Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A, B ) Anti-rootletin immunofluorescent staining (green) is not evident at centrosomes after rootletin ( CROCC ) siRNA. Centrosomes were co-stained with anti-NEDDl antibody (red), in multiple cell types. Anti-rootletin staining (green) is present after non-targeting siRNA negative control (nt) treatment. Arrows have been annotated manually to indicate centrosomes. Imaging conditions and brightness and contrast settings are consistent between control and siRNA treated samples. ( C ) Anti-rootletin antibody stains eGFP-rootletin over expressed from a cDNA transgene. Anti-rootletin staining is shown in red and GFP-rootletin fluorescence is shown in green. ( D ) Anti-rootletin bands are not detected by western blot of whole cell lysate after rootletin ( CROCC ) siRNA, demonstrating antibody specificity in multiple cell types.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Staining, Negative Control, Imaging, Control, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) Anti-rootletin staining was imaged systematically in different cell types by Airyscan imaging (green). Pericentriolar material (PCM) is costained with anti-NEDD1 (red). Staining and imaging conditions are the same throughout. Confocal slices are shown. Scale bar 1μm. ( B ) Pairwise co-staining of rootletin (green) and other centrosomal genes (red), which are either in the PCM or centrioles as indicated. Maximum intensity projections, scale bar 1 μm. ( C ) Quantification of the ratio of rootletin immunostaining area relative to GFP-Centrin1 area from maximum intensity projected Airyscan images. ( D ) Rootletin immunofluorescent staining is equal in unreplicated centrosomes and diplosomes. Centrosomes were classified based on GFP-Centrin1 foci number (either two or four) and anti-rootletin staining was segmented. Scale bar 1μm. The mean is shown as + and the median as a horizontal bar. n.s., t-test. N=21 cells. Note that roots are shown in red in this panel. ( E ) Cells were arrested in prometaphase with either STLC (Eg5 inhibition) or BI2536 (PLK1 kinase inhibition), before being forced into interphase by RO-3306 (CDK1 inhibition), without the completion of mitosis. ( F ) Cells expressing GFP-centrin1 (green) were treated as depicted in ( E ) before staining with anti-rootletin antibody (red). Rootletin staining was not detected on prometaphase arrested cells. Maximum intensity projections are shown. Scale bar 1μm. ( G ) Root area per cell was quantified by direct segmentation of rootletin staining from images obtained as described in ( F ). * p=0.0006, t-test.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Staining, Imaging, Immunostaining, Inhibition, Expressing

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) Representative maximum intensity z-projection Airyscan image of over-expressed eGFP-rootletin fibres (green), co-stained with the PCM marker PCNT (red). Scale bar 5μm. ( B ) FRAP of a single eGFP-rootletin fibre in the location denoted by the arrow. The graphs show the fluorescence intensity along a line profile at each timepoint, denoted by the dashed line in timepoint −20s. Scale bar 3μm.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) eGFP-rootletin fibres progressively assemble following transfection. The images are timepoints from a single cell, taken by live cell 3D confocal time-lapse imaging. The arrows point to a fusion event of two pre-existing fibres. Scale bar 3μm. See also Video S1 for the full timecourse. ( B ) Representative images from single cell three colour 3D confocal time-lapse imaging of rootletin-meGFP (green), NEDD1-mRuby3 (red; marking the PCM) and DNA (blue; marked by SiR-hoechst). Rootletin-meGFP (green) is visible at the centrosome during interphase but not during mitosis. Images were smoothed for display purposes here using a two-pixel median filter, but not for analysis. Scale bar 1 μm. See also Video S2 . ( C ) Cell cycle dependent changes in rootletin-meGFP centrosomal fluorescence intensity. Centrosomes were automatically tracked based on NEDD1-mRuby3 as described in methods, to obtain the intensity of rootletin-meGFP in individual cycling cells. Traces were manually aligned relative to anaphase onset based on SiR-hoechst staining of DNA (time 0) to create a plot of the mean +/− SD, N=17 cells. ( D ) An example of rootletin-meGFP (green) during centrosome separation in early mitosis. NEDD1-mRuby3 is shown in red as centrosomes move apart. Scale bar 2μm. ( E ) FRAP recovery curve over 15 hours, plotting the mean +− SD centrosomal intensity of rootletin-meGFP from 3D confocal imaging after fluorescence bleaching, in thymidine arrested cells. Centrosome position was efficiently tracked independently of rootletin-meGFP fluorescence intensity through the use of simultaneous NEDD1-mRuby3 imaging in a spectrally distinct channel. N=11 cells.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Transfection, Imaging, Fluorescence, Staining

