Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A ) Pyrene-actin polymerization assay in the absence or presence of profilin, CapZ, and/or zyxin-VASP or vinculin-VASP condensates. Error bars represent the standard deviations of three independent experiments. ( B ) Schematic of the experimental setup for condensate-mediated actin polymerization on SLBs. ( C and D ) Representative images showing G-actin partitioning into zyxin-VASP (C) and vinculin-VASP (D) condensates. Scale bars, 10 μm. ( E ) Time-lapse image series showing actin polymerization by zyxin-VASP and vinculin-VASP condensates. Scale bars, 1 μm. ( F ) Representative images of actin filaments nucleating outside condensates; arrows indicate filament origin sites, and circles mark condensate positions. Scale bars, 1 μm. ( G ) Box plot quantifying the fraction of actin filaments originating from condensates compared to those originating outside condensates. Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range; P < 0.05 is reported. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Zyxin is visualized with zyxin-mCherry, and actin is visualized with 12.5% actin-ATTO488. Condensates are imaged by epifluorescence microscopy and actin filaments by TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Polymerization Assay, MANN-WHITNEY, Epifluorescence Microscopy, Microscopy

Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A ) Representative image of an actin network generated by zyxin-VASP condensates after 20 min, showing prominent actin bundles. Scale bar, 10 μm. ( B ) Representative image of an actin network generated by vinculin-VASP condensates after 20 min, showing predominantly single filaments. Scale bar, 10 μm. ( C ) Time-lapse image series showing zyxin-VASP–generated filaments forming a bundle upon filament-filament encounter; arrows mark the interaction point. Scale bar, 1 μm. ( D ) Time-lapse image series showing vinculin-VASP–generated filaments crossing over each other upon encounter; arrows mark the interaction point. Scale bar, 1 μm. ( E ) Box plot quantifying actin bundle line density (number of bundles intersecting a line). Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range; P < 0.05 is reported. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Actin is visualized with actin-ATTO488. Actin images are imaged using TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Generated, MANN-WHITNEY, Microscopy

Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A and B ) Time-lapse image series illustrating changes in condensate shape upon actin polymerization. Scale bars, 5 μm. ( C and D ) Histograms showing changes in condensate eccentricity as a function of actin polymerization. ( E ) Representative FRAP image series of zyxin-VASP and vinculin-VASP condensates. Scale bars, 1 μm. ( F ) Normalized FRAP recovery curves and extracted parameters [fast half-time of recovery ( t 1 / 2 _ fast ) and mobile fraction] for zyxin-VASP and vinculin-VASP condensates. Data are shown as the means ± standard deviation. Error bars represent the standard deviations of three independent experiments. ( G ) Representative image of an actin network generated by zyxin-VASP and subsequently contracted by NMMII in the presence of α-actinin after 60 min, showing clustered actin filaments and condensates. Scale bar, 10 μm. ( H ) Time-lapse image series showing actin network contraction and concomitant clustering of zyxin-VASP condensates in the presence of NMMII and α-actinin. Scale bar, 5 μm. ( I ) Box plot quantifying the number of zyxin-VASP condensates before and after actin network contraction in the presence of NMMII and α-actinin. Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Zyxin is visualized with zyxin-mCherry, vinculin is visualized with 10% vinculin-AF555, and actin is visualized with actin-ATTO488. Condensates are imaged using epifluorescence microscopy, and actin filaments are imaged using TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Standard Deviation, Generated, MANN-WHITNEY, Epifluorescence Microscopy, Microscopy

Journal: ACS Applied Bio Materials

Article Title: Hydrogel-Based Extracellular Vesicle Isolation Method from Various Biological Solutions

doi: 10.1021/acsabm.5c01838

Figure Lengend Snippet: Cellular uptake of isolated MSC-derived EVs isolated using the HAS method in different cell types. (A) Internalization of EVs in dermal papilla cells. EV membranes were labeled with ExoSparkler EX01 (green) and cell nuclei were stained with DAPI (blue). (B) Internalization of EVs in skin cells. EV proteins were labeled with ExoSparkler EX05 (red), and cell nuclei and cytoskeleton were stained with DAPI (blue) and phalloidin (green).

Article Snippet: Proteins within MSC-EVs were labeled with ExoSparkler EX05 (Dojindo Laboratories).

Techniques: Isolation, Derivative Assay, Labeling, Staining