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

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells under basal conditions (solvent) and 0, 5, 10, 30 or 60 minutes after cytochalasin D treatment (0.5 µmol/L). Similar results were obtained in 2 additional cell batches. Scale bar: 50 µm . B, Representative confocal images and quantification of F-actin in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL) treated with solvent or cytochalasin D (0.5 µmol/L, 20 minutes) for 0, 30 and 180 minutes. The quantification shows the extent of endothelial monolayer disruption measured as counts of low intensity pixels in F-actin immunofluorescence images. n=5 independent cell batches (two-way ANOVA and Holm-Šídák’s multiple comparisons test). Scale bar: 50 µm. C, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and Arp3. Scale bar: 10 µm. Similar results were obtained in 2 additional cell batches. D, Western blot analysis and quantification of Actin-related protein 2 (Arp2), Actin-related protein 3 (Arp3), Vasodilator-stimulated phosphoprotein (VASP), WASP-family verprolin homologous protein 1 (WAVE1), Ras homolog family member A (RhoA) and Cortactin expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches (unpaired Student’s t-test). E, Representative confocal images and quantification of mean fluorescence intensity (MFI) per cell of DiI-LDL uptake in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL), treated with either control inhibitor CK689 or Arp2/3 inhibitor CK666. n=4 independent cell batches (two-way ANOVA and Holm-Šídák’s multiple comparisons test). Scale bar: 50 µm.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: AP2A1 (1:1000, Novus Biologics, Centennial, United States, NB600-1545), AP2M1 (1:1000, Cell Signalling, Danver, United States, #68196), Arp2 (1:2000, Proteintech, Rosemont, United States, 10922-1-AP), Arp3 (1:1000, Abcam, Cambridge, United Kingdom, ab49671), β-actin (1:1000, Sigma-Aldrich, Burlington, United States, A1978), CAV1 (1:1000, Cell Signaling, #3238), CHC (1:2000, BD Transduction, Franklin Lakes, United States, 610499), Cortactin (1:10000, Abcam, ab81208), DAAM1 (1:2000, Proteintech, 67287-1-Ig), GAPDH (1:300, Millipore, Burlington, United States, MAB374), LDLR (1:2000, Invitrogen, Waltham, United States, PA5-46985), Phospho-AP2M1 (1:1000, Cell Signaling, #7399), RhoA (1:1000, Santa Cruz, Dallas, United States, sc-179) and VASP (1:1000, BD Transduction, V40020).

Techniques: Solvent, Disruption, Immunofluorescence, Proximity Ligation Assay, Western Blot, Expressing, Control, Fluorescence