Fig 2: TARA recruits DYRK2 and GSK3ß to induce sequential phosphorylation of NDEL1 S336/S332.(A) In vitro phosphatase assay. Calf intestinal alkaline phosphatase (CIP) was treated in vitro after IP of FLAG-tagged NDEL1. Additional bands of NDEL1 disappeared upon CIP treatment indicating that these additional bands are caused by the multiple phosphorylation. (B) MS/MS spectrum for the phosphorylated fragments of NDEL1 peptide including S332 and S336 residues. NDEL1 proteins from HEK293 cells over-expressing NDEL1 and TARA were subjected to LC-MS/MS analysis. The sequence of the peptide (aa 306–345) and all detected fragment ions are shown above. The b- and y-ions annotated in the spectrum include the sizes of y12 and b33 ions indicative of a single phosphorylation at either S335 or S336. (C) TARA-induced NDEL1 phosphorylation S336 and S332. TARA increased sequential phosphorylation of NDEL1, first at S336 followed by S332. (D) The protein-protein interaction between DYRK2 and TARA. Endogenous DYRK2 was co-precipitated by IP of endogenous TARA from HEK293 cell lysates. Rabbit IgG was used as a negative control. At input lanes, 1% and 10% of lysates were loaded for anti-DYRK2 and anti-TARA blots, respectively. (E) The protein-protein interaction between GSK3ß and TARA. Endogenous TARA was co-precipitated by IP of endogenous GSK3ß proteins from HEK293 cell lysates. Mouse IgG was used as a negative control. (F) Co-IP among DYRK2, GSK3ß, and TARA. Over-expression of TARA increased an amount of GSK3ß proteins co-precipitated by IP of DYRK2, implying that TARA scaffolds these kinases to form a DYRK2-GSK3ß-TARA tripartite complex. (G) Colocalization of DYRK2, GSK3ß, and TARA in mouse hippocampal neurons. GFP-hDYRK2, FLAG-hGSK3ß, and MYC-hTARA colocalized at the soma (above) and the growth cone (bottom) regions. See also Figure 4—figure supplements 1 and 2. The following figure supplement is available for Figure 4.

Fig 3: Phosphorylation of NDEL1 S336/S332 regulates dendritic arborization of cortical pyramidal neurons.(A–E) Suppression of NDEL1 S336/S332 phosphorylation disrupted dendritic arborization of layer II/III pyramidal neurons. All constructs were electroporated in utero to E15 mouse brain and then P14 brain was subjected for analysis. All of NDEL1 over-expressing constructs here contain an shRNA-resistant mutation. (A) Representative images of the brain slices with the tracked neuron (above) and the overlapped dendritic structures of five independent neurons (bottom). Sholl analysis plots (B), the total length of dendrites (C), the total number of branches (D), and the number of primary/secondary/tertiary dendrites (E) were analyzed by using Simple neurite tracer plug-in of ImageJ software. White arrowheads in (A) indicate neurons with migration defect. (F–J) NDEL1 S336/S332 phosphorylation induced by DYRK2-GSK3ß kinases increased dendritic arborization of layer II/III pyramidal neurons. All constructs were electroporated in utero to E15 mouse brain and P14 brain was subjected for analysis. (F) Representative images of the brain slices with the tracked neuron (above) and the overlapped dendritic structures of five independent neurons (bottom). Sholl analysis plots (G), the total length of dendrites (H), the total number of branches (I), and the number of primary/secondary/tertiary dendrites (J) were analyzed by using Simple neurite tracer plug-in of ImageJ software. Each n number is shown at the bottom of the bar of the graph. Scale bars represent 100 µm. All results are presented as means ± SEM. *p<0.05, **p<0.01, and ***p<0.001 from one-way ANOVA for (C), (D), (H), and (I) and two-way ANOVA for (B), (E), (G), and (J). All brain samples for each group were collected from offspring of at least three independent in utero electroporation surgeries. See also Figure 3—figure supplement 1, Figure 3—videos 1 and 2, and Figure 3—source data 1 and 2.Figure 3—source data 1.Source data for dendritic arborization of NDEL1 knockdown and rescue groups.Figure 3—source data 2.Source data for dendritic arborization of NDEL1 and kinases over-expression groups.

