Journal: bioRxiv

Article Title: MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

doi: 10.1101/2020.09.21.306522

Figure Lengend Snippet: A In vitro pyrene actin assays using 12.5 nM of the indicated recombinant Arp2/3 reveal Arp3 and Arp3B containing complexes are equally efficient at nucleating actin regardless of the ARPC1A/ARPC5 and ARPC1B/ARPC5L background. B In vitro TIRF microscopy images to visualize branched actin formation. The top row shows image stills taken from TIRF assays using 2.5 nM Arp2/3 complexes containing Arp3B, ARPC1A and ARPC5 at the indicated times (see Movie S1) Scale bar = 15 μm. The bottom row shows the same panels after automatic detection of filament branches (yellow nodes) and ends (red nodes) with AnaMorf ImageJ plugin. C Quantification of mean branch number and length. D Quantification of the halflife of photoactivated GFP PA -β-actin in actin tails in Arp3B RNAi treated cells. These data were collected at same time as the data in . E Quantification of the half-life of Arp3 and Arp3B in vaccinia actin tails after activation of the GFP PA tag. The data in D and E from 3 independent experiments were combined and error bars represent SEM. for the indicated number of tails. Tukey’s multiple comparisons test was used to determine statistical significances, where **** P <0.0001.

Article Snippet: The other antibodies used in this study are listed below: ARPC2/p34-Arc polyclonal (Millipore, 07-227, 1:1000 dilution), ARPC5 monoclonal (Synaptic Systems, 305011, clone 323H3, 1:1000 dilution), Arp2 monoclonal (Abcam, ab129018, clone EPR7979, 1:1000 dilution), ARPC5L monoclonal (Abcam, ab169763, clone EPR10274, 1:1000 dilution), ARPC1A polyclonal (Sigma, HPA004334, 1:250 dilution), Arp3 monoclonal (Sigma, A5979, clone FMS338, 1:1000 dilution), ARPC1B polyclonal (Bethyl Laboratories, A302-781, 1:1000 dilution), ARPC3 monoclonal (BD Transduction labs, 612234, clone 26/p21-Arc, 1:1000 dilution), ARPC4 polyclonal (Sigma, SAB1100901, 1:1000 dilution), β-actin monoclonal (Sigma, A5316, clone AC-74, 1:1000 dilution), Vinculin monoclonal (Sigma, V4505, 1:1000 dilution), coronin-1C polyclonal (Invitrogen, PA5-21775, 1:500 dilution), a-Actinin monoclonal (Sigma, A5044, 1:1000 dilution), GAPDH monoclonal (Santa Cruz, sc-32233, 1:1000 dilution), Cortactin monoclonal (Millipore, 05-180, clone 4F11, 1:1000 dilution), MICAL1 polyclonal (Proteintech, 14818-1-AP, 1:1000 dilution), GFP monoclonal (custom made by CRUK, 1:1000 dilution).

Techniques: In Vitro, Recombinant, Microscopy, Activation Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Integrated Mendelian Randomization and Single‐Cell Transcriptomics Analysis Identifies Critical Blood Biomarkers and Potential Mechanisms in Epilepsy

doi: 10.1111/cns.70172

Figure Lengend Snippet: Single‐cell transcriptomic analysis reveals cellular heterogeneity in the NC and TLE group. (A) UMAP visualization of the single‐cell RNA‐seq data from normal and TLE hippocampus samples, displaying diverse cell populations. The horizontal and vertical axes represent components after dimensionality reduction. Each point in the figure represents a cell, with cells in close proximity considered to be of the same type. Different cell groups are distinguished by different colors. (B) UMAP plots highlighting the distribution of 12 identified cell types in NC (four samples, left) and TLE groups (four samples, right). The horizontal and vertical axes represent components after dimensionality reduction. Each point in the figure represents a cell, with cells in close proximity considered to be of the same type. Different cell groups are distinguished by different colors. (C) Pie charts showing the proportion of different cell types in the NC (left) and TLE (right) groups, illustrating the changes in cellular composition. (D) Bar graph depicting the count or numbers of each cell type in NC and TLE groups. (E) Visualization of the expression of eight key genes ( Cdc25b , Dnmt1 , Fgd3 , Gzma , Mtx1 , Raf1 , Sh3bp5l , and Ssh2 ) across various cell types in the hippocampal tissue via a UMAP plot of gene expression. It is observed that, except for Fgd3 and Ssh2 which are specifically highly expressed in microglia, and Gzma in a small population of T cells, other genes do not exhibit cell type‐specific expression and are generally distributed across cell types. (F) Bubble chart of pathways significantly enriched in DEGs from single cell RNA sequence analysis of microglia comparing TLE and NC groups. Top 20 key pathways are listed on the y ‐axis, with the rich factor ( x ‐axis) indicating enrichment strength. Bubble size symbolize the count of genes involved, while color intensity reflects the significance of enrichment, with darker red indicating higher statistical significance. (G) KEGG pathway enrichment analysis while comparing microglia in TLE versus NC, for the target gene set ( Fgd3 and Ssh2 ). This KEGG pathway enrichment analysis delineates the regulation of the actin cytoskeleton, with a specific emphasis on the roles of GPCRs ( Bdkrb2 ), FGD1/3 ( Fgd3 ), IQGAP ( Iqgap1 ), Rac ( Rac2 ), PAK ( Pak2 ), SSH ( Ssh2 ), and the Arp2/3 ( Arpc1b ) complex. Arrow (→) indicates a promoting or activating effect. T‐shaped line (⊣) indicates an inhibitory or blocking effect. All entities with colored backgrounds in the diagram are part of the KEGG annotation results for genes/transcripts under TLE versus NC comparison. Yellow background indicates known genes/transcripts, while green background represents new genes/transcripts (none). Red borders denote upregulated genes, and blue borders indicate downregulated genes (none). Genes encircled in pink and blue ellipses are those for which protein expression validation was conducted subsequently (Figure E). Pink signifies that the corresponding gene's protein level in the TLE hippocampus is significantly upregulated, consistent with predictions, whereas blue indicates no significant upregulation was found. (H) Capillary‐based immunoblots showing the expression of key proteins upstream and downstream of Fgd3 and Ssh in the regulation of the actin cytoskeleton pathway. (I) Quantification of Western blot analysis of the protein bands, using relative chemiluminescence signal values compared across NC and TLE hippocampus samples. (* p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant). (TLE, temporal lobe epilepsy; NC, normal control.)

