rescue with KIF1A deletion constructs (N = 3, n = 26–30). Data are displayed as mean ± SEM. Unpaired t test (G, I, and K) and Mann–Whitney U test (B–E, M, and O), *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 10 µm (L and N) and 20 µm (J).

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