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Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment - PubMed

  • ️Fri Jan 01 2016

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment

Amélie Fréal et al. J Neurosci. 2016.

Abstract

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon.

Significance statement: Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity.

Keywords: ankyrin-G; axon initial segment; end-binding protein; microtubule; neuronal polarity.

Copyright © 2016 the authors 0270-6474/16/364421-13$15.00/0.

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Figures

Figure 1.
Figure 1.

480AnkG associates with MT plus ends in an EB-dependent manner via its specific tail domain. A, Table illustrating the different AnkG constructs used in this study and their ability to track MT plus ends. Regions specific to 480AnkG, shared by 480 and 270Ank or by all AnkG isoforms are indicated in red, blue, and black respectively. Asterisks pinpoint the position of the SxIP motifs and amino acids at the beginning and the end of each construct are numbered. B, Identification of 10 conserved SxIP motifs (boxed) between the mouse (Mus) and human (Hum) sequences of 480AnkG. Motifs in red are specific to 480AnkG, motifs in blue are shared by 480AnkG and 270AnkG. Bold motifs are repeated in tandem. C, D, COS-7 cells overexpressing the indicated constructs and stained for GFP (green) and tyrosinated tubulin (C, red) or EB1 (D, red). E, Mean velocity of 480AnkG-GFP (n = 30), 480Tail-GFP (n = 37), and EB3-RFP (n = 46) comets in COS-7 cells. p > 0.05, two-tailed unpaired t test. Error bars are SEM. F, Maximum t projection from a time-lapse recording of COS-7 cells expressing EB3-RFP (red) and 480AnkG-GFP (green). G, H, Representative stills (G) and kymograph (H) from a time-lapse recording of a single MT plus end colabeled by EB3-RFP (red) and 480AnkG-GFP (green) in COS-7 cells. Vertical and horizontal scale bars represent 60 s and 12 μm, respectively. I, DIV1 rat hippocampal neurons transfected at DIV0 with 480AnkG-GFP or 480AnkG-NN-GFP and stained for GFP (green) and EB1 (red). Right, Enlargements of the boxed areas in the left panel.C–I, Arrows point to MT plus ends. Scale bars: C, D, F, 15 μm; I, 10 μm; magnifications: C, D, 5 μm; I, 1 μm.

Figure 2.
Figure 2.

480AnkG is required for 270- and 190AnkG clustering at the AIS during neuronal development. A, Representative scheme of 270- and 480AnkG, with the epitope positions of the three antibodies used in this study. B, Western-blot analysis of protein extracts from COS-7 cells expressing or not 480AnkG-GFP using 480AnkG or GFP antibodies. β2-karyopherin was used as a loading control. C, Western blot of DIV10 cortical neurons extracts using 480AnkG or Pan(SR)AnkG antibodies. D, Immunolabeling of adult mouse spinal cord (sp. cord) and hippocampus (hippo., CA3 region) sections with Pan(SBD)AnkG (red) and 480AnkG (green) antibodies. E, Immunolabeling of embryonic mouse spinal cord (at E9.5) and hippocampus (E14.5) sections with 480AnkG (in red) and Pan(SBD)AnkG (in green) antibodies. The spinal cord is delimited by a dashed line. F, G, Western blot of E13.5, E16.5, and P1 mouse spinal cords (F) and extracts from rat cortical neurons at DIV1, DIV3, DIV5, DIV7, and DIV10 (G) stained with Pan(SR)AnkG. Ratios of 480/270AnkG are quantified at indicated stages. The 480AnkG bands used for the quantifications are indicated by brackets. H, Immunolabeling of COS-7 cells transfected with 190-, 270-, or 480AnkG-GFP constructs using GFP (green) and 480AnkG (red) antibodies. Right, The border of transfected cells is framed by a dashed line and the asterisks pinpoint nontransfected cells. I, Western blot on protein extracts from COS-7 cells cotransfected with 270AnkG-GFP or 480AnkG-GFP and control- or 480AnkG-shRNA using Pan(SR)AnkG antibody. β2-Karyopherin was used as a loading control. The specific bands for 480AnkG are shown by a bracket. J, Average percentage of DIV5 and DIV8 neurons cotransfected at DIV1 with GFP and control (white bars), or 480AnkG-shRNA (gray bars) showing 480AnkG, Pan(SR)AnkG, or Pan(SBD)AnkG staining at the AIS (at least 60 neurons were analyzed per condition). K, DIV5 mouse hippocampal neurons cotransfected at DIV1 with GFP (green) and control- or 480AnkG-shRNA and stained with 480AnkG (red) or Pan(SR)AnkG (red) and Pan(SBD)AnkG (gray) antibodies. Right, Top, Higher-magnifications of the boxed regions in the left panels. L, Mean fluorescence intensity profile of 480AnkG, Pan(SR)AnkG and Pan(SBD)AnkG staining along the axon of untransfected neurons (gray line; n = 7, 7, and 6, respectively) and along the longest neurite of neurons coexpressing GFP and 480AnkG-shRNA (black line; n = 7, 7, and 6, respectively). F, G, J, L, Error bars represent SEM. D, E, K, Arrowheads point to the AIS, whereas arrows indicate the longest neurite, and the open arrowhead highlights the AIS of a non-transfected neuron. Scale bars: K (top, left), 30 μm; all other panels, 15 μm.

