Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: (A) Schematic of experimental set-up. Chromatin was in vitro reconstituted and imaged by HS-AFM in fluid (0.67M NaCl + 2 mM MgCl 2 ) and individual nucleosomes were tracked and analyzed. CENP-A nucleosomes were tracked in fluid for ∼120 seconds by HS-AFM at 0.5 Hz alone (B) or in the presence of CENP-C CD (C). Representative images from the movie are shown (B, C), as well as the individual nucleosome trajectories (B, C). (D) The average mean square displacement is shown with standard error as a function of the time interval. (E) The diffusion constants obtained from the MSD curves. (F) The average single frame step-size. (G) The distributions of the average single frame step-sizes for each individual tracked nucleosome (grey points). Red dots represent the overall mean (one-way ANOVA; significance was determined at p <0.05).

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques: In Vitro, Diffusion-based Assay

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: Representative images of three HS-AFM movies and their respective traces for CENP-A alone or CENP-A + CENP-C CD .

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques:

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: A) Individual traces for CENP-A and CENP-A + CENP-C CD nucleosomes as summarized in in the main text. (B) and (C) Single step-width was computed for CENP-A alone and CENP-A + CENP-C CD (red dots) and fitted to the predicted step-width from the diffusion constant measured through MSD (blue line).

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques: Diffusion-based Assay

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: (A) Quantification of RNAP2 levels that pulled down with either CENP-C or sequential ACA nChIP. (B) Quantification of CENP-A levels that pulled down with either CENP-C or sequential ACA nChIP. (C) Quantification of consensus α-satellite transcription in mock-transfected (WT) and CENP-C overexpression (CENP-C OE ) (two-sided t-test; significance was determined at p <0.05).

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques: Transfection, Over Expression

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: Representative western blot for RNAP2 and CENP-A levels. HeLa cells were transfected with an empty vector (WT) or overexpressing CENP-C (CENP-C OE ) and synchronized to early G1 when centromeric transcription is at its highest, similar to what we reported earlier ( Melters et al 2019 PNAS). We probed for RNAP2, CENP-C, and CENP-A.

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques: Western Blot, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Colocalized immunofluorescent signals for CENP-A and TMR-Star are collected and the intensity of both foci is measured as well as background directly neighboring the foci to determine the ratio of the TMR-star signal over total CENP-A signal. (C) De novo CENP-A incorporation was assessed by quench pulse-chase immunofluorescence. After old CENP-A was quenched with TMR-block, newly loaded CENP-A was stained with TMR-Star and foci intensity was measured over total CENP-A foci intensity. Inset is a 2x magnification of the dotted box in each respective image. (D) Quantification of de novo CENP-A loading by measuring the ratio of TMR-Star signal over total CENP-A signal (one-way ANOVA; significance was determined at p <0.05).

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques: Pulse Chase, Immunofluorescence, Blocking Assay, Staining

Journal: bioRxiv

Article Title: Mobility of nucleosomes is regulated by their binding partners

doi: 10.1101/2022.01.04.474986

Figure Lengend Snippet: Under wildtype conditions, we propose that CENP-A chromatin not bound by CENP-C (yellow box) and the kinetochore is readily accessible to the transcriptional machinery, because of the intrinsic material properties of CENP-A nucleosomes. In contrast, when CENP-C binds CENP-A nucleosomes, it alters its material properties and quenches CENP-A’s mobility resulting in clustered CENP-A chromatin (darker red), restricting progression of the transcriptional machinery and subsequent loading of new CENP-A molecules. Similarly, H1.5 restricts H3 nucleosome mobility (darker blue), impacting RNAP2 transcriptional competency.

Article Snippet: In vitro reconstitution of CENP-A (CENP-A/H4 cat#16-010 and H2A/H2B cat#15-0311, EpiCypher, Research Triangle Park, NC) and H3 (H3/H4 cat#16-0008 and H2A/H2B cat#15-0311, EpiCypher Research Triangle Park, NC) nucleosomes were performed as previously described ( ; ).

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Summary of all cases used in this study. See supplementary data for subgroup analyses

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Primary antibody clones used in this study

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Clone Assay

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Primary motor cortex and spinal cord single-IHC results

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Pathology of the ALS primary motor cortex is variable both within and across the genotypic spectrum of disease. Relatively little pTDP-43 aggregation was found in a single TARDBP mutation case ( a ), but this was severe in an OPTN mutation case (d; see also supp. Figure ). Insets highlight variance of pTDP-43 morphology between genotypes. Average highest pTDP-43 deposition was seen in sporadic cases, which was statistically higher than in C9-ALS ( g ). Quantification of p62 ( b , e , h ). Levels of p62 correlated with pTDP-43 in sporadic cases but less so in C9ORF72 disease, reflective of the existence of p62-positive dipeptide repeat protein species unique to C9-ALS ( h and j ). Cortical microglial activation was highly variable between genotypes ( i ), and in some cases there was evidence of severe nodular neuronophagia surrounding layer V neurons ( f ). Grey matter CD68 correlated with the extent of pTDP-43 deposition ( k ) in both sporadic and C9ORF72 cases, but this relationship was not recapitulated using p62 and CD68 in SOD1 / FUS cases ( l ). Arrows highlight pathology, asterisks highlight Betz cells. r correlations = Pearson ( j ) or Spearman ( k , l ), results as on figure. Bars in ( i ) represent means and SEM. Best-fit lines are manually added for illustrative purposes. All scale bars = 50 μm

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Mutagenesis, Activation Assay

Journal: Acta Neuropathologica Communications

Article Title: Quantitative patterns of motor cortex proteinopathy across ALS genotypes

doi: 10.1186/s40478-020-00961-2

Figure Lengend Snippet: Spatial and morphological distribution of proteinopathy across genotypes in the ALS primary motor cortex. Sporadic ALS exhibits NCI and oligodendroglial pathology across layers I-VI as well as the subcortical white matter ( a - d ). The distribution pattern of pTDP-43 pathology was not entirely dissimilar between sporadic and C9-ALS ( e - f ), although there was a slight preponderance towards oligodendrocyte inclusions in C9 cases (Fig. g and h). FUS mutation cases demonstrated infrequent, compact p62-positive NCI with occasional granular inclusions, as well as occasional oligodendroglial pathology (i-l). SOD1 mutation cases, by contrast, exhibited granular p62 staining confined to the middle cortical layers with infrequent NCI in layer V, without obvious glial pathology. Arrows highlight respective proteinopathy. Scale bar applicable to all panels = 50 μm

Article Snippet: SOD1 d , StressMarq Bio , SPC-206 , Rabbit , Polyclonal , 1:1000 , –.

Techniques: Mutagenesis, Staining