Fig 2: Pk2 binds to AnkG, leading to MT bundling.(A) Schematic of the AnkG270, the AnkG190 isoform domains of AnkG, and the short AnkG-MBD. N, N terminus; C, C terminus; MBD, membrane-binding domain; SBD, spectrin-binding domain. (B and C) coIP of AnkG with Flag-Pk2. Both AnkG isoforms interact with Pk2 but not with immunoglobulin G (IgG)–R serum. (D) coIP of the short AnkG-MBD with Flag-Pk2. (E) Schematic of the full-length Pk2 protein with the different domains and the corresponding amino acid numbers and the GST constructs used. (F) GST pulldown experiments showing that Pk2 interacts with AnkG via the LIM and the C-terminal domains, the latter including the C2 region. (G) Pk2 coimmunoprecipitates with AnkG in vivo but not with IgG control serum (Ig). Asterisks indicate the two bands immunoprecipitated. (H and I) COS7 cells transfected with Flag-Pk2 (H) or AnkG480-GFP (I). (J to J?) Flag-Pk2 (magenta) and AnkG480-GFP (green) promote the bundling of MTs, strongly immunoreactive to tyrosinated tubulin [cyan in (J?)]. Note the relocalization of Pk2 and AnkG to the MT bundles. (K to L?) MT bundling is lost upon coexpression of Flag-Pk2 with AnkG480NN, an AnkG480 construct that can no longer bind to MTs. Scale bars, 10 µm. Statistics, Table 1.

Fig 3: Ablation of exon 8 increases TRIM46 protein and leads to enhanced AnkG localization to the proximal axon.a Schematic CRISPR/Cas9 strategy to convert parental WT ESC to mutant exon 8-KO. Graphical representation of Trim46 locus showing sgRNAs targeting introns 7 and 8 to generate exon 8 deletion. F5 and R5 denote approximate location of genotyping primers to detect removal of exon 8. Arrow represents edited locus showing change from WT to mutant Trim46?E8/?E8 or (E8-KO). b Genotyping results of WT and Trim46?E8/?E8 ESC. Band sizes are 739 bp and 409 for WT and Trim46?E8/?E8, respectively from 5 independent experiments. c Sanger sequencing of PCR product from genomic DNA confirms exon 8 deletion in E8-KO ES cell line. Top panel shows sequence alignment between WT and E8-KO lines and the bottom panel shows chromatogram of E8-KO mutant highlighting sequence of intron 7 preceding the 330 bp deletion of exon 8 and the immediate downstream sequences. d–g. Western blot highlights upregulated TRIM46 protein expression in exon 8 deletion mutants from ESC-derived neurons on DIV 3 and DIV 5. Protein quantification for TRIM46L protein between WT and E8-KO neurons on DIV 3 and DIV 5; n = 4 independent experiments; Data is represented as mean ± SEM. Statistics: t-test, two-tailed, paired; (DIV 3: WT v. E8-KO, *p = 0.02447; DIV 5: WT v. E8-KO, *p = 0.02008). h RT-qPCR for Trim46 total mRNA levels between WT and E8-KO from EB day 8, DIV 1, and DIV 7. Data is represented as mean ± SD; Statistics: t-test, two-tailed, unpaired: WT v. E8-KO; EB D8, ns = 0.02091317, DIV 1, ns = 0.27058758, DIV 7, ns = 0.65857523; WT, n = 3 biological replicates. i ESC-derived neurons of WT and E8-KO on DIV 3 stained for TRIM46 (green) and AnkG (red). Arrowhead denotes TRIM46 signal at the proximal axon and asterisk (*) denotes nonspecific TRIM46 somatic staining. Scale bars, 10 µm. j, k. Quantification between WT and E8-KO neurons on DIV 3 for TRIM46 length (µm), (**p = 0.001351) and normalized TRIM46 mean intensity in arbitrary units (AU), (*p = 0.04061). l The deletion effect of Trim46 exon 8 between WT and E8-KO neurons on DIV 3 for percentage of AnkG-positive neurons at the AIS, *p = 0.02048; m, AnkG length (µm), *p = 0.01743; and n normalized AnkG mean intensity in arbitrary units (AU), ns = 0.7097. Data is represented as mean ± SEM; WT, n = 130 and E8-KO, n = 154 which represent 3 independent differentiation experiments; Statistics: t-test, two tailed, paired.

Fig 4: In vivo deletion of Pk2 impairs AIS formation in neocortical neurons.(A) Schematic of IUE at E16.5 in rat embryos. (B and C) Low magnifications of coronal sections from P6 control and shPk2-electroporated brains immunolabeled for anti-GFP to identify GFP-positive neurons. In (C), note that some Pk2-depleted neurons remain stuck close to the ventricular zone (vent) (circled in green), while in the control, they all migrate dorsally. (D to E?) In GFP-positive control neurons (D to D?) (green circles), Pk2 and AnkG colocalize at the AIS (arrows), while in Pk2-depleted neurons, almost no Pk2 or AnkG remains (E to E?). (F) Percentage of Pk2-positive neurons in control and shPk2 brain sections as compared to the surrounding, nonelectroporated cells. (G) Percentage of AnkG-positive neurons in control and shPk2 brain sections as compared to the surrounding, nonelectroporated cells. (H) AnkG levels at the AIS in the shPk2 neurons with a positive AnkG labeling. Scale bars, 200 µm (A to C) and 20 µm (D to E?). Statistics, Table 1.

Fig 5: Pk2 depletion impairs the AIS cytoskeleton network.(A and B) Immunolabeling for GFP in control (A) and Pk2-depleted neurons (B). (C) Average thickness of AnkG-positive axons. In both proximal [orange arrows in (A) and (B)] and distal [blue arrows in (A) and (B)], Pk2 depletion leads to a severe reduction in thickness. (D and E) STORM imaging of F-actin shows a loss of actin periodicity at the AIS of Pk2-depleted (E) neurons compared to control (D) at DIV7. (F to G?) TRIM46 immunolabeling is reduced at the AIS of Pk2-depleted neurons (G to G?) compared to control (F to F?). (H) Fluorescence intensity levels of TRIM46 at the AIS from neurons in (F) to (G?). (I to J?) MAP2 immunolabeling is decreased in Pk2-depleted neurons (J to J?) compared to control (I to I?) and restricted to the somatodendritic domain at DIV7. Note the MAP2 staining of the dendrite of a nontransfected cell comparison in (J?) (asterisk). Arrows indicate the multiple AISs. (K) PI of AnkG in DIV7 neurons. (L and M) Illustration of live neurons expressing MACF-RFP (green) and labeled with fluorescent-tagged NF-186 antibody (magenta). Right: Kymographs of control axons and dendrites and shPk2 NF-186–positive (axons) and NF-186–negative neurites at DIV7. Yellow asterisks indicate the severing position. (N) Percentage and identity of comets in axons and dendrites after laser cut. (O) MT growth speed in control (black) versus shPk2 (magenta) neurons, in axon and dendrites. (P) Number of comets displayed by control (black) and shPk2 (magenta) neurons, in axon and dendrites. Scale bars, 4 µm (A and B), 2 µm and 0.5 µm [for higher magnification (D and E)], and 10 µm (F to G?, I to J?, L, and M). Bracket indicates the AIS. Statistics, Table 1.