Figure 3.
Figure 3.

480AnkG is required for the AIS formation and the maintenance of axonal identity. A, DIV5 mouse hippocampal neurons cotransfected at DIV1 with GFP (green) and control-shRNA and stained for βIV-spectrin (red), Pan(SBD)AnkG (gray), and MAP2 (blue). B, DIV5 mouse hippocampal neurons cotransfected at DIV1 with GFP (green) and 480AnkG-shRNA and stained for βIV-spectrin (red) and Pan(SBD)AnkG (gray) or Pan(SBD)AnkG (red) and MAP2 (blue). C, Average percentage of DIV5 or DIV8 mouse neurons cotransfected at DIV1 with GFP and control- (white bars) or 480AnkG-shRNA (gray bars) showing the somatodendritic restriction of MAP2 and the accumulation of βIV-spectrin at the AIS (at least 60 neurons were analyzed per condition). D, Mean fluorescence intensity profile of βIV-spectrin and MAP2 along the axons of untransfected neurons (gray line; n = 6 and 5, respectively) and along the longest neurite of neurons coexpressing GFP and 480AnkG-shRNA (black line; n = 6 and 5, respectively). E, DIV8 mouse hippocampal neurons cotransfected at DIV1 with GFP (green) and control-shRNA and stained for βIV-spectrin (rabbit antibody, red) and Pan(SBD)AnkG (gray). F, DIV8 mouse hippocampal neurons cotransfected at DIV 1 with GFP (green) and 480AnkG-shRNA and stained for βIV-spectrin (chicken antibody, red), 480AnkG (gray) and MAP2 (blue). G, Mean fluorescence intensity profile of 480AnkG, βIV-spectrin and MAP2 along the axons of DIV8 untransfected neurons (gray line; n = 5 for each condition) and along the longest neurite of neurons cotransfected with GFP and 480AnkG-shRNA (black line; n = 5 for each condition). A, B, E, F, Arrowheads and open arrowheads point to the AIS of transfected and nontransfected neurons respectively. Arrows indicate the longest neurite of transfected neurons. Dashed lines delineate the cell body of transfected neurons, whereas asterisks pinpoint the soma of untransfected neurons. Scale bars: F, 30 μm; all other panels, 15 μm. Errors bars are SEM.

Figure 4.
Figure 4.

