Fig 2: TAOK2 is an ER-protein kinase(A) Transmembrane Hidden Markov Model (TMHMM v2.0) prediction plot shows the posterior probabilities (y axis) of inside/outside(magenta)/TM helix (red) along the length of TAOK2 sequence (x axis). Hydrophobic region following the sixth predicted transmembrane domain (TMD) was analyzed through AMPHIPASEEK which predicted residues 1,146–1,162 to be amphipathic. Charged (blue) and hydrophobic (yellow) residues are indicated in the inset. HeliQuest was used to calculate hydrophobicity , hydrophobic moment <µH> and net charge Z.(B) Schematic representation of TAOK2 isoforms a, ß and TAOK1. Coiled coils (CC), six transmembrane domains (TM), and amphipathic helix (AH) predicted in TAOK2 are depicted. The asterisk indicates the epitope used to generate the TAOK2a-specific antibody.(C) SoRa (super resolution by optical reassignment) images of HeLa cells expressing GFPTAOK2 (cyan) and ER marker ER-mRFP (red). Scale bar: 5 µm. Grayscale images of TAOK2 and ER-RFP are shown separately. Scale bar: 1 µm.(D) Manders’ overlap coefficient for co-localization of expressed GFP-TAOK2 and ER-mRFP and for endogenous TAOK2 and EGFP-Sec22b are plotted. Values indicate mean ± SEM; n = 10 for each condition.(E) Western blot (n = 3) of cell homogenate fractionated into ER membrane and cytosol probed with antibodies against TAOK2, ER-membrane proteins Stim1 and Rtn3, mitochondrial protein Tom20, and tubulin.(F) Western blot (n = 3) of cell homogenate fractionated into membrane pellet (P) and cytosolic supernatant (S) components, in the absence (control) or presence of detergent, probed with antibodies against TAOK2 and ER protein Rtn3a.(G) GFP-tagged TAOK2 deletion constructs used in (H).(H) Confocal images of cells transfected with distinct GFP-tagged TAOK2 deletion constructs (green) and ER-mRFP (magenta). Scale bar: 3 µm.See also Figure S1.

Fig 3: TAOK2 is an ER-MT tethering protein kinase(A) Schematic representation of TAOK2 with cytoplasmic-facing kinase domain, membrane bound 6 transmembrane helices and amphipathic region, and C-terminal tail directly bound to MT.(B) Cells transfected with GFP-TAOK2 (yellow), ER-mRFP (magenta), and MT dye (cyan). Scale bar: 1 µm.(C) Magnified images displays individual TAOK2 (yellow) punctae on ER tubules in contact with MT (cyan). Scale bar: 1mm.(D) Percentage of TAOK2 punctae co-localized with ER and MTs in HEK293T cells. Values indicate mean ± SEM; n = 10 cells with at least 50 punctae per cell were analyzed. ****p < 0.0001(E) Western blot of lysate from WT and TAOK2 KO cell lines generated using CRISPR/Cas9 gene editing.(F) WT and TAOK2 KO cells transfected with ER-mRFP (green) and mEmerald-ensconsin (magenta) to visualize MTs. Peripheral ER is magnified and shown for both WT and TAOK2 KO cells. Scale bar: 5 µm.(G) Ratio of peripheral ER area in contact with MT in 100-µm2 region for WT and TAOK2 KO cells. Values indicate mean SEM; n = 6 cells from 3 experiments, t test with Welch’s correction. **p < 0.01(H) ER density measured in WT and TAOK2 knockout cells. Values indicate mean ± SEM, n = 6 cells from 3 experiments, t test with Welch’s correction.(I) Topology of TAOK2 deletion constructs. KO cells expressing indicated TAOK2 constructs are shown where TAOK2 (yellow), ER-mRFP (magenta) and MTs (cyan) are shown in merged and grayscale. Scale bar: 5 µm. See also Figure S2.

Fig 4: Direct binding of TAOK2 to MTs(A) Confocal images of cells transfected with indicated GFP-tagged TAOK2 constructs immunostained for a-tubulin. Scale bar: 3µm. Magnified images highlight overlap of GFP-tagged TAOK2 with MTs. Scale bar: 5 µm.(B) GFP-tagged deletion constructs, coiled-coil (CC), transmembrane (TMD), amphipathic helix (AH) domains are marked. Domain necessary and sufficient for MT localization is indicated by red box.(C) Coomassie-stained SDS-page gel shows co-sedimentation of indicated proteins with polymerized tubulin. MT binding assay (n = 3) performed with 5 µg of GST or GST-TAOK2-C (1,187–1,235) protein in presence of Taxol stabilized MT.(D) Percentage of protein bound to MT plotted for indicated protein. Error bars: SEM; n = 3; p < 0.0001, one-way ANOVA.(E) Coomassie-stained SDS-page gel shows amount of purified GST-TAOK2-(1,187–1,235) unbound (S) or co-sedimented with polymerized MT (P).(F) Dissociation constant KD derived by the Michaelis-Menten equation. Error bars: SEM; n = 3 replicates. See also Figure S2.

Fig 5: TAOK2 regulates ER TAC movement and interacts with EB1 via SxIP motif(A) Single z-frame confocal images of WT and TAOK2 KO cells expressing ER marker Sec61-BFP (blue), STIM1-YFP (green) and EB3-mcherry (red). Scale bar: 3 µm.(B) Maximum projection of single z frames shown in (A) over a period of 20 s to visualize movement and paths of EB3 and STIM1 over time in WT and TAOK2 KO cells. STIM1-YFP (green) and EB3-mcherry (red). Scale bar: 3 µm.(C) EB3 comets were classified as (1) comets interacting with STIM1/ER and performing TAC, (2) comets interacting with STIM1 transiently but not performing TAC, and (3) comets not contacting STIM1/ER. Values indicate mean ± SEM; n = 7 cells from 3 experiments; two-way ANOVA. *p < 0.05.(D) Schematic depicts the SxIP motif of MT plus tip-interacting proteins that associate with EB proteins.(E) HeLa cells immunostained for EB1 (magenta) and TAOK2 (blue). Scale bar: 1 µm.(F) Western blot (n = 3) shows coimmunoprecipitation of TAOK2 and STIM1 with EB1 using anti-EB1 antibodies; 1% input is shown for comparison.(G) TAOK2 KO cells transfected with EB3-mcherry (red) along with GFP (1,196–1,235) or GFP (1,162–1,235) containing the TRIP (SxIP) motif. Scale bar: 3 µm.(H) Bar graph represents fractions of EB1-positive TAOK2 punctae to total TAOK2 punctae, number of TAOK2-positive EB1 punctae compared to total EB1 punctae, number of EB1 puncta on ER compared with total EB1 punctae, and number of TAOK2-positive EB1 punctae on ER compared with the total number of EB1s on the ER. Values indicate mean ± SEM; n = 10 cells from 3 experiments. See also Figure S4.