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rhodamine conjugated fibronectin  (Cytoskeleton Inc)


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    Cytoskeleton Inc rhodamine conjugated fibronectin
    Mechanical confinement of mitotic cells in fibrous environments. a) (i) Representative 3D isometric views of single fixed mitotic cells on top of fibers (unconfined) or trapped between two fibers (confined), actin (cell cortex), histone H2B (CHs) and the fibers <t>(fibronectin</t> coating) are indicated in red, cyan, and green respectively. Inset cartoons highlight the outward bowing of fibers during ECM confinement. (ii) Representative side views ( yz cross-section) of fixed unconfined and confined cells with the fibers as green dots (yellow arrowheads). H represents the cell height taken at metaphase, and h is the height from the fiber plane to the bottom of the cell. Scale bars are 10 µm. (iii) Histogram showing relative occurrence of the different levels of confinement (quantified by h / H , N = 10, n = 73). (iv) Metaphase cell height increases with confinement ( h / H ) ( R 2 = 0.9, P = 0.0132; N = 10, n = 73). Representative cross-sectional images of fixed mitotic cells with very low h / H (∼0.1) and high h / H (∼0.5). Scale bars are 10 µm. b) (i) Time-lapse images of a representative live mitotic confined cell undergoing cell division. (ii) Nanonet Force Microscopy–based force profiles of confined and unconfined cells transitioning from interphase to mitosis, with inset images showing a live single cell at different stages. By convention, mitosis forces are shown as negative to represent outward deflection of fibers. The dashed rectangle box shows the average force profiles for confined cells rounding during mitosis which is normalized for time taken from NEBD to cytokinesis ( N = 4, n = 23) along with representative top views of fixed cells undergoing various stages of division. Scale bars are (i) 50 µm and (ii) 20 µm. c) Representative top ( xy ) and front ( xz ) views of fixed, confined and unconfined cells demonstrating the 3D tilt of the MP. The yellow arrow represents the MP rotation. Cells are stained with actin cortex (magenta), microtubules (cyan), KTs (green), and CHs (red). Scale bars represent 10 µm. d) Representative fixed images showing the measurement of inter-KT separation and the quantitation showing an increase in the average inter-KT distance with confinement. Scale bars represent 5 µm. e) Confinement causes a drop in the mitosis duration, N = 5, n = 47.
    Rhodamine Conjugated Fibronectin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 94/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rhodamine+conjugated+fibronectin/Rhodamine+fibronectin/pmc12236158-232-13-15
    Average 94 stars, based on 149 article reviews
    rhodamine conjugated fibronectin - by Bioz Stars, 2026-09
    94/100 stars

    Images

    1) Product Images from "Confinement in fibrous environments positions and orients mitotic spindles"

    Article Title: Confinement in fibrous environments positions and orients mitotic spindles