480AnkG controls EB accumulation and MT polarity orientation in the proximal axon. A, B, Staining of 480AnkG (green) and EB1 (red) in rat DIV2 (top) and DIV5 (bottom) hippocampal neurons (A) and corresponding fluorescence intensity profile along their axons (B). Arrowheads point to the proximal accumulation of 480AnkG and EB1. C, Percentage of DIV2 neurons showing an accumulation of EB1 and/or 480AnkG in their proximal axon. n = 189 neurons. D, DIV5 rat hippocampal neuron cotransfected at DIV1 with GFP (green) and control, (top) or 480AnkG-shRNA (bottom) and stained for EB1 (red) and 480AnkG (gray). Arrowheads highlight the AIS, whereas arrows indicate the longest neurite of transfected neurons. Asterisks and open arrowheads pinpoint the soma and the AIS of nontransfected neurons respectively. Dashed lines delimit cell bodies. E, F, Percentage of neurons with an accumulation of EB1 at AIS (E) and EB1 fluorescence intensity ratio (F) in the soma of neurons cotransfected at DIV1 with GFP and a control, (white bars; n = 41 in E and 35 in F) or a 480AnkG-shRNA (gray bars; n = 50 in E and 37 in F) normalized to neighboring nontransfected neurons. Unpaired t test, p = 4.95 × 10−5 in E and 1.01 × 10−14 in F. G, Fluorescence intensity profile of endogenous EB1 along the axon (black line) and longest neurite (gray line) of the neuron transfected with control (D, top) and 480AnkG-shRNA (D, bottom). Arrowheads show the position of the AIS. H, I, Mean fluorescence intensity profile of GFP, EB1, and 480AnkG along the axon (H) or the longest neurites (I) of neurons cotransfected with GFP and a control, (H) or 480AnkG-shRNA (I). n = 9 neurons per condition. J, Representative stills (J, top) and kymographs (J, bottom) from a time-lapse recording of mouse DIV5–DIV6 hippocampal neurons cotransfected with EB3-GFP and control (left), or 480AnkG-shRNA (right) and corresponding fluorescence intensity profile (K) along the axon (black line) or the longest neurite (gray line) of the control (J, left) or the 480AnkG-depleted neurons (J, right). Vertical and horizontal bars represent 60 s and 5 μm, respectively. Arrowheads highlight the position of the AIS, whereas arrows show the longest neurite of transfected neurons. L, Percentage of DIV5–DIV6 neurons transfected with EB3-GFP and control, (n = 24) or 480AnkG-shRNA (n = 18) showing an accumulation of GFP in the proximal axon. M, Percentage of anterogradely (black bars) and retrogradely moving (white bars) comets in the proximal axon (n = 18) versus the proximal dendrites (n = 100) of DIV5–DIV6 neurons cotransfected with EB3-GFP and control-shRNA, or in the longest (n = 18) versus other neurites (n = 67) of neurons coexpressing EB3-GFP and 480AnkG-shRNA. ANOVA, *p = 0.044. N, COS-7 cells cotransfected with Kv-Nav and 480AnkG-GFP (left) or 480AnkG-NN-GFP (right) and stained for GFP (green) and EB1 (red). O, Mean fluorescence intensity ratio of GFP (top) and EB1 (bottom) staining within membrane patches versus the rest of the cell, in COS-7 cells expressing Kv-Nav together with 480AnkG-GFP (n = 39) or 480AnkG-NN-GFP (n = 53). Unpaired t test; top, p = 0.276; bottom, p = 5.87 × 10−9. Arrowheads and arrows indicate EB accumulation and MT plus ends, respectively. A.U., Arbitrary units. n.s., nonsignificant. Scale bars: A, D, N, 15 μm; A, D, N (zoomed), 5 μm. Error bars are SEM. *p < 0.05; ****p < 0.0001.

Figure 5.
Figure 5.

Interaction of 480AnkG with EBs is critical for its restricted localization and stabilization at the AIS. A, DIV5 rat neurons cotransfected with GFP and control- or EB-shRNA and stained for GFP (green) and EB1 (red). Enlargement of the boxed areas are shown for transfected (a, c) and nontransfected (b, d) neurons. The soma of transfected neurons is delimited by dashed lines. B, C, EB1 fluorescence intensity ratio (B) and mean number of EB1 comets per micrometer squared (C) in the soma of neurons cotransfected at DIV1 with GFP and control- (white bars) or EB-shRNA (gray bars). B, Mann–Whitney test, n = 26 neurons at least per condition p = 4.03 × 10−5. C, Unpaired t test, n = 32 neurons at least per condition, p = 7.79 × 10−15. D, E, DIV5 rat neurons cotransfected at DIV1 with GFP and control- (D) or EB-shRNA (E) and stained for GFP (green), 480AnkG (red), MAP2 (blue), and Tau-1 (E, gray). E, Right, Enlargements of the left panel boxed areas. F, Fluorescence intensity of 480AnkG staining at the AIS of DIV5 neurons cotransfected at DIV1 with GFP and control- (n = 34) or EB-shRNA (n = 26), normalized to neighboring untransfected neurons. Unpaired t test, p = 0.0094. G, Percentage of DIV5 neurons cotransfected at DIV1 with GFP and control- (n = 94) or EB-shRNA (n = 92) showing endogenous 480AnkG in dendrites. Unpaired t test, p = 0.0013. H, DIV5 hippocampal neurons cotransfected at DIV1 with 480AnkG-GFP (green) and control- (top) or EB-shRNA (bottom) and stained for PanNav (red) and MAP2 (blue). I, Fluorescence intensity of PanNav staining at the AIS of DIV5 neurons transfected with 480AnkG-GFP and control- (n = 21) or EB-shRNA (n = 24) normalized to neighboring untransfected neurons. Unpaired t test, p = 0.14. J, Percentage of DIV5 neurons cotransfected with 480AnkG-GFP and control- (n = 53) or EB-shRNA (n = 47) showing PanNav immunoreactivity in dendrites. Unpaired t test, p = 0.0059. K, Polarity index of GFP in DIV5 neurons coexpressing 480AnkG-GFP together with control- (n = 21) or EB-shRNA (n = 23). Mann–Whitney test, p = 7.00 × 10−7. L, Average percentage of DIV5 neurons transfected with 480AnkG-GFP and control- (n = 97) or EB-shRNA (n = 76) showing a dendritic localization of GFP. Unpaired t test, p = 0.029. M–O, FRAP experiments in the proximal axon of DIV5–DIV6 hippocampal neurons cotransfected at DIV0 with 480AnkG-GFP or 480AnkG-NN-GFP and control-shRNA or AnkG-shRNA. Representative stills of corresponding time-lapse videomicroscopy recording (M), percentage of GFP fluorescence recovery after FRAP (N), and percentage of normalized GFP fluorescence recovery (O), at least 10 neurons per condition were analyzed. One-way ANOVA. Arrowheads and empty arrowheads indicate the AIS of transfected and nontransfected neurons respectively. Arrows highlight the ectopic localization of AIS components. Scale bars, 20 μm and 5 μm in the zooms (A, E). Error bars are SEM. n.s., Nonsignificant. **p < 0.01, ****p < 0.0001.

