arp2 3  (Bioss)

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

Journal: bioRxiv

Article Title: Biochemical Functions of the Membrane-Binding Domain of CARMIL

doi: 10.64898/2026.01.05.697744

Figure Lengend Snippet: Asymmetric tails of actin filament networks generated by incubating Ni-functionalized and fluorescent lipid-coated beads with His-VVCA (N-WASP), followed by 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 inhibited actin growth (row labeled 2000 nM).

Article Snippet: Porcine brain Arp2/3 complex from Cytoskeleton (Cat. No. RP01P, Denver, CO) was reconstituted per manufacturer instructions and used within one month.

Techniques: Generated, Labeling

Journal: bioRxiv

Article Title: Biochemical Functions of the Membrane-Binding Domain of CARMIL

doi: 10.64898/2026.01.05.697744

Figure Lengend Snippet: 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-minute time points) similar to His-CBR126 wt. B. Untethered CBR126 associates with the lipid beads via the MB domain and displays more robust asymmetric actin growth than tethered His-CBR126. MB mutants do not associate with the lipid beads and do not show the same effects on the actin network.

Article Snippet: Porcine brain Arp2/3 complex from Cytoskeleton (Cat. No. RP01P, Denver, CO) was reconstituted per manufacturer instructions and used within one month.

Techniques:

Journal: bioRxiv

Article Title: Biochemical Functions of the Membrane-Binding Domain of CARMIL

doi: 10.64898/2026.01.05.697744

Figure Lengend Snippet: 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.

Article Snippet: Porcine brain Arp2/3 complex from Cytoskeleton (Cat. No. RP01P, Denver, CO) was reconstituted per manufacturer instructions and used within one month.

Techniques: Binding Assay