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) Schematic of guide RNAs targeting the STOP codon of CROCC , and a donor plasmid containing meGFP and homology arms. ( B ) Clones were screened sequentially by FACS sorting, fluorescence microscopy and junction PCR. ( C ) Example overlapping PCR screen of clones expressing rootletin-meGFP. Clone 4_1 was used in this study since it hashomozygous tagging of rootletin. Clones 4_7 and 20 are examples of heterozygous and negative clones respectively. ( D ) Representative fluorescence microscopy screening of clones expressing endogenous rootletin-meGFP. The bottom panel shows centrosomal fluorescence in positive clones. Scale bar 5μm. ( E ) Rootletin-meGFP centrosomal fluorescent signal closely resembles anti-rootletin antibody staining. The image shows clone 4_1 stained with anti-rootletin antibody (red) and imaged by Airyscan imaging. Scale bar 1μm.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Plasmid Preparation, Clone Assay, Fluorescence, Microscopy, Expressing, Staining, Imaging

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) Quantification of centrosome cohesion in the interphase of various cell types through systematic immunofluorescent staining. The images show representative staining of PCNT (red; marking centrosomal PCM) and DNA (blue; hoechst 44432). Scale bar 5μm. The right panel shows representative segmentation of centrosomes (red), nuclei (blue) and cytoplasm (white) in Cal51 cells. The yellow asterisk denotes a cell containing two centrosome foci. The bar graph shows the mean % of cells with centrosomes separated by >1.5μm, from a minimum of 500 cells of each cell type. Error bars show SEM from two experiments. ( B - E ) Selected frames showing centriole splitting in live 3D confocal time-lapse imaging. Centrosomes are marked by either GFP-Centrin1 or NEDD1-mRuby3. Arrows denote centriole splitting events. The time intervals between frames are 12 minutes ( B, C ), 24 minutes ( D ) or 8 minutes ( E ). Scale bar 5μm. See also Videos S3-S5 . ( F ) Centrosome cohesion in HeLa cells -/+ overexpression of eGFP-rootletin, measured by automated imaging and analysis. Horizontal bars show the mean of two experiments +− SEM. * p<0.001 by Fischer’s exact test. More than 1000 cells were measured for each sample. ( G ) Opposing models of root behaviour during centriole splitting, termed “Stable contact” or “Disentangle”. ( H ) Representative image of root disentanglement after centriole splitting. Scale bar 1μm. ( I ) Root linkage plotted as a function of centriole spacing distance. ( J, K ) High resolution Airyscan time-lapse imaging of endogenous rootletin-meGFP and NEDD1-mRuby3 during a centriole split ( J ) and when remaining stably cohered ( K ) in Cal51 cells. Scale bar 2μm. See also Videos S6 and S7 .

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Staining, Imaging, Over Expression, Stable Transfection

Journal: bioRxiv

Article Title: Stable centrosomal roots disentangle to allow interphase centriole independence

doi: 10.1101/199166

Figure Lengend Snippet: ( A ) Root fibre area is significantly lower in split versus cohered centrioles (p<0.0001, t-test). Anti-rootletin immunofluorescent staining was Airyscan imaged and segmented, N=36 cells from two experiments. ( B ) Rootletin immunofluorescent staining (green) is the same at both mature centrioles (n.s., t-test). Centrioles were identified based on GFP-Centrin fluorescence (red), and then classified according to age, identifying the older centriole by CEP164 positivity (white). The arrow denotes a CEP164 positive centriole. N=21 cells per sample. Scale bar lμm. ( C ) PCNT immunofluorescent staining (of the PCM) is the same on either mature centriole (n.s., t-test). Cells were imaged and analysed as described in B , except segmenting PCNT staining. N=21 cells. ( D ) Cells with four mature centrioles might either maintain them as separate pairs or cohere them together. ( E ) The pie chart shows the proportion of each GFP-Centrin1 centriole configuration in cells with four centrioles, produced as depicted in - by sequential arrest in mitosis by STLC treatment, followed by induction into interphase with RO-3306. The images are representative of each configuration and the text denotes the configuration, e.g. 40 indicates all four centrioles cohered in one location. ( F ) Cells expressing rootletin-meGFP were stained with CellTrace Violet dye and then fused with cells stably expressing NEDD1-mRuby3. ( G ) Root arrangement in three different fused cells, produced as described in ( F ). Note that four NEDD1-mRuby3 foci are visible due to dynamic exchange of NEDD1 between the centrosome and cytosol. Scale bar lμm. ( H ) Interphase centriole pairs contain large bifurcating fibres which disentangle when centrioles move apart >1.5μm relative to each other. Root dissolution begins prior to mitotic centrosome separation and chromosome condensation. At the time of centrosome separation, roots are diminished in quantity and ripped apart during poleward movement of centrosomes. Roots form slowly over many hours from anaphase, as diffusionally stable fibres. PLK1 dependent modification of procentrioles allows root formation on mature centrioles in the ensuing cell cycle.

Article Snippet: Multiple lines of evidence indicate that a commercially available anti-rootletin antibody (Novus Biologicals NBP1-80820) specifically recognises the product of the CROCC gene. siRNA depletion of rootletin ( CROCC ) using RNA interference removed signal by both immunofluorescence and western blot in multiple cell types ( , S1B and S1D ).

Techniques: Staining, Fluorescence, Produced, Expressing, Stable Transfection, Dissolution, Modification