Journal: bioRxiv

Article Title: Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

doi: 10.64898/2026.07.31.742066

Figure Lengend Snippet: The structural basis for AnkA-ABM peptide binding to actin and the inhibition of actin polymerisation by AnkA. A. Cartoon representation of the AnkA-ABM:actin complex. The sub-domains of actin are numbered,1-4. AnkA-ABM binds within the cleft formed between sub-domains 1 and 3 at the barbed end of actin. B. Surface representation of the AnkA-ABM:actin complex demonstrating shape complementarity. C. Comparisons between AnkA-ABM:actin and the published structures of Wave2:actin (PDB:2A40; ) and VASP:actin (PDB: 2PBD, ). D. Alignment of selected WH2 motifs with AnkA-ABM sequences. The defining WH2 residues are highlighted green for hydrophobic, blue for basic, and yellow for small. E. Actin-pyrene was used in actin polymerisation assays in which actin polymerisation was initiated by the addition of KCl, Mg 2+ , and ATP. The addition of AnkA-CTD protein caused a decrease in the rate of actin polymerisation. F. Actin pyrene assays including Arp2/3 and the Wave2-VCA protein. The addition of Arp2/3 plus Wave2-VCA initiated rapid actin polymerisation which was inhibited by the inclusion of AnkA-CTD protein. Results are representative of two independent experiments.

Article Snippet: Pyrene-labelled actin and pure Arp2/3 complex were purchased (Cytoskeleton).

Techniques: Binding Assay, Inhibition

Journal: Communications Biology

Article Title: CDK1-mediated phosphorylation of EPLIN is crucial for mitotic spindle positioning

doi: 10.1038/s42003-026-10416-4

Figure Lengend Snippet: A , B The in vitro actin polymerization assays were carried out with pyrene-label actin in the presence or absence of purified His-EPLIN proteins (30 nM), as described in Materials and Methods. The elution buffer for His-tagged protein purification was used as a control. The fluorescence intensity of the reaction was monitored at 1-min intervals for 70 min using a spectrofluorometer ( A ). A.U., arbitrary units. The relative maximum polymerization slope was calculated ( B ). C , D The in vitro Arp2/3-mediated actin polymerization assays were carried out with pyrene-label actin in the presence of the Arp2/3 complex and the WASP-VCA domain, as described in Materials and Methods. His-EPLIN proteins (30 nM) were added to the assay to evaluate their effect on the Arp2/3-mediated actin polymerization. The elution buffer for His-tagged protein purification was used as a control. The fluorescence intensity of the reaction was monitored at 1-min intervals for 70 min using a spectrofluorometer ( C ). A.U., arbitrary units. The relative maximum polymerization slope was calculated ( D ). ( E ) The products of the Arp2/3-mediated actin polymerization assays as described in ( C ) were stained with phalloidin (red) and anti-EPLIN antibody (green), and then visualized under an epifluorescence microscope. Note that His-EPLIN S203D reorganizes short branched actin filaments into astral actin structures (arrowhead), whereas His-EPLIN S203A mainly induces long filamentous F-actin (arrow). The areas selected by the white frame are enlarged. The further enlarged spokes (from the dashed white frame) show that the S203D is associated with the F-actin spokes at ~0.48-μm intervals. Scale bars: 10 μm (overviews), 1 μm (zoom-ins) and 0.5 μm (enlarged spokes). The green fluorescence intensity in the images of the control “-“ was enhanced to visualize F-actin; the red fluorescence intensity in the images of “EPLIN S203A” was enhanced to visualize spokes. F – I Images were quantified from ( E ) to determine the number of asters (in ( F ), n ≥ 13 random images), the number of spokes of an aster (in ( G ), n ≥ 15 asters), the length ( H ) and width ( I ) of the spoke ( n ≥ 138 spokes, from n ≥ 15 asters). Data information: In ( B , D), values (means ± SD) are from three independent experiments. Values in ( F , G , H and I ) are presented as the box-whisker plot. Statistical significance of differences is assessed with Student’s t -test.

Article Snippet: General Actin Buffer (G Buffer, BSA01-010), pyrene-labeled muscle actin (pyrene-actin, AP05), rabbit muscle actin protein (AKL95), Arp2/3 protein complex (RP01P), and WASP-VCA Domain-GST protein (VCG03) were purchased from Cytoskeleton.

