Fig 2: chTOG localizes to kinetochores during mitosis.(a) Immunofluorescence images of chTOG subcellular localization during mitosis, as visualized in HCT116 cells expressing endogenously epitope-tagged chTOG-EGFP. Anti-centromere protein antibody (ACA) staining marks the centromere-binding proteins and representative images are shown with inlays of kinetochore proximal chTOG at each stage of mitosis. (b) Quantification of chTOG kinetochore association. Each data point represents mean chTOG-EGFP fluorescence intensity at all kinetochores in a single cell normalized to the mean value of the anaphase population (c) HEK-293T cells with either an empty vector control or overexpressed chTOG-3Flag were immunoprecipitated using anti-Flag antibodies. Immunoblots of the input (left) or Flag IP (right) show that the endogenous Hec1/Ndc80 protein specifically co-purified with chTOG. Endogenous and epitope tagged chTOG cannot be individually resolved by anti-chTOG immunoblotting because the 3Flag tag increases the protein’s predicted MW by only 3%. Anti-GAPDH served as a non-specific control. (d) Immunoblotting with anti-Hec1 antibodies was performed on samples of mock-depleted lysate that were diluted to contain the indicated percent of total protein and compared to a lysate prepared from a population of HCT116 cells treated with Hec1 siRNA. Greater than 75% of Hec1 protein was depleted in the siRNA-treated cells. Anti-GAPDH is a loading control. (e) Kinetochore localization of chTOG-EGFP in Hec1 depleted HCT116 cells was determined by fluorescence microscopy. Representative images are shown and were quantified on the right to show endogenously tagged chTOG-EGFP signal at kinetochores decreased by ~70% in siRNA-treated HCT116 cells. Each data point represents mean fluorescence intensity at all kinetochores in a single cell normalized to the mean value of the mock depleted population. (f) Same as (e) but cells were treated with 10 µM nocodazole for 1 hr prior to fixation to preclude any chTOG bound to the microtubule tips from this analysis. All scale bars are 5 µm; contrast on inlays was adjusted independently; data points on graphs are grouped from three experimental replicates and colored according to each replicate with median and 95% confidence intervals displayed in black. p-Values determined by an unpaired Mann-Whitney test.

Fig 3: fEPSPs and LTP in CA1 of control and TOG cKO hippocampal brain slices.A. Infrared differential interference contrast micrograph of a hippocampal slice showing bipolar tungsten electrode (left) and glass recording electrode (right) placement. B – C. Electrical recording of field responses from control (B) and TOG cKO (C) slices at various stimuli intensities. Presynaptic fiber volley and field excitatory postsynaptic potential (fEPSP) are marked by arrows. Input output curves generated from control (n = 7 slices from 5 animals) and TOG cKO (n = 7 slices from 4 animals) plotted using fEPSP amplitudes (left) and rising slopes (right). Error bars represent standard deviation of the mean. D – E. Field responses and input output curves in the presence of GABAA antagonist (GABAzine, 5 µM) for the same specimens as in B and C. Error bars represent standard deviation of the mean. F. Electrical recording of field responses in control and TOG cKO hippocampal slices, in presence of GABAzine (5 µM), during baseline (BL) as well as 5 and 60 minutes after theta burst stimulation (TBS, 10 bursts in 2 sec). G. Rising slopes of fEPSPs during BL, and 5 and 60 minutes after TBS, from control (n = 5 animals) and TOG cKO (n = 4 animals). * denotes significant difference from BL (repeated measures ANOVA followed by Newman-Keuls multiple comparison test, p<0.05). H. Group time-course before and after TBS, normalized to the rising slopes of its corresponding BL. Error bars represent standard deviation of the mean.* denotes significant difference from BL period in control (repeated measures ANOVA followed by Bonferroni's multiple comparison test, p<0.05).

Fig 4: The chTOG basic pair mutant regulates microtubule dynamics, spindle structure, and localizes to kinetochores.(a) Images isolated from live-cell TIRF microscopy were used to generate kymographs of EB1-mCherry in chTOGWT and chTOGKK/AA-expressing cells with average EB1 track speed (µm/min) in bottom left and quantifications shown below. Each data point represents the mean EB1 track speed per cell and is grouped from three experimental replicates and colored according to each replicate with median and 95% confidence intervals displayed in black. p-Values were determined with an unpaired Mann-Whitney test. (b) Representative images of each spindle phenotype observed in mitotic chTOG-depleted, chTOGWT, or chTOGKK/AA expressing cells. While bipolar spindles exhibited two distinct phenotypes, we first quantified the fraction of cells exhibiting multipolar or bipolar spindles. Mean values and 95% confidence interval for three replicates were reported, p values calculated with a paired t test. (c) Representative images (left) and quantification (right) of chTOGWT and chTOGKK/AA localization to kinetochores in the absence of endogenous chTOG. Cells were treated with nocodazole to eliminate microtubules. Each data point represents the mean chTOG-EGFP fluorescence intensity at all kinetochores in a single cell normalized to the mean value of chTOGWT-expressing cells. Data are grouped from three experimental replicates and colored according to each replicate with mean values and 95% confidence displayed in black. p-Value was determined with an unpaired t test. All scale bars are 5 µm.

Fig 5: TOG knockout in hippocampal neurons.A. Diagram of the portion of the TOG/CKAP5 gene that is targeted for excision, and the targeting construct. The bottom panel shows the out-of-frame deletion after Cre excision. B. Western blot of brain homogenates from +/+ (control) and +/null mice stained with rabbit anti-TOG and mouse anti-ß-actin; error bars indicate standard deviations; n = 6 animals for each genotype (t-test *p<0.05). C. Western blot of homogenates from hippocampus, cerebellum and cortex of 2 month old control (wild type) and TOG cKO mice stained with rabbit anti-TOG, mouse anti-aCaMKII and mouse anti-ß-actin; n = 3 animals for each genotype.