Investigating Uncertainties in Single-Molecule Localization Microscopy Using Experimentally Informed Monte Carlo Simulation

Single-molecule localization microscopy (SMLM) enables the visualization of cellular nanostructures in vitro with sub-20 nm resolution. While substructures can generally be imaged with SMLM, the structural understanding of the images remains elusive. To better understand the link between SMLM images and the underlying structure, we developed a Monte Carlo (MC) simulation based on experimental imaging parameters and geometric information to generate synthetic SMLM images. We chose the nuclear pore complex (NPC), a nanosized channel on the nuclear membrane which gates nucleo-cytoplasmic transport of biomolecules, as a test geometry for testing our MC model. Using the MC model to simulate SMLM images, we first optimized our clustering algorithm to separate >106 molecular localizations of fluorescently labeled NPC proteins into hundreds of individual NPCs in each cell. We then illustrated using our MC model to generate cellular substructures with different angles of labeling to inform our structural understanding through the SMLM images obtained

Additional file 1: of A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63

Includes the supplementary Figures S1–4. Figure S1. BAF is essential for epidermal gene induction. (a) Quantitative RT-PCR analysis demonstrating the knockdown efficiency of BRG1/BRM siRNAs and the suppression of differentiation gene expression in BAF loss compared to control. (b) GO analysis of the significant changed genes (fold change > 3, FDR < 0.01) with BAF knockdown in RNA-seq. (c) Western blot analysis showing the time course of 4-day differentiation induction in primary human keratinocytes, comparing BRG1/BRM loss with control. BRG1/BRM siRNA efficiently knocked down BRG1 and BRM protein levels. The induction of Keratin 1 is significantly impaired with BAF loss, although no significant changes were detected with p63 and p53 protein level relative to tubulin loading control. (d–f) Distribution of total ATAC-seq peaks, and the BAF-dependent ATAC-seq peaks relative to gene promoter, exon, intron and distal regulatory regions. Figure S2. BAF maintains open chromatin regions with p63 binding sites. (a) Scatter plot demonstrating correlation between BAF binding and open chromatin across the genome. (b) RNA expression levels (RPKM) of p53 family transcription factors in human keratinocytes. (c) Pie chart demonstrating the percentage of p63 motif sites at p63 binding sites that became inaccessible with BAF knockdown. (d) RNA expression levels of all expressed TFs in keratinocytes. Gray dots indicate the expression levels of 809 TFs (RPKM > 1 in differentiating human keratinocytes) listed in GO. Representative TFs known to be functional in epidermal differentiation along with CTCF are highlighted in brown and red. (e) ATAC-seq accessibility in KLF4 motif sites in KLF4 binding sites comparing control vs BAF loss conditions. (f) Heatmap showing the fold changes of the shared 236 genes (fold change > 3, FDR < 0.01) controlled by both BAF and p63. (g) Representative RNA-seq data tracks of BAFi, p63i, and CTRLi replicates. Figure S3. BAF loss does not affect nucleosome positioning or genome accessibility at CTCF binding regions. (a) ATAC-seq fragment size distribution. Gray shaded area represents nucleosome-free fragments (<100 bp), and blue shaded area represents mononucleosome fragments (180–247 bp). Schematic illustration of these ATAC-seq fragments is shown on the right. (b, c) V-plot analysis demonstrating the nucleosome positioning at CTCF motif regions comparing control versus BAF loss. (d) Average diagram of nucleosome-free ATAC-seq fragments at CTCF motif regions comparing control and BAF loss. (e) Average diagram of mononucleosome ATAC-seq fragments at CTCF motif regions comparing control and BAF loss. (f) Average diagram of predicted nucleosome binding probability based on DNA sequences using same number of shuffled genomic regions as in p63 motif nucleosome probability analysis. Figure S4. BAF loss impairs p63 binding to its target sites. (a, b) Single nucleotide ATAC accessibility analysis with 1-bp resolution at p63 and CTCF motif regions in their ChIP-seq binding sites, comparing control and BAF loss. (c) Summit-centered heatmap comparing p63 ChIP-seq peaks in control and BAF loss. (d, e) Average diagram of p63 ChIP-seq signal enrichment in the peaks that are overlapped or unique in control. (f) Average diagram of BAF ChIP-seq signal at CTCF sites comparing p63 loss with control conditions. (PDF 3349 kb

Additional file 2: Table S1. of A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63

Genes changed >3-fold with BRG1/BRM knockdown. Table S2 Shared genes controlled by both BAF and p63 with fold change > 3. Table S3 Sequencing depth of the ATAC-seq, RNA-seq, and ChIP-seq data generated for this study. (XLSX 78 kb

14-3-3 antibody labeling of salivary gland chromosomes and nuclei.