Techniques: In Vitro, Purification, Protein Purification, Control, Fluorescence, Staining, Microscopy, Whisker Assay

Journal: Communications Biology

Article Title: CDK1-mediated phosphorylation of EPLIN is crucial for mitotic spindle positioning

doi: 10.1038/s42003-026-10416-4

Figure Lengend Snippet: A The in vitro actin polymerization assay was carried out in the presence of His-EPLIN S203A or S203D for 70 min. The reaction products were centrifuged at 8000 × g for 20 min. The supernatant (S) and pellet (P) were fractionated by SDS-PAGE and stained with Coomassie blue to visualize actin or analyzed by immunoblotting with anti-His antibody. The actin-bundling activity was analyzed by measuring the extent of actin in the pellet fraction relative to the control (i.e., the reaction without His-EPLIN). B The in vitro Arp2/3-mediated actin polymerization assays were carried out in the presence of His-EPLIN S203A or S203D for 20 min. The products of the reaction were subjected to centrifugation at 8000 × g for 20 min, and then the pellets were analyzed by immunoblotting with the indicated antibodies. The relative level of co-sedimentation of actin and His-EPLIN (S203A vs S203D) was measured. Values in ( A , B ) (means ± SD) are from three independent experiments. Statistical significance of differences is assessed with Student’s t -test.

Article Snippet: General Actin Buffer (G Buffer, BSA01-010), pyrene-labeled muscle actin (pyrene-actin, AP05), rabbit muscle actin protein (AKL95), Arp2/3 protein complex (RP01P), and WASP-VCA Domain-GST protein (VCG03) were purchased from Cytoskeleton.

Techniques: In Vitro, Polymerization Assay, SDS Page, Staining, Western Blot, Activity Assay, Control, Centrifugation, Sedimentation

Journal: Communications Biology

Article Title: CDK1-mediated phosphorylation of EPLIN is crucial for mitotic spindle positioning

doi: 10.1038/s42003-026-10416-4

Figure Lengend Snippet: A Purified His-EPLIN S203A or S203D proteins (60 nM) were used in the in vitro pyrene-actin polymerization assay in the presence of Arp2/3 complex and WASP-VCA domain. After 20 min of reaction, the product was stained for F-actin (by phalloidin, green), EPLIN (red) and Arp3 (white). The images were taken by an epifluorescence microscope. The cores of astral structures were enlarged from the white squares. Scale bars, 10 μm. B , C The intensities of Arp3 and EPLIN at the astral core (a circle of 2.074 μm 2 ) were analyzed from images as described in ( A ). The levels of Arp3 at the cores of asters were shown in (B) (S203A, n = 21; S203D, n = 78). The intensities of EPLIN and Arp3 at the cores were analyzed in the correlation analysis by Excel, as shown in ( C ). D , E Control HeLa cells and those with EPLIN-depletion (sh-EPLIN) were synchronized in metaphase and then treated with (+) or without (-) the Arp2/3 inhibitor CK-666 (50 μM) for 2 h. The cells were fixed and stained for F-actin (red, by phalloidin), α-tubulin (white), and DNA (blue). Representative images acquired using epifluorescence microscopy are shown ( D ). Scale bars, 10 μm. The difference in the “pole to cortex” ( n ≥ 104 cells) was measured ( E ). Values (means ± SD) are from three independent experiments. Statistical significance of differences is assessed with Student’s t -test.

Article Snippet: General Actin Buffer (G Buffer, BSA01-010), pyrene-labeled muscle actin (pyrene-actin, AP05), rabbit muscle actin protein (AKL95), Arp2/3 protein complex (RP01P), and WASP-VCA Domain-GST protein (VCG03) were purchased from Cytoskeleton.

Techniques: Purification, In Vitro, Polymerization Assay, Staining, Microscopy, Control, Epifluorescence Microscopy

Journal: Communications Biology

Article Title: CDK1-mediated phosphorylation of EPLIN is crucial for mitotic spindle positioning

doi: 10.1038/s42003-026-10416-4

Figure Lengend Snippet: Phosphorylation of EPLIN at Ser203 by CDK1 promotes its capability to reorganize branched actin filaments into astral structures by bundling F-actin and interacting with the Arp2/3 complex. Such actin asters may sequester F-actin bundles from the turnover of actin filaments and thereby shut down the supply of G-actin for de novo actin polymerization. In the meantime, the sequestration of the Arp2/3 complex in the actin asters prevents excessive Arp2/3-mediated actin polymerization in mitotic cells. In the absence of EPLIN, no such actin asters are formed, which unleashes the Arp2/3 complex to promote branched F-actin networks, and causes abnormal cortical actin accumulation and aberrant cortical contractility, leading to cortical blebs and spindle deviation.

