Fig 2: Characterization of mGFP-EB1 and mCherry-PRC1 expressing hTERT-RPE1 cells.(A) PCR test for correct mGFP gene insertion at the genomic EB1 gene locus. The schematic (top) indicates where the primers (small arrows) bind to the genomic DNA (black arrowhead indicates the position of primer binding). One primer binds inside the mGFP gene, the other primer outside the left homology arm. Agarose gel (bottom) shows DNA fragments amplified from genomic DNA of control (Ctrl) and gene-edited single cell clones (clones A1–A5). The detection of a 2 kb band for the single cell clones demonstrates the correct insertion of the mGFP gene at the 5' end of the endogenous EB1 genes. (B) PCR test for determining the number of modified EB1 alleles. The schematic (top) indicates where the primers bind to the genomic DNA for this PCR test. One primer binds to the region outside the LHA and the other primer binds inside the right homology arm. Agarose gel (bottom) showing DNA fragments amplified from control and gene-edited cells. The absence of the 1.2 kb band and the presence of a 2 kb band for the single cell clones A1-5 demonstrates that both EB1 alleles are modified in the gene-edited cells. (C) Western blot of cell lysates using an anti-EB1 antibody (top) showing the expression levels of unmodified EB1 in control cells and mGFP-EB1 in the gene-edited single cell clones A1-5 (left). Densitometry of the bands demonstrates that clones A1, A2, A3, A4, and A5 express 0.47, 0.45, 0.60, 0.33 and 0.0.27 times the amount of mGFP-EB1, respectively, compared to the amount of EB1 in unmodified control cells (n = 2), data are normalized with respect to the tubulin control. Western blot of the same samples using an anti-tubulin antibody (bottom) as a loading control. Single-cell clone A3, expressing mGFP-EB1 from its endogenous locus was selected for further gene editing to additionally tag endogenous PRC1 with mCherry. (D, E, F) PCR tests and Western blots, as indicated, for cells expressing both mCherry-PRC1 and mGFP-EB1 from their endogenous loci. (D, E) All gene-edited clones (B1 and B2) show apparently correct mCherry gene insertion at the 5' end of the PRC1 gene (D) and have both PRC1 alleles tagged (E). (F) Both clones B1 and B2 show correct protein expression (F). Clones B1, and B2, show expression levels of mCherry-PRC1 are 0.60, and 0.3 times of the expression levels of PRC1 in only EB1 modified control cells and unmodified control cells (n = 2). Clone B1 was selected for further studies.

Fig 3: Live-cell imaging of endogenously expressed mGFP-EB1 and mCherry-PRC1 during mitosis.(A, B) Spinning disk confocal microscopy of endogenous mGFP-EB1 in RPE1 cells (also expressing mCherry-PRC1). (A) Image of an interphase cell (left) and a time stack from a time-lapse movie of the same cell (right) showing EB1 microtubule end tracking traces color-coded as a function of time. Scale bar = 10 µm. See also Video 1. (B) Confocal microscopy images of gene-edited RPE1 cells coexpressing mGFP-EB1 (green) and mCherry-PRC1 (magenta) from their endogenous loci. Chromosomes are stained by Hoechst (blue). Images were acquired at four frames per second. Scale bar = 10 µm. Time is in min:s. See also Videos 2 and 3. (C) Representative kymographs (space-time plots) generated from a mitotic cell movie showing chromosomes (blue), EB1 (green), and PRC1 (magenta) distribution along the spindle (pole-to-pole) axis. Images were acquired every 5 s from metaphase to telophase. Scale bars = 10 µm (horizontal) and 1 min (vertical). See also Video 2. (D) A representative kymograph (left) generated from a late anaphase movie along the pole-to-pole axis. Internal (i.e., in between the poles) EB1 comets tracks and the EB1 tracks along astral microtubules are visible. (Right) overlay of the kymograph on the left and EB1 tracks, shown in different colors, as detected using the KymoButler software (76). Scale bars = 10 µm (horizontal) and 10 s (vertical). (E) EB1 comet speed behaviour in interphase and from metaphase to telophase. EB1 average comet speeds were calculated from the first and last point of each trajectory. Data points are median values and error bars represent the standard error of the mean. The number of EB1 comets (n) and cells (N) considered in the analysis is: metaphase (M) n = 52, N = 11; early anaphase (EA) n = 51, N = 5; mid-anaphase (MA) n = 50, N = 8; late anaphase (LA) n = 125, N = 8; telophase (T) n = 87, N = 12.

Fig 4: HCR regulates microtubule organization by recruiting CEP72 to the centrosome. A Microtubule regrowth assay of negative control, astrin, HCR, and CEP72 siRNA-treated HeLa cells co-stained with gamma-tubulin (green), alpha-tubulin (red), and DAPI (blue). The quantified analysis was based on alpha-tubulin staining length. Error bars represent the mean ± SD of cells (n = 100 per group) from three independently performed experiments; ****P < 0.0001 (Student’s t test); scale bars, 10 µm. B Negative control, astrin, HCR, and CEP72 siRNA-treated HeLa cells were co-stained with EB1 (green) and DAPI (blue) for immunofluorescence detection. Quantitative analysis was based on the staining length of EB1. Error bars represent the mean ± SD of cells (n = 100 each group) from three independently performed experiments. ****P < 0.0001 (Student’s t test); scale bars, 10 µm. C GFP-tagged HCR deletion fragments (green) were transfected into HeLa cells and co-stained with CEP72 (red) and DAPI (blue). Arrows represent the area of the centrosome; scale bars, 10 µm. D Microtubule regrowth assay of vector- or HCR-3-GFP-transfected HeLa cells co-stained with alpha-tubulin (red), gamma-tubulin (cyan), and DAPI (blue); scale bars indicate 10 µm

Fig 5: EB1 localization in different cell lines.(A, B, C) Control experiments: (A) Immunofluorescence microscopy of EB1 in unmodified RPE1 cells. (B) Immunofluorescence microscopy of EB1 in gene-edited RPE1 cells expressing only mGFP-PRC1 from its endogenous locus. (C) Immunofluorescence microscopy of EB1 in gene-edited RPE1 cells expressing only mCherry-PRC1 from its endogenous locus. These experiments show the typical “comet”-like localization of EB1 to microtubule ends in RPE1 cells, similar to the EB1 localization in gene-edited RPE1 cells expressing mGFP-EB1 and mCherry-PRC1 from their endogenous locus (Fig 1). DNA is always stained by Hoechst and shown in blue. Scale bars = 10 µm.