Article Snippet: For protein capillary electrophoresis, the primary antibodies used were IQGAP1 (#ab133490, 1:50, Abcam), BKRB2 (#YN2508; 1:50, Immunoway), PAK2 (#2608S, 1:50, CST), ARPC1B (#28368‐1‐AP, 1:50, Proteintech), and RAC2 (#10735‐1‐AP; 1:50, Proteintech).

Techniques: RNA Sequencing, Expressing, Gene Expression, Sequencing, Blocking Assay, Comparison, Biomarker Discovery, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Single-cell sequencing reveals homogeneity and heterogeneity of the cytopathological mechanisms in different etiology-induced AKI

doi: 10.1038/s41419-023-05830-z

Figure Lengend Snippet: A Pseudotime analysis of the differentiation trajectory of PTCs. PTCs gradually progressed to two cell fate as AKI progressed. B Cell number distribution of 7 PTC celltypes on the pseudotime trajectory. Maladaptive-PTCs, Proliferative PTCs and part of the Krt20 NewPT were mainly distributed on the trajectory of cellfate1, and the remaining NewPT and InjuredS3 were mainly distributed on the trajectory of cellfate2. InjuredS1 PTCs were mainly distributed on the bifurcation of cellfate1 and cellfate2, indicating that these cells may be the earliest corrupted PTC cell subpopulation. C BEAM heatmap analysis identified genes associated with cellfate1 and cellfate2 differentiation. A total of three clusters are identified. The genes in Cluster1 are related to cellfate2 and Ferroptosis pathway were activated in this cluster. The genes in Cluster2 are related to cellfate1 and Necroptosis pathway were activated in this cluster. The Cluster3 lies at the bifurcation of the Pseudotime trajectory, and the heatmap reveals that a large number of genes are progressively highly expressed toward the Cellfate1 trajectory. Notably, the regulation of the cytoskeleton is a widespread feature of the PTC injury process. D – F Expression of Top genes of Cluster1 ( D ), Cluster2 ( E ), and Cluster3 ( F ) in the BEAM heatmap. G GSEA analysis of differentially expressed proteins in the phosphoproteomics, and the most significantly enriched pathway in the KEGG was the regulation of actin cytoskeleton pathway, in which Arpc1b is also involved. H , I In a combined analysis of the UUO mouse kidney proteomics and single-cell sequencing data of AKI-PTCs, a protein was identified that was simultaneously highly expressed in transcriptional data, quantitative protein data, and phosphorylated protein data: Arpc1b, and its phosphorylation site was determined. J Co-staining of sections from Control, cisplatin (CP), folic acid (FA), sodium oxalate (SO), ischemia reperfusion injury (IRI), and unilateral ureteral obstruction (UUO) kidneys for Arpc1b (red) and Megalin (green). Scale bars = 100 μM. Quantitative analysis of protein immunofluorescence intensity in Supplemental Fig. S .

Article Snippet: Mouse TMSB4X (EIAM-TMSB4X-1, RayBiotech), ARPC1B (ABX502254, BIOZOL), and S100A6 (ELM-S100A6-1, RayBiotech) ELISA Kit were used for detection of their urine levels by ELISA, respectively, in accordance with manufacturers’ instructions.

Techniques: Expressing, Phospho-proteomics, Sequencing, Staining, Control, Immunofluorescence