Article Snippet: General Actin Buffer (G Buffer, BSA01-010), pyrene-labeled muscle actin (pyrene-actin, AP05), rabbit muscle actin protein (AKL95), Arp2/3 protein complex (RP01P), and WASP-VCA Domain-GST protein (VCG03) were purchased from Cytoskeleton.

Techniques: Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: CARMIL membrane-binding domain regulates capping protein and actin assembly

doi: 10.1016/j.jbc.2026.111484

Figure Lengend Snippet: Actin filament network assembly on lipid-coated beads with CP, V-1, and CBR126. Asymmetric tails of actin filament networks generated by incubating Ni-functionalized and fluorescent lipid-coated beads with His-VVCA (N-WASP), followed by the addition of 100 nM Arp2/3 complex, 5 μM profilin-actin, and 50 nM CP for 30 min ( top row ). Addition of 500 nM V-1 to the reaction mixture resulted in F-actin growing from the bead surface as a symmetric ring and a diffuse cloud around the bead ( second row ). Addition of low concentrations of His-CBR126 resulted in asymmetric F-actin tail growth from the bead (rows labeled 35 nM and 50 nM), and higher concentrations of CBR126 inhibited actin growth (row labeled 2000 nM). CP, capping protein; CBR, CP-binding region.

Article Snippet: Porcine brain Arp2/3 complex from cytoskeleton (Cat. No. RP01P) was reconstituted per manufacturer instructions and used within 1 month.

Techniques: Generated, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: CARMIL membrane-binding domain regulates capping protein and actin assembly

doi: 10.1016/j.jbc.2026.111484

Figure Lengend Snippet: Membrane-binding domain (MB) effects on CP activity. A , His-tagged MB mutants cause actin network to grow asymmetrically from the bead surface in a mixture of 100 nM Arp2/3 complex, 5 μM profilin-actin, 50 nM CP, and 500 nM V-1 (30-min time points) but to a lesser extent than His-CBR126 wt. B - E , three data sets are plotted as different colors and shapes . The data analyzed are from experiments with an optimal concentration of His-CBR, one that produced the highest numbers of beads with asymmetric actin growth for each data set. The horizontal black bar is the median, and p values are calculated from a Mann-Whitney nonparametric analysis. The following parameters were measured and plotted: B , the area of brightest fluorescence near the bead surface; C , circularity of the region of brightest fluorescence near the bead surface; D , the total area of fluorescence, including the diffuse cloud surrounding the beads, and E , the total fluorescence of the actin network. CP, capping protein; CBR, CP-binding region.

Article Snippet: Porcine brain Arp2/3 complex from cytoskeleton (Cat. No. RP01P) was reconstituted per manufacturer instructions and used within 1 month.

Techniques: Membrane, Binding Assay, Activity Assay, Concentration Assay, Produced, MANN-WHITNEY, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: CARMIL membrane-binding domain regulates capping protein and actin assembly

doi: 10.1016/j.jbc.2026.111484

Figure Lengend Snippet: Model of regulatory cycles for CP actin capping. 1 , CP bound to V-1 in the cytoplasm is inactive. 2 , CP/V-1 binding to CARMIL promotes V-1 dissociation. 3 , Free CP binds barbed ends and promotes Arp2/3-nucleated polarized actin growth at the bead surface. 4 & 5 , Near the bead surface, CARMIL can a) promote uncapping of a capped barbed end to allow filament growth or b) capture a capped actin filament. Dynamic association of CP with barbed end - “loose/leaky” capper. 6 , Dynamic association of CARMIL with lipid: CARMIL can leave the bead surface and stay bound to CP as the actin filament network grows and flows away from the bead surface. CP, capping protein.

Article Snippet: Porcine brain Arp2/3 complex from cytoskeleton (Cat. No. RP01P) was reconstituted per manufacturer instructions and used within 1 month.

