human fascin  (Novus Biologicals)

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Schematic showing the comparison between RNA expression in OV90 and mesothelial cells. ( B and C ) PCA plot of (B) OV90 and (C) HPMCs. ( D ) Volcano plot and clustering of RNA expression changes in OV90. The red line indicates an adjusted P value <0.05. ( E ) Volcano plot and clustering of RNA expression changes in HPMCs. The red line represents an adjusted P value <0.05. The right side of the volcano plot represents a fold change. ( F ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( G ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( H ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( I ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( J and K ) PROGENy pathway activity analysis of the ascites samples of the Zheng et al. EOC scRNA-seq dataset revealed high TGF-β pathway activity in both EOC and mesothelial cells. ( L ) Bar plot showing the concentration of TGF-β1 in the supernatant from HPMCs, TGF-β1–stimulated HPMCs, and OV90 cells. ( M ) Scheme of an invadopodium in a mesothelial cell. ( N ) Immunofluorescence images of a single cell invading the collagen layer using invadopodium formation. Green, cortactin; red, phalloidin. Scale bars, 10 μm. ( O ) The number of invadopodia was significantly higher in TGF-β1–stimulated mesothelial cells. ( P ) Strategy to detect candidates with a high invasion ability in mesothelial cells. ( Q ) Western blot analysis of fascin-1 and several proteins related to invadopodium formation. ( R ) Immunofluorescence images of fascin-1 or myosin X (green) in TGF-β1–stimulated mesothelial cells. Scale bars, 5 μm. FACS, fluorescence-activated cell sorting; FC, fold change. *** P < 0.001.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: Comparison, RNA Expression, Activity Assay, Concentration Assay, Immunofluorescence, Single Cell, Western Blot, Fluorescence, FACS

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A and B ) Violin plots showing the expression of invadopodium-related genes across cell components in ascites on the basis of two different scRNA-seq datasets from Izar et al. and Zheng et al. . In the dataset of (A), mesothelial cells are classified as fibroblasts. ( C ) Collagen degradation assay. The thickness represents the cell invasion ability. Scale bars, 400 μm. ( D ) Bar graph showing the thickness of remnant collagen 48 hours after incubation. ( E ) Bar graph showing the number of invadopodia. sh-Fascin-1 or sh-myosin X inhibited invadopodium maturation. ( F and G ) 3D images and bar graph showing that spheroids invade collagen with shRNA-induced mesothelial cells (green) and OV90 (red). The invasion ability of mesothelial cells was significantly inhibited by sh- FSCN1 or sh- MYO10 . Scale bars, 200 μm. ( H ) Scheme of the malignant ascites in vivo model using shRNA-treated HPMCs. ( I and J ) Images and bar graph showing the differences in the metastasis area on the omentum from mice 1 week after the injection of OV90 with or without sh-induced mesothelial cells. Scale bars, 1 mm. ( K ) Representative IHC image of mouse tissue with fascin-1. Invasive stromal cells strongly expressed fascin-1. Scale bar, 100 μm. ( L ) IHC of metastasis samples in clinical samples. Fascin-1–positive stromal cells were present in the tumor-invasive regions. Scale bar, 100 μm. ( M ) Kaplan-Meier plot showing the patient’s progression-free survival depending on fascin-1 expression in stromal cells or cancer cells. Fascin-1 expression in stromal cells in metastasis samples was significantly related to a worse prognosis ( P = 0.030). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: Expressing, Degradation Assay, Incubation, shRNA, In Vivo, Injection

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: Almost all the EOC cells identified in the ascites were in a spheroids formation and 65% were accompanied by mesothelial cells, referred to as ACMSs. The formation of ACMSs enabled EOC cells to alter the RNA expression profiles of mesothelial cells via TGF-β related pathway. These alternations increased the expression of fascin-1 in this pathway, which caused invadopodia formations in mesothelial cells to mature, and this degraded collagen with MMP14. Mesothelial cells interacted with EOC cells, which aggressively invaded the collagen and mesothelial layer. These results show that EOC cells can induce peritoneal metastasis without direct dynamic RNA expression changes. EOC cells then followed the route created by the mesothelial cells. This model explains that EOC cells control the unique tumor microenvironment in ascites to rapidly induce abdominal dissemination.

