Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A–B ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 (centriole and cilia marker) and the distal appendage marker CEP164 ( A ) or the subdistal appendage marker ODF2 ( B ). Percentages represent cells with indicated marker at the mother centriole for 3 N’s. ( C ) Selected tomographic slices of RPE1 D21, T21, and Q21 cells serum depleted for 24 hr showing microtubule triplets and distal appendages (top panel) and subdistal appendages (bottom panel). ( D ) 3D models of structures at the centrosome in electron tomograms. Top row shows mother centriole (yellow), daughter centriole (magenta), microtubule minus ends (cyan spheres), vesicles (red), and smooth tubular membranes (blue-green). Bottom row shows microtubules (green), vesicles (red), and smooth membranes (blue-green) surrounding the centriole pair. Tomograms and models shown are of cells prior to ciliary vesicle formation. Tables on right display quantitation of the number of small red vesicles and smooth tubular membrane surface area measurements for D21, T21, and Q21 cells. N=2 reconstructed cells per cell line. Scale bars are 500 nm. Movies of the complete volume and rotating models can be found in .

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Microscopy, Staining, Marker, Quantitation Assay, Membrane

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A, B ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the distal appendage marker CEP83 ( A ) or centrin and the subdistal appendage marker Ninein (NIN) ( B ). Percentages represent cells with indicated marker for 3 N’s.

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Microscopy, Staining, Marker

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A ) Representative structured illumination microscopy images from time course experiments of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 2, 4, and 24 hr. Cells were stained with GT335 to label centrioles and MYO5A. ( B–D ) Distribution of MYO5A intensities around the centrosome for 2 ( B ), 4 ( C ), and 24 ( D ) hr timepoints. All values were normalized to D21 at 0 µm. Inset in ( B ) shows MYO5A intensity distribution in D21 cells prior to normalization. Graphs show mean ± SD. N=3. ( E ) Quantitation of centrosomal MYO5A intensity in 0–1.2 µm region around the centrosome for control and siPCNT treated D21, T21, and Q21 cells. Cells were treated with siControl or siPCNT for 24 hr concurrent with serum depletion. All values were normalized to the D21 siControl average. Graph show mean ± SD. N=3. Mann-Whitney U test. ( F ) Quantitation of pericentrosomal MYO5A intensity in 1.2–5 µm region around centrosome for control and siPCNT treated D21, T21, and Q21 cells. Cells were treated with siControl or siPCNT for 24 hr concurrent with serum depletion. All values were normalized to the D21 siControl average. Elevated MYO5A levels are distinct from the reduction observed in the distribution analyses ( D ) and are likely the result of the unique conditions for each experiment. Graph show mean ± SD. N=3. Mann-Whitney U test. Figure 3—source data 1. Values for biological and technical replicates for graphs and .

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Microscopy, Staining, Quantitation Assay, Control, Serum Depletion, MANN-WHITNEY

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A ) Quantitation of whole cell MYO5A intensity for D21, T21, and Q21 cells. Graph shows mean ± SD. N=3. Mann-Whitney U test. ( B ) Western Blot probing for MYO5A (top panel) and India Ink stain for loading control (bottom panel) on D21, T21, and Q21 whole cell lysates. ( C ) Quantitation of MYO5A Western Blot band intensities normalized to D21. Graph shows mean ± SD. N=3. Mann-Whitney U test. ( D–F ) Distribution of centrosomal MYO5A intensities moving away from the centrosome for 2 ( D ), 4 ( E ), and 24 ( F ) h timepoints. Values were normalized to the D21 average. Note that the FIJI radial analysis plugin does not plot the intensity at the centroid, thus the first point on the graph is the average intensity within the first concentric circle from the centroid. Graph shows mean ± SD. N=3. ( G ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were either treated with control or PCNT siRNA during the 24 hr serum depletion. Cells were stained with GT335 and MYO5A. ( H ) Quantitation of PCNT intensities in a 5 µm radial circle around the centrosome in control and PCNT siRNA treated cells normalized to the D21 siControl average. Graph shows mean ± SD. N=3. Mann-Whitney U test. ( I, J ) Correlation analysis between PCNT and MYO5A levels in 5 μm radius for D21, T21, and Q21 cells 24 hr after serum depletion. N=3. Pearson r value is shown. ( K ) Representative confocal images of RPE1 D21, T21, and Q21 cells stably expressing GFP-EHD1 grown on coverslips and serum depleted for 24 hr. Cells were stained for PCNT and Actub. Percentages represent cells with indicated phenotype across 3 N’s. Figure 3—figure supplement 1—source data 1. Uncropped blots and protein gels related to .