Techniques: Binding Assay

Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Extracellular Vesicles Carrying APOE Downregulate Filament Actin Polymerization Signaling to Inhibit Synapse Formation in Alzheimer's Disease

doi: 10.1002/jev2.70248

Figure Lengend Snippet: APOE in APPNEVs impairs synapses by downregulating F‐actin polymerization signaling. (A and B) DIV14 primary neurons are treated for 48 h with DMSO (Vehicle), 10 µM Rac1 inhibitor NSC23766, 5 µM N‐WASP inhibitor Wiskostatin, 30 µM Arp2/3 inhibitor CK666, or 10 µg/mL APPNEVs. Synaptic integrity is assessed by measuring PSD95 protein expression through western blot analysis (A) and immunofluorescence staining (B). (C and D) To further investigate the role of APOE in APPNEVs on actin cytoskeleton regulation, DIV14 primary neurons are treated for 48 h with DMSO (Vehicle), 1 µM EZ‐482, 10 µg/mL APPNEVs, or a combination of 10 µg/mL APPNEVs and 1 µM EZ‐482 (pre‐incubated for 1 h). (C) Rac1 activation is evaluated using a pull‐down assay to isolate Rac1‐GTP, followed by western blot analysis to quantify the levels of active GTP‐bound Rac1 and total Rac1. (D) To assess Arp2/3 complex activation, neurons are immunostained for phosphorylated Arp2 (p‐Arp2). The cytoskeletal structure is visualized using phalloidin staining, while nuclei are stained with DAPI. Phosphorylation levels and cytoskeletal organization are analyzed using confocal microscopy. (E) Neurons are treated for 6 h with 10 µg/mL APPNEVs, with or without 25 nM Rac1 activator ML‐099, and p‐Arp2 levels are detected by western blot. (F) Neurons are incubated with APPNEVs for 48 h, followed by treatment with or without 20 nM Jasplakinolide for the final 20 min. F‐actin in neurons is visualized using phalloidin staining and z‐stack confocal imaging, and the total number of dendritic spines is quantified. Data are presented as mean ± SEM from n = 3–5 independent experiments per condition. Statistical comparisons are performed using one‐way ANOVA followed by Tukey's post‐hoc test, with statistical significance indicates as * p < 0.05 and ** p < 0.01. “ns” indicates no significant change.

Article Snippet: Neurons were cultured for 14 DIV (days in vitro) and treated with 10 μg/mL APPNEVs or WTNEVs, 1 μM of the APOE inhibitor EZ‐482 (HY‐103706, MCE), 10 μM of the Rac1 inhibitor NSC23766 (HY‐15723A, MCE), 5 μM of the N‐WASP inhibitor Wiskostatin (HY‐12534, MCE), 30 μM of the Arp2/3 inhibitor CK666 (HY‐16926, MCE), 25 nM Rac1 activator ML‐099 (HY‐124306; MedChemExpress), or vehicle control.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Incubation, Activation Assay, Pull Down Assay, Phospho-proteomics, Confocal Microscopy, Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Extracellular Vesicles Carrying APOE Downregulate Filament Actin Polymerization Signaling to Inhibit Synapse Formation in Alzheimer's Disease

doi: 10.1002/jev2.70248

Figure Lengend Snippet: Schematic model of APPNEVs carrying APOE downregulate F‐actin polymerization signaling to inhibit synapse formation in AD. During AD progression, EVs derived from APP/PS1 neurons transport APOE into healthy neurons, potentially interacting with neuronal APOE receptors (LRP1, LDLR, VLDLR) to transduction the signaling. This signaling inhibits Rac1‐GTP activation and subsequently downregulates F‐actin polymerization through the Rac1–N‐WASP–Arp2/3 pathway. Disruption of this pathway impairs mature synapse formation, ultimately converting healthy neurons into synaptically damaged neurons and exacerbating AD progression.

Article Snippet: Neurons were cultured for 14 DIV (days in vitro) and treated with 10 μg/mL APPNEVs or WTNEVs, 1 μM of the APOE inhibitor EZ‐482 (HY‐103706, MCE), 10 μM of the Rac1 inhibitor NSC23766 (HY‐15723A, MCE), 5 μM of the N‐WASP inhibitor Wiskostatin (HY‐12534, MCE), 30 μM of the Arp2/3 inhibitor CK666 (HY‐16926, MCE), 25 nM Rac1 activator ML‐099 (HY‐124306; MedChemExpress), or vehicle control.

Techniques: Derivative Assay, Transduction, Activation Assay, Disruption