Fig 4: NDEL1 S336/S332 phosphorylation induced by TARA increases neurite outgrowth and dendritic arborization.(A–C) Induction of NDEL1 S336/S332 phosphorylation by TARA increased neurite outgrowth. Each DNA construct was transfected at DIV 2 and neurites of transfected neurons were analyzed at DIV 5. (A) Representative images of transfected neurons. The total neurite length (B) and the longest neurite length (C) were measured by using ImageJ software. (D–F) TARA?413-499 could not increase neurite outgrowth. Each DNA construct was transfected at DIV 2 and neurites of transfected neurons were analyzed at DIV 5. (D) Representative images of transfected neurons. The total neurite length (E) and the longest neurite length (F) were measured by using ImageJ software. (G–K) Up-regulated NDEL1 S336/S332 phosphorylation increased dendritic arborization of layer II/III pyramidal neurons. All constructs were electroporated in utero to E15 mouse brain and P14 brain was subjected for analysis. (G) Representative images of the brain slices with the tracked neuron (above) and the overlapped dendritic structures of five independent neurons (bottom). Sholl analysis plots (H), the total length of dendrites (I), the total number of branches (J), and the number of primary/secondary/tertiary dendrites (K) were analyzed by using Simple neurite tracer plug-in of ImageJ software. Each n number is shown at the bottom of the bar of the graph. Scale bars represent 100 µm. All results are presented as means ± SEM. **p<0.01 and ***p<0.001 from one-way ANOVA for (B), (C), (E), (F), (I), and (J) and two-way ANOVA for (H) and (K). All neurite outgrowth experiments for (A–C) and (D–F) were independently repeated for at least three times. All brain samples for each group of (G–K) were collected from offspring of at least three independent in utero electroporation surgeries. See also Figure 5—figure supplement 1, Figure 5—video 1, and Figure 5—source data 1 and 2.Figure 5—source data 1.Source data for axon/dendrite outgrowth of NDEL1 and TARA over-expression groups.Figure 5—source data 2.Source data for dendritic arborization of NDEL1 and TARA over-expression groups.

Fig 5: Phosphorylation of NDEL1 S336/S332 increases neurite outgrowth of primary cultured hippocampal neurons.(A–C) Suppression of NDEL1 S336/S332 phosphorylation inhibited neurite outgrowth of hippocampal neurons. Each DNA construct was transfected at 9 hr after neuronal culture and neurites of transfected neurons were analyzed at DIV 3. All of NDEL1 over-expressing constructs here contain an shRNA-resistant mutation. (A) Representative images of transfected neurons. The total neurite length (B) and the longest neurite length (C) were measured by using ImageJ software. (D–F) NDEL1 S336/S332 phosphorylation induced by DYRK2-GSK3ß co-expression is enough to increase neurite outgrowth of hippocampal neurons. Each DNA construct was transfected at DIV 2 and neurites of transfected neurons were analyzed at DIV 5. (D) Representative images of transfected neurons. The total neurite length (E) and the longest neurite length (F) were measured by using ImageJ software. (G–I) KI of phospho-deficient mutations resulted in neurite outgrowth defects. DNA constructs for KI were transfected at DIV 1 and neurites of transfected neurons were analyzed at DIV 4. (G) Representative images of transfected neurons. The total neurite length (H) and the longest neurite length (I) were measured by using ImageJ software. Each sample number is shown at the bottom of the bar of the graph. Scale bars represent 100 µm. All results are presented as means ± SEM. *p<0.05, **p<0.01, and ***p<0.001 from one-way ANOVA for (B), (C), (E), and (F) and Student’s t-test for (H) and (I). All experiments were independently repeated at least three times. See also Figure 2—figure supplements 1 and 2 and Figure 2—source data 1, 2 and 3.Figure 2—source data 1.Source data for axon/dendrite outgrowth of NDEL1 knockdown and rescue groups.Figure 2—source data 2.Source data for axon/dendrite outgrowth of NDEL1 and kinases over-expression groups.Figure 2—source data 3.Source data for axon/dendrite outgrowth assay of NDEL1 S332/336A KI.