    Journal: PNAS Nexus

    doi: 10.1093/pnasnexus/pgaf201

    Mechanical confinement of mitotic cells in fibrous environments. a) (i) Representative 3D isometric views of single fixed mitotic cells on top of fibers (unconfined) or trapped between two fibers (confined), actin (cell cortex), histone H2B (CHs) and the fibers (fibronectin coating) are indicated in red, cyan, and green respectively. Inset cartoons highlight the outward bowing of fibers during ECM confinement. (ii) Representative side views ( yz cross-section) of fixed unconfined and confined cells with the fibers as green dots (yellow arrowheads). H represents the cell height taken at metaphase, and h is the height from the fiber plane to the bottom of the cell. Scale bars are 10 µm. (iii) Histogram showing relative occurrence of the different levels of confinement (quantified by h / H , N = 10, n = 73). (iv) Metaphase cell height increases with confinement ( h / H ) ( R 2 = 0.9, P = 0.0132; N = 10, n = 73). Representative cross-sectional images of fixed mitotic cells with very low h / H (∼0.1) and high h / H (∼0.5). Scale bars are 10 µm. b) (i) Time-lapse images of a representative live mitotic confined cell undergoing cell division. (ii) Nanonet Force Microscopy–based force profiles of confined and unconfined cells transitioning from interphase to mitosis, with inset images showing a live single cell at different stages. By convention, mitosis forces are shown as negative to represent outward deflection of fibers. The dashed rectangle box shows the average force profiles for confined cells rounding during mitosis which is normalized for time taken from NEBD to cytokinesis ( N = 4, n = 23) along with representative top views of fixed cells undergoing various stages of division. Scale bars are (i) 50 µm and (ii) 20 µm. c) Representative top ( xy ) and front ( xz ) views of fixed, confined and unconfined cells demonstrating the 3D tilt of the MP. The yellow arrow represents the MP rotation. Cells are stained with actin cortex (magenta), microtubules (cyan), KTs (green), and CHs (red). Scale bars represent 10 µm. d) Representative fixed images showing the measurement of inter-KT separation and the quantitation showing an increase in the average inter-KT distance with confinement. Scale bars represent 5 µm. e) Confinement causes a drop in the mitosis duration, N = 5, n = 47.
    Figure Legend Snippet: Mechanical confinement of mitotic cells in fibrous environments. a) (i) Representative 3D isometric views of single fixed mitotic cells on top of fibers (unconfined) or trapped between two fibers (confined), actin (cell cortex), histone H2B (CHs) and the fibers (fibronectin coating) are indicated in red, cyan, and green respectively. Inset cartoons highlight the outward bowing of fibers during ECM confinement. (ii) Representative side views ( yz cross-section) of fixed unconfined and confined cells with the fibers as green dots (yellow arrowheads). H represents the cell height taken at metaphase, and h is the height from the fiber plane to the bottom of the cell. Scale bars are 10 µm. (iii) Histogram showing relative occurrence of the different levels of confinement (quantified by h / H , N = 10, n = 73). (iv) Metaphase cell height increases with confinement ( h / H ) ( R 2 = 0.9, P = 0.0132; N = 10, n = 73). Representative cross-sectional images of fixed mitotic cells with very low h / H (∼0.1) and high h / H (∼0.5). Scale bars are 10 µm. b) (i) Time-lapse images of a representative live mitotic confined cell undergoing cell division. (ii) Nanonet Force Microscopy–based force profiles of confined and unconfined cells transitioning from interphase to mitosis, with inset images showing a live single cell at different stages. By convention, mitosis forces are shown as negative to represent outward deflection of fibers. The dashed rectangle box shows the average force profiles for confined cells rounding during mitosis which is normalized for time taken from NEBD to cytokinesis ( N = 4, n = 23) along with representative top views of fixed cells undergoing various stages of division. Scale bars are (i) 50 µm and (ii) 20 µm. c) Representative top ( xy ) and front ( xz ) views of fixed, confined and unconfined cells demonstrating the 3D tilt of the MP. The yellow arrow represents the MP rotation. Cells are stained with actin cortex (magenta), microtubules (cyan), KTs (green), and CHs (red). Scale bars represent 10 µm. d) Representative fixed images showing the measurement of inter-KT separation and the quantitation showing an increase in the average inter-KT distance with confinement. Scale bars represent 5 µm. e) Confinement causes a drop in the mitosis duration, N = 5, n = 47.

