Figure 1.
gAnkB suppresses axon branching through regulating axonal MT dynamics.(A) Schematic of 220-kD AnkB and 440-kD gAnkB. Asterisks indicate the binding sites of antibody against total AnkB (tAnkB Ig) or giant AnkB (gAnkB Ig). NSD is highlighted in red. (B) Fluorescence images of DIV4 cultured hippocampal neurons stained with gAnkB Ig (green), tAnkB Ig (red), and dendritic marker MAP2 (Dend; blue). A merged picture of gAnkB (green) and MAP2 (blue) staining is displayed on the left panel with high-magnification pictures of axon or dendrite for gAnkB staining to the right. The same neuron stained with tAnkB (red) is displayed in the right panel with high-magnification images of axon or dendrite for tAnkB staining to the right. (scale bar, 50 µm for whole neuron images; 5 µm for axon/dendrite high-magnification images). (C) Cultured hippocampal neurons from AB37f/fNes-Cre− (WT, top) or AB37f/fNes-Cre+ (bottom) mice were transfected with GFP at DIV3 and fixed, then stained for MAP2 at DIV4. Blue arrowheads point to the cell body of the transfected neurons (scale bar, 50 µm). A zoomed-in image of the yellow-boxed axon region is displayed below for better visualization of branches (marked with red asterisks; scale bar, 50 µm). The number of branches longer than 10 µm is quantified (mean ± SEM; ****P < 0.0001, t test; n = 17 for WT; n = 20 for AB37f/f Nes-Cre+ from three biological replicates). (D) Top left: A single-frame image and a stacked 2-min time-lapse series image of an axon from an AB37f/fNes-Cre− neuron (Video 1) or an AB37f/fNes-Cre+ neuron (Video 2; EB3-tdTM transfected, DIV4; scale bar, 5 µm). Red asterisks indicate EB3 comets and blue arrowheads indicate nonaxis EB3 comets. Nonaxis EB3 comet means the EB3 comet’s moving direction deviated from the axis direction of the axon. Bottom left: The steps of MT invasion into a newly formed filopodium in the axon shaft of AB37f/fNes-Cre+ neurons (Video 3; scale bar, 5 µm). Blue arrowheads track the movement of one EB3 comet. Right: Quantification of the number of nonaxis EB3 comets or the number of invading (into filopodia) EB3 comets (mean ± SEM; t test; **P = 0.0024 for nonaxis EB3; *P = 0.016 for invading EB3; n = 10 cells in all conditions from three biological replicates).

gAnkB suppresses axon branching through regulating axonal MT dynamics. (A) Schematic of 220-kD AnkB and 440-kD gAnkB. Asterisks indicate the binding sites of antibody against total AnkB (tAnkB Ig) or giant AnkB (gAnkB Ig). NSD is highlighted in red. (B) Fluorescence images of DIV4 cultured hippocampal neurons stained with gAnkB Ig (green), tAnkB Ig (red), and dendritic marker MAP2 (Dend; blue). A merged picture of gAnkB (green) and MAP2 (blue) staining is displayed on the left panel with high-magnification pictures of axon or dendrite for gAnkB staining to the right. The same neuron stained with tAnkB (red) is displayed in the right panel with high-magnification images of axon or dendrite for tAnkB staining to the right. (scale bar, 50 µm for whole neuron images; 5 µm for axon/dendrite high-magnification images). (C) Cultured hippocampal neurons from AB37f/fNes-Cre− (WT, top) or AB37f/fNes-Cre+ (bottom) mice were transfected with GFP at DIV3 and fixed, then stained for MAP2 at DIV4. Blue arrowheads point to the cell body of the transfected neurons (scale bar, 50 µm). A zoomed-in image of the yellow-boxed axon region is displayed below for better visualization of branches (marked with red asterisks; scale bar, 50 µm). The number of branches longer than 10 µm is quantified (mean ± SEM; ****P < 0.0001, t test; n = 17 for WT; n = 20 for AB37f/f Nes-Cre+ from three biological replicates). (D) Top left: A single-frame image and a stacked 2-min time-lapse series image of an axon from an AB37f/fNes-Cre− neuron (Video 1) or an AB37f/fNes-Cre+ neuron (Video 2; EB3-tdTM transfected, DIV4; scale bar, 5 µm). Red asterisks indicate EB3 comets and blue arrowheads indicate nonaxis EB3 comets. Nonaxis EB3 comet means the EB3 comet’s moving direction deviated from the axis direction of the axon. Bottom left: The steps of MT invasion into a newly formed filopodium in the axon shaft of AB37f/fNes-Cre+ neurons (Video 3; scale bar, 5 µm). Blue arrowheads track the movement of one EB3 comet. Right: Quantification of the number of nonaxis EB3 comets or the number of invading (into filopodia) EB3 comets (mean ± SEM; t test; **P = 0.0024 for nonaxis EB3; *P = 0.016 for invading EB3; n = 10 cells in all conditions from three biological replicates).

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