Article Snippet: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

Techniques: RNA Expression, Expressing, Control

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration of the GUV content and the two macromolecular reactions at membrane level: the MinDE self-assembly mechanism behind pattern formation and the diffusiophoresis-mediated transport of neutravidin-bound actomyosin bundles by Min proteins. The active flux of MinDE proteins on the vesicle membrane interacts non-specifically via frictional forces with membrane-bound neutravidin inducing the transport and positioning of these molecules, and consequently the actomyosin bundles linked to them, towards areas of low MinD density. b 3D projections of confocal images showing the 4 phenotypes of actin architectures obtained after encapsulating 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 10 µm. c Bar graphs with the frequencies of the four actomyosin phenotypes observed at different vesicle diameters when encapsulation experiments were performed at 0.25 and 0.5 fascin/actin molar (M/M) ratio in the presence and absence of Min proteins and protein/crowding conditions specified in b. Experiments performed per condition n = 3, total number of GUVs analysed per condition = 150. d 3D projections of time-lapse confocal images depicting the reorganization and stacking of actomyosin bundles towards the vesicle equator driven by the diffusiophoretic transport of Min pole-to-pole oscillations. Yellow arrows indicate the perpendicular orientation of MinDE oscillations with respect to actomyosin bundles, which get antagonistically positioned at mid-cell. Kymographs generated at the vesicle equator (blue dashed circle) are meant to visually define the position of fluorescent features at this region over time. Orange dotted lines depict the approximate distribution of actin bundles on the membrane at two time points. Vesicle content as specified in b. Scale bars: 10 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Encapsulation, Generated

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration behind the mechanism of membrane deformation. Contractile actomyosin bundles positioned by MinDE proteins at mid-cell induce furrow-like membrane invaginations. 3D projections and 2D confocal images show an actomyosin ring constricting the vesicle at its equator. Orange lines indicate the major (a) and minor (b) axes measured to calculate the aspect ratio of the deformed vesicle (for spherical vesicles: aspect ratio = 1). Inner solution mix: 4 µM actin, 2 µM fascin (fascin/actin molar ratio = 0.5), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10µm. b Schematic illustration, 3D projections and 2D confocal images of a vesicle containing a soft web of actomyosin bundles at the vesicle centre being positioned by pole-to-pole Min oscillations. The contractile actomyosin band formed causes the deformation of the vesicle (aspect ratio < 1). Inner solution mix: 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10µm. c Schematic illustration, 3D projection and 2D confocal image of a vesicle with a non-positioned contractile actomyosin assembly due to the loss in pole-to-pole MinDE oscillations. Constriction of the actomyosin bundles results in the deformation of the vesicle membrane into an asymmetric dumbbell shape. Scatter plot depicts the aspect ratio of the vesicle at different time points. Inner reaction mix: 4 µM actin, 2 µM fascin (fascin/actin molar ratio = 0.5), 0.05 µM myosin II, 20 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bar: 10 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration depicting the change in vesicle shape due to MinDE chaotic oscillations. Min proteins attach to areas delimited by soft actomyosin bundles and deform the membrane generating dynamic bleb-like protrusions. Fluorescence and brightfield confocal time-series show a blebbing vesicle. After bleb retraction, the reduction in bilayer tension generates an outward lipid bud (blue arrows). Encapsulation conditions: 2.4 µM actin, 0.6 µM fascin, 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 10 µm. b Confocal cross-section images at two time points of the vesicle in section a. Peripheral actomyosin anchoring creates a delimiting area which deforms upon MinDE binding. Additionally, MinDE diffusiophoretic transport changes the position of actomyosin bundles and the shape of the membrane area available for Min protein recruitment (blue arrows). Fluorescence intensity line plots of EGFP-MinD (green) and ATTO647-actin (magenta) demonstrate the demixing of both protein systems at the membrane perimeter (orange dotted line). Scale bars: 10 µm. c Schematic illustration of the proposed mechanism behind MinDE-induced blebbing. The recruitment of MinDE proteins to the compartmentalized inner leaflet of the bilayer generates the effect of a membrane outward protrusion in bleb form. d Schematic illustration depicting the radius of curvature R C used to calculate the curvature (Κ = 1/R C ) of the blebs. 3D projection and 2D time-lapse confocal images show a vesicle with diverse bleb-like deformations emerging over time. Orange arrow points at a bleb with Κ = 0.73 µm -1 . Blue arrow, Κ = 0.27 µm -1 . Magenta arrow, Κ = 0.10 µm -1 . Encapsulation mix: 4 µM actin, 2 µM fascin, 0.05 µM myosin II, 50 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 20 µm.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Fluorescence, Encapsulation, Binding Assay

Journal: bioRxiv

Article Title: Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

doi: 10.1101/2024.06.17.599291

Figure Lengend Snippet: a Schematic illustration (top) and 3D confocal image (bottom) show the membrane composition employed to generate phase-separated vesicles and the domains obtained. Scale bar: 10 µm. b 3D projections and 2D confocal images depict a blebbing phase-separated vesicle. MinDE proteins bind and oscillate on Ld domains. Actomyosin bundles remain at lipid-phase boundaries as Min proteins transiently deform Ld domains (orange arrows). Inner encapsulation mix: 2.4 µM actin, 0.6 µM fascin (fascin/actin molar ratio = 0.25), 0.05 µM myosin II, 20 g/L Ficoll70, 3 µM MinD, 3 µM MinE and 5 mM ATP. Scale bars: 20 µm. c Schematic illustration of the proposed mechanism behind the dynamic deformation of Ld domains by MinDE protein oscillations.

Article Snippet: Fascin (human, recombinant) was purchased from Cytoskeleton Inc (Tebubio GmbH, Offenbach, Germany) and HYPERMOL (Germany).

Techniques: Membrane, Encapsulation