    Techniques Used: Microscopy, Staining, Quantitation Assay

    Computational modeling recapitulate experimentally observed MP tilt with increasing confinement. a) (i, ii) Representative images of fixed unconfined and confined cells with top ( xy ), side ( yz ), and front ( xz ) views shown, actin, histone H2B, and fibronectin are shown in red, cyan, and green to identify the cell cortex, MP, and the fibers, respectively. Scale bars are 10 µm. (iii) MP tilt as a function of the extent of confinement ( h / H ). Inset images show front and side views of fixed unconfined ( h / H ∼0.1) and confined ( h / H ∼0.5) cells. The angle is measured from a normal drawn to the fiber axis (green dashed lines shown for confined and unconfined cases) ( N = 10, n = 73). Scale bars are 10 µm. b) (i) Schematic showing the quantification of the RF coverage. (ii) RF coverage decreases with h / H . Inset images showing RF organization in representative front views of fixed, confined and unconfined cells. Scale bars are 10 µm ( N = 5, n = 44). c) (i) Intensity heat map demonstrating the average projection of yz side views in actin-stained cells. Scale bars are 10 µm. Band-like arrangement emerges for RF organization in confined cells, while unconfined cells have triangular forms of RF regions that extends downwards from the mid-cortical level, corresponding adopted RF regions for the computational simulations. (ii) Computational model of the mitotic cell between the two external fibers (marked in green). CSs attract each other ( f CS - CS ) or KTs ( f CS - KT ) and are attracted to the cortex region devoid of RFs ( f CS − CRTX ) or coupled to RFs ( f CS − RF ). CSs repel CH arms ( f CS − CH ). All relevant components of the mitotic machinery are marked and indexed below. f CS − RF attraction is considered several times stronger than f CS − CRTX . (iii) Computational data for MP tilt as a function of h / H without cortex deformation, representative snapshots showing the front and side views of spherical unconfined and confined cells. Average MP tilt is estimated from ∼500 simulations with different random initializations for each condition. Error bars represent the SEM measured with respect to the corresponding mean values of the data. Note that crosses in a(iii) and c(iii) show the low and high MP tilt angles observed at high confinement along with representative images. The average of these two angles is plotted in the main figure, and the origin of two configurations is explained in Fig. .
    Figure Legend Snippet: Computational modeling recapitulate experimentally observed MP tilt with increasing confinement. a) (i, ii) Representative images of fixed unconfined and confined cells with top ( xy ), side ( yz ), and front ( xz ) views shown, actin, histone H2B, and fibronectin are shown in red, cyan, and green to identify the cell cortex, MP, and the fibers, respectively. Scale bars are 10 µm. (iii) MP tilt as a function of the extent of confinement ( h / H ). Inset images show front and side views of fixed unconfined ( h / H ∼0.1) and confined ( h / H ∼0.5) cells. The angle is measured from a normal drawn to the fiber axis (green dashed lines shown for confined and unconfined cases) ( N = 10, n = 73). Scale bars are 10 µm. b) (i) Schematic showing the quantification of the RF coverage. (ii) RF coverage decreases with h / H . Inset images showing RF organization in representative front views of fixed, confined and unconfined cells. Scale bars are 10 µm ( N = 5, n = 44). c) (i) Intensity heat map demonstrating the average projection of yz side views in actin-stained cells. Scale bars are 10 µm. Band-like arrangement emerges for RF organization in confined cells, while unconfined cells have triangular forms of RF regions that extends downwards from the mid-cortical level, corresponding adopted RF regions for the computational simulations. (ii) Computational model of the mitotic cell between the two external fibers (marked in green). CSs attract each other ( f CS - CS ) or KTs ( f CS - KT ) and are attracted to the cortex region devoid of RFs ( f CS − CRTX ) or coupled to RFs ( f CS − RF ). CSs repel CH arms ( f CS − CH ). All relevant components of the mitotic machinery are marked and indexed below. f CS − RF attraction is considered several times stronger than f CS − CRTX . (iii) Computational data for MP tilt as a function of h / H without cortex deformation, representative snapshots showing the front and side views of spherical unconfined and confined cells. Average MP tilt is estimated from ∼500 simulations with different random initializations for each condition. Error bars represent the SEM measured with respect to the corresponding mean values of the data. Note that crosses in a(iii) and c(iii) show the low and high MP tilt angles observed at high confinement along with representative images. The average of these two angles is plotted in the main figure, and the origin of two configurations is explained in Fig. .

    Techniques Used: Staining

    Related Articles

    Clinical Proteomics:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Briefly, Sylgard 184 Silicone Elastomer base (Dow Corning) was mixed with Sylgard 184 Silicone Elastomer Curing Agent at a 10:1 ratio, then poured on the silicon wafer, degassed in a vacuum chamber, and cured in the oven at 65 °C for 2 h. After curing, the PDMS was removed and cut into approximately 1 cm × 1 cm stamps that were placed in a petri dish with the feature side up. .. The surface of the PDMS stamps was treated with a low-frequency plasma cleaner unit (PlasmaEtch, Inc., PE-25) for 2 min before 100 μl of rhodamine-conjugated fibronectin (Cytoskeleton) at 0.1 mg/ml was applied to the surface for 1 h to allow adsorption of rhodamine fibronectin onto the surface of the stamps. ..