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Quantitation Assay, MANN-WHITNEY, Western Blot, Staining, Control, Microscopy, Serum Depletion, Stable Transfection, Expressing

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the centriole capping protein CP110. Percentages represent cells with indicated phenotype across 3 N’s. ( B ) Quantitation of CP110 centriole capping for control and siPCNT treated D21, T21, and Q21 cells. Graph show mean ± SD. N=3. Two-tailed unpaired t-test. ( C ) Selected tomographic slices and 3D models showing ciliary vesicle formation in D21, T21, and Q21 cells. T21 and Q21 have smaller ciliary vesicles (arrows) and the ciliary vesicle in Q21 cell is offset from the center of the mother centriole. Models show mother centriole (yellow), daughter centriole (magenta) ciliary vesicle (large red structure at the distal end of the mother centriole), smaller vesicles (red spheres), and smooth tubular membranes (blue-green). ( D ) Quantitation of membrane surface area at the centrosome in electron tomograms for D21, T21, and Q21 cells. D21 cells showed increased small vesicles (red spheres), smooth membranes (blue-green), and ciliary vesicle membrane (red structures) compared to T21 and Q21 cells. Ciliary vesicles in Q21 cells were not quantified due to low frequency (n of 1) and unusual offset of that ciliary vesicle. N=2 reconstructed cells per cell line. Scale bars are 200 nm. Movies of the complete volume and rotating models can be found in . Figure 4—source data 1. Values for biological and technical replicates for graphs in and .

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Microscopy, Staining, Quantitation Assay, Control, Two Tailed Test, Membrane

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the centriole capping protein CEP97. Percentages represent cells with indicated phenotype across 3 N’s. ( B ) Quantitation of CP110 centriole capping for D21, T21, and Q21 cells 24 hr after serum depletion. Graph shows mean ± SD. N=3. Two-tailed paired t-test. ( C ) Quantitation of CEP97 centriole capping for D21, T21, and Q21 cells. Graph shows mean ± SD. N=3. Two-tailed paired t-test. ( D ) Quantitation of PCNT intensities in a 5 µm radial circle around the centrosome in control and PCNT siRNA treated cells normalized to the D21 average. Graph shows mean ± SD. N=3. Mann-Whitney U test. ( E ) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 2, 4, 8, 24, and 48 hr. Cells were stained with γ-tubulin, Actub, and RAB8. Percentages represent cells with indicated phenotype across 3 N’s. ( F ) Quantitation of cells with RAB8 ciliary vesicle throughout the time course. Graph shows mean ± SD. N=3. ( G ) Representative confocal images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with Hoechst 33342, PCNT, Golgin97, and RAB8. ( H ) Quantitation of RAB8 Golgi intensity normalized to the D21 average. Graph shows mean ± SD. N=3. Mann-Whitney U test.

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Microscopy, Staining, Quantitation Assay, Serum Depletion, Two Tailed Test, Control, MANN-WHITNEY

Journal: eLife

Article Title: Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development

doi: 10.7554/eLife.78202

Figure Lengend Snippet: ( A ) Representative confocal images of RPE1 D21 and T21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335, PCNT, and the transition zone protein CEP290. Yellow arrows point to the CEP290 transition zone population and cyan arrows point to CEP290 satellites that colocalize with PCNT. ( B ) Quantitation of CEP290 transition zone intensity. Graph show mean ± SD. N=3. Mann-Whitney U test. ( C ) Quantitation of pericentrosomal CEP290 intensity in the 1.2–5 µm region around the centrosome. Graph show mean ± SD. N=3. Mann-Whitney U test. ( D, F, H ) Representative structured illumination microscopy images of RPE1 D21 and T21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the transition zone proteins RPGRIP1L ( D ), NPHP4 ( F ), or TMEM67 ( H ). ( E, G, I ) Quantitation of indicated transition zone protein intensities from confocal images. All values were normalized to the D21 average. Mean intensity values are indicated on graphs. Graphs show mean ± SD. N=3. Mann-Whitney U test. ( J ) 3D models from D21 and T21 cells that contained a primary cilium. An increase in vesicles (red spheres) and tubular membranes (blue-green) at the distal end of the mother centriole is evident in the D21 cell. A procentriole was observed at the T21 daughter centriole (violet). Scale bars are 200 nm. Movies of the complete volume and rotating models can be found in . ( K ) Quantitation of total membrane surface area at the centrosome in electron tomograms for D21 and T21 cells. N=1 reconstructed cell per cell line. Figure 5—source data 1. Values for biological and technical replicates for graphs in and .

Article Snippet: Super resolution imaging was performed on a Nikon 3D structured illumination microscopy system (Ti 2 Eclipse) with a 100 x TIRF objective (NA 1.45).

Techniques: Staining, Quantitation Assay, MANN-WHITNEY, Microscopy, Membrane