<p>(A–B) 14-3-3 antibody labeling of male polytene squash preparations before and after heat shock treatment. (A) Wild-type squash preparations labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type squash preparations after heat shock treatment labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red), Pol IIo^ser5 antibody (in green), and Hoechst (DNA, in blue/gray). No or little specific labeling above background of either 14-3-3 antibody was discernable. (C–E) 14-3-3 localizes to the nuclear matrix surrounding the chromosomes. (C–D) Confocal sections of whole-mount salivary gland nuclei labeled with 14-3-3 (SZ) or 14-3-3 (CS) antibody (in red) and Hoechst (DNA in blue). (E) Confocal section of a live salivary gland nuclei from a 14-3-3ε-GFP (in green) enhancer trap line co-expressing histone H2Av-RFP (in red).</p

Tethering of LacI-JIL-1 is not associated with upregulation of either H3S28ph or 14-3-3 at the <i>LacO</i> insertion site.

<p>(A–C) Triple labelings with LacI antibody (in green), H3S10ph antibody (A) or H3S28ph antibody (UP) (B) or 14-3-3 (SZ) antibody (C) (in red), and Hoechst (DNA in blue/gray) of polytene squash preparations from larvae homozygous for the <i>lacO</i> repeat line P11.3. There is robust labeling by the H3S10ph antibody at the insertion site; however, there was no discernable signal above background levels when the preparations were labeld with either H3S28ph or 14-3-3 antibody.</p

Polytene chromosomes from wild-type and <i>JIL-1</i> null salivary glands labeled with two different H3S28ph antibodies after heat shock treatment.

<p>(A) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (CS) antibody (in red), Pol IIo^ser5 antibody (in green), and Hoechst (DNA, in blue/gray). There was no obvious labeling by the antibody above background of the heat shock puffs although they were robustly labeled by the Pol IIo^ser5 antibody. (B) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (UP) antibody (in red), Pol IIo^ser5 antibody (in green), and Hoechst (DNA, in blue/gray). This antibody weakly labeled heat shock puffs above background levels; however, such labeling was also observed in the <i>JIL-1</i> null mutant background.</p

Histone H3S10ph and H3S28ph antibody labelings of male salivary gland nuclei smush preparations.

<p>(A) Double labeling with H3S10ph antibody (in red) and JIL-1 antibody (in green) demonstrating co-localization and the characteristic upregulation of JIL-1 and H3S10ph labeling on the male X chromosome (X). (B) Double labeling with H3S28ph (CS) antibody (in red) and JIL-1 antibody (in green). (C) Double labeling with H3S28ph (UP) antibody (in red) and JIL-1 antibody (in green). In contrast to the labeling of the H3S10ph antibody there was no discernable labeling above background of the autosomes or the male X chromosome by either of the two H3S28ph antibodies.</p

Immunoblot characterization of two different H3S28ph antibodies.

<p>(A–D) Immunoblots of protein extracts from salivary glands (SG) or the CNS from wild-type (wt) or <i>JIL-1^z2/JIL-1^z2</i> (<i>z2/z2</i>) larvae labeled with H3S28ph (UP) antibody (A), H3S28ph (CS) antibody (B), and H3S10ph antibody (C). Labeling with histone H3 (H3) antibody was used as a loading control (D) and as a marker for the relative migration of histone H3 (lane 6). The relative migration of molecular size markers is indicated in kD.</p

Polytene squash preparations from male wild-type and <b><i>JIL-1</i></b><b> null salivary glands labeled with H3S28ph antibodies.</b>

<p>(A) Wild-type and homozygous <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (UP) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (B) Wild-type and <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) squash preparations labeled with H3S28ph (CS) antibody (in red), JIL-1 antibody (in green), and Hoechst (DNA, in blue/gray). (C) Polytene squash preparation from a <i>JIL-1^z2/JIL-1^z2</i> null (<i>z2/z2</i>) salivary gland expressing a CFP-tagged JIL-1-CTD construct (JIL-1-CTD-CFP) labeled with H3S28ph (CS) antibody (in red); GFP/CFP antibody (in green), and Hoechst (DNA, in blue/gray). The male X chromosome is indicated by an X and the nucleolus by an n. Examples of interband labeling by the H3S28ph (CS) antibody are indicated by arrowheads in (B) and (C).</p