Figure 6.
Figure 6.

480AnkG exclusively orchestrates AIS assembly through its interaction with EBs. A, DIV5 rat hippocampal neurons cotransfected at DIV0 with AnkG-shRNA and 480AnkG-GFP (green) and stained for Nfasc (top, red) or PanNav (bottom, red), and MAP2 (blue). B, Mean fluorescence intensity profile of GFP, NFasc and PanNav staining along the axons of untransfected neurons (gray lines) and neurons coexpressing AnkG-shRNA and 480AnkG-GFP (black lines, at least 7 neurons per condition). C, DIV5 rat hippocampal neurons cotransfected at DIV0 with AnkG-shRNA and 480AnkG-NN-GFP (green), and stained for Nfasc (top, red) or PanNav (bottom, red) and MAP2 (blue). D, Mean fluorescence intensity profile of GFP, NFasc, and PanNav staining along the axon of untransfected neurons (gray lines) and along the longest neurite of neurons coexpressing AnkG-shRNA and 480AnkG-NN-GFP (black lines, at least 7 neurons per condition). E, DIV5 rat hippocampal neurons cotransfected at DIV0 with AnkG-shRNA and 270AnkG-GFP (green), and stained for Nfasc (top, red) or PanNav (bottom, red) together with MAP2 (blue). F, Mean fluorescence intensity profile of GFP, NFasc, PanNav, and 480AnkG along the axon of untransfected neurons (gray lines) and along the longest neurite of neurons coexpressing AnkG-shRNA and 270AnkG-GFP (black lines, at least 6 neurons per condition). G, H, Average percentage of neurons cotransfected with AnkG-shRNA and 480AnkG-GFP (n = 56), 480AnkG-NN-GFP (n = 48), or 270AnkG-GFP (n = 81) showing an accumulation of NFasc (dark gray) or PanNav (light gray) at the AIS (G) and GFP staining restricted to the AIS (H). One-way ANOVA. I, Distal limit of MAP2 expression in the longest neurite of neurons cotransfected with AnkG-shRNA and 480AnkG-GFP (n = 16), 480AnkG-NN-GFP (n = 13), or 270AnkG-GFP (n = 13). Kruskal–Wallis test. J, Western blot analysis of protein extracts from COS-7 cells cotransfected with control- or AnkG-shRNA and rat or mouse 270AnkG-GFP using Pan(SR)AnkG antibody. β2-karyopherin was used as a loading control. Arrowheads and open arrowheads show the AIS of transfected and nontransfected neurons respectively and arrows point the lack of AIS in transfected neurons. Scale bars: A, C, E (top),15 μm; E (bottom), 30 μm. n.s., Nonsignificant; A.U., arbitrary unit. Error bars represent SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

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