    Article Title: Macrophage migration is differentially regulated by distinct ECM components
    Article Snippet: α6 integrin antibody (GoH3, ThermoFischer, #14-0495-82); p-Cofilin Ser3 antibody (77G2, Cell Signaling, #3313); p-MLC Ser20 (for western blot) (AWBMyl9F6 (F-6), ThermoFischer, #MA5-27983); GAPDH antibody (clone 6C5, ThermoFisher, #AM4300); Myosin IIA antibody (Polyclonal, Cell Signaling, #3403); Phospho-Myosin Light Chain 2 antibody (for immunofluorescence) (Polyclonal, Cell Signaling, #3674); Goat anti-Rabbit IgG Rhodamine Red-X (RRX) secondary antibody (Polyclonal, Jackson Immunoresearch, 111-295-144); Goat anti-Mouse IgG RRX secondary antibody (Polyclonal, Jackson Immunoresearch, 115-295-166); Goat anti-Rabbit IgG Alexa 488 secondary antibody (Polyclonal, Jackson Immunoresearch, 111-545-144); Goat anti-Mouse IgG Alexa 488 secondary antibody (Polyclonal, Jackson Immunoresearch, 115-545-166); Goat anti-Rat IgG RRX secondary antibody (Polyclonal, Jackson Immunoresearch, 112-295-167); Goat anti-Rat IgG Alexa 488 secondary antibody (Polyclonal, Jackson Immunoresearch, 112-545-003); Goat anti-mouse IgG, HRP-conjugated (Jackson Immunoresearch, 115-035-146); Goat anti-rabbit IgG, HRP-conjugated (Jackson immunoresearch, 111-035-144) .. Poly-L-lysine (Sigma-Aldrich, #P8920); Rat tail collagen, type I (ThermoFisher, #A1048301); Fibronectin, human plasma (ThermoFisher, #33016015); Laminin 111, mouse (ThermoFisher, #23017015); Vitronectin, human plasma (Sigma-Aldrich, #5051); Rhodamine-conjugated fibronectin (Cytoskeleton Inc., FNR01-A); HiLite 488-labeled laminin (Cytoskeleton Inc., LMN02-A) .. CellTrackerTM Green CMFDA Dye (Invitrogen, #C7025); CellBrite® Orange: Ex/Em 549/565 nm (Biotium, #30022); Alexa FluorTM 647 Phalloidin (Invitrogen, #A22287); Alexa FluorTM 488 Phalloidin (Invitrogen, #A12379)

    Adsorption:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Briefly, Sylgard 184 Silicone Elastomer base (Dow Corning) was mixed with Sylgard 184 Silicone Elastomer Curing Agent at a 10:1 ratio, then poured on the silicon wafer, degassed in a vacuum chamber, and cured in the oven at 65 °C for 2 h. After curing, the PDMS was removed and cut into approximately 1 cm × 1 cm stamps that were placed in a petri dish with the feature side up. .. The surface of the PDMS stamps was treated with a low-frequency plasma cleaner unit (PlasmaEtch, Inc., PE-25) for 2 min before 100 μl of rhodamine-conjugated fibronectin (Cytoskeleton) at 0.1 mg/ml was applied to the surface for 1 h to allow adsorption of rhodamine fibronectin onto the surface of the stamps. ..

    Imaging:

    Article Title: Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic.
    Article Snippet: Current basement membrane (BM) mimics used for modeling endothelial and epithelial barriers in vitro do not faithfully recapitulate key in vivo physiological properties such as BM thickness, porosity, stiffness, and fibrous composition.. Here, we use networks of precisely arranged nanofibers to form ultra-thin (∼3 μm thick) and ultra-porous (∼90%) BM mimics for blood–brain barrier modeling.. We show that these nanofiber networks enable close contact between endothelial monolayers and pericytes across the membrane, which are known to regulate barrier tightness.

    Article Title: Mitotic outcomes and errors in fibrous environments.
    Article Snippet: Nanofiber networks were first sterilized with 70% ethanol for 10 min, followed by functionalization with 4 μg/mL fibronectin in PBS (Invitrogen, Carlsbad, CA). .. For select imaging experiments, fibers were coated with rhodamine- conjugated fibronectin (Cytoskeleton Inc.). ..

    Concentration Assay:

    Article Title: Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic.
    Article Snippet: Current basement membrane (BM) mimics used for modeling endothelial and epithelial barriers in vitro do not faithfully recapitulate key in vivo physiological properties such as BM thickness, porosity, stiffness, and fibrous composition.. Here, we use networks of precisely arranged nanofibers to form ultra-thin (∼3 μm thick) and ultra-porous (∼90%) BM mimics for blood–brain barrier modeling.. We show that these nanofiber networks enable close contact between endothelial monolayers and pericytes across the membrane, which are known to regulate barrier tightness.

    Incubation:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization.
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mmglass dishes (WorldPrecision Instruments). .. The dishes were coated with 0.1mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mm glass dishes (World Precision Instruments). .. The dishes were coated with 0.1 mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Immunofluorescence:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization.
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mmglass dishes (WorldPrecision Instruments). .. The dishes were coated with 0.1mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mm glass dishes (World Precision Instruments). .. The dishes were coated with 0.1 mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Staining:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization.
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mmglass dishes (WorldPrecision Instruments). .. The dishes were coated with 0.1mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mm glass dishes (World Precision Instruments). .. The dishes were coated with 0.1 mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Microscopy:

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization.
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mmglass dishes (WorldPrecision Instruments). .. The dishes were coated with 0.1mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..

