Journal: The Journal of Cell Biology

Article Title: MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes

doi: 10.1083/jcb.202102043

Figure Lengend Snippet: Arp3- and Arp3B-containing complexes have the same actin nucleating activity. (A) In vitro pyrene actin assays using 12.5 nM of the indicated recombinant Arp2/3 complexes reveal Arp3 and Arp3B containing complexes nucleate actin equally efficient, regardless of the ARPC1A/ARPC5 and ARPC1B/ARPC5L background. The error bars represent SEM from n = 2 independent experiments. (B) The top row shows representative in vitro TIRF microscope images of branched actin formation using 2.5-nM Arp2/3 complexes containing Arp3B, ARPC1A, and ARPC5 at the indicated times (see ). n = 2 independent experiments. 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 filament length for the indicated Arp2/3 complexes from one of the two independent TIRF experiments. Each data point represents the average of 14 and 15 actin “structures” for Arp3B-C1B-C5L and Arp3-C1B-C5L, respectively, although there are fewer structures to analyze at the beginning of the experiment than at the end.

Article Snippet: The other antibodies used in this study were ARPC2/p34-Arc rabbit polyclonal (Millipore; 07–227, 1:1,000 dilution), ARPC5 mouse monoclonal (Synaptic Systems; 305011, clone 323H3, 1:1,000 dilution), Arp2 rabbit monoclonal (Abcam; ab129018, clone EPR7979, 1:1,000 dilution), ARPC5L rabbit monoclonal (Abcam; ab169763, clone EPR10274, 1:1,000 dilution), ARPC1A rabbit polyclonal (Sigma; HPA004334, 1:250 dilution), Arp3 mouse monoclonal (Sigma; A5979, clone FMS338, 1:1000 dilution), ARPC1B rabbit polyclonal (Bethyl Laboratories; A302-781, 1:1,000 dilution), ARPC3 mouse monoclonal (BD Transduction Laboratories; 612234, clone 26/p21-Arc, 1:1,000 dilution), ARPC4 rabbit polyclonal (Sigma; SAB1100901, 1:1,000 dilution), β-actin mouse monoclonal (Sigma; A5316, clone AC-74, 1:1,000 dilution), Vinculin mouse monoclonal (Sigma; V4505, 1:1000 dilution), coronin 1C rabbit polyclonal (Invitrogen; PA5-21775, 1:500 dilution), α-Actinin mouse monoclonal (Sigma; A5044, 1:1,000 dilution), GAPDH mouse monoclonal (Santa Cruz Biotechnology; sc-32233, 1:1,000 dilution), cortactin mouse monoclonal (Millipore; 05–180, clone 4F11, 1:1,000 dilution), MICAL1 rabbit polyclonal (Proteintech; 14818–1-AP, 1:1,000 dilution), GFP mouse monoclonal (custom made by Cancer Research UK; 1:1,000 dilution).

Techniques: Activity Assay, In Vitro, Recombinant, Microscopy

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