    Article Title: Matrix stiffness drives drop like nuclear deformation and lamin A/C tension-dependent YAP nuclear localization
    Article Snippet: Micropost stamps were peeled off and used to make upright microposts on 35-mm glass dishes (World Precision Instruments). .. The dishes were coated with 0.1 mg/ml rhodamine-conjugated fibronectin (Cytoskeleton) for 1 h at room temperature, washed three times with PBS, and then cells were passaged on the microposts, incubated overnight at 37 °C and processed for immunofluorescence staining and microscopy. ..



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    Mechanical confinement of mitotic cells in fibrous environments. a) (i) Representative 3D isometric views of single fixed mitotic cells on top of fibers (unconfined) or trapped between two fibers (confined), actin (cell cortex), histone H2B (CHs) and the fibers (fibronectin coating) are indicated in red, cyan, and green respectively. Inset cartoons highlight the outward bowing of fibers during ECM confinement. (ii) Representative side views ( yz cross-section) of fixed unconfined and confined cells with the fibers as green dots (yellow arrowheads). H represents the cell height taken at metaphase, and h is the height from the fiber plane to the bottom of the cell. Scale bars are 10 µm. (iii) Histogram showing relative occurrence of the different levels of confinement (quantified by h / H , N = 10, n = 73). (iv) Metaphase cell height increases with confinement ( h / H ) ( R 2 = 0.9, P = 0.0132; N = 10, n = 73). Representative cross-sectional images of fixed mitotic cells with very low h / H (∼0.1) and high h / H (∼0.5). Scale bars are 10 µm. b) (i) Time-lapse images of a representative live mitotic confined cell undergoing cell division. (ii) Nanonet Force Microscopy–based force profiles of confined and unconfined cells transitioning from interphase to mitosis, with inset images showing a live single cell at different stages. By convention, mitosis forces are shown as negative to represent outward deflection of fibers. The dashed rectangle box shows the average force profiles for confined cells rounding during mitosis which is normalized for time taken from NEBD to cytokinesis ( N = 4, n = 23) along with representative top views of fixed cells undergoing various stages of division. Scale bars are (i) 50 µm and (ii) 20 µm. c) Representative top ( xy ) and front ( xz ) views of fixed, confined and unconfined cells demonstrating the 3D tilt of the MP. The yellow arrow represents the MP rotation. Cells are stained with actin cortex (magenta), microtubules (cyan), KTs (green), and CHs (red). Scale bars represent 10 µm. d) Representative fixed images showing the measurement of inter-KT separation and the quantitation showing an increase in the average inter-KT distance with confinement. Scale bars represent 5 µm. e) Confinement causes a drop in the mitosis duration, N = 5, n = 47.

    Journal: PNAS Nexus

    Article Title: Confinement in fibrous environments positions and orients mitotic spindles

    doi: 10.1093/pnasnexus/pgaf201

    Figure Lengend Snippet: Mechanical confinement of mitotic cells in fibrous environments. a) (i) Representative 3D isometric views of single fixed mitotic cells on top of fibers (unconfined) or trapped between two fibers (confined), actin (cell cortex), histone H2B (CHs) and the fibers (fibronectin coating) are indicated in red, cyan, and green respectively. Inset cartoons highlight the outward bowing of fibers during ECM confinement. (ii) Representative side views ( yz cross-section) of fixed unconfined and confined cells with the fibers as green dots (yellow arrowheads). H represents the cell height taken at metaphase, and h is the height from the fiber plane to the bottom of the cell. Scale bars are 10 µm. (iii) Histogram showing relative occurrence of the different levels of confinement (quantified by h / H , N = 10, n = 73). (iv) Metaphase cell height increases with confinement ( h / H ) ( R 2 = 0.9, P = 0.0132; N = 10, n = 73). Representative cross-sectional images of fixed mitotic cells with very low h / H (∼0.1) and high h / H (∼0.5). Scale bars are 10 µm. b) (i) Time-lapse images of a representative live mitotic confined cell undergoing cell division. (ii) Nanonet Force Microscopy–based force profiles of confined and unconfined cells transitioning from interphase to mitosis, with inset images showing a live single cell at different stages. By convention, mitosis forces are shown as negative to represent outward deflection of fibers. The dashed rectangle box shows the average force profiles for confined cells rounding during mitosis which is normalized for time taken from NEBD to cytokinesis ( N = 4, n = 23) along with representative top views of fixed cells undergoing various stages of division. Scale bars are (i) 50 µm and (ii) 20 µm. c) Representative top ( xy ) and front ( xz ) views of fixed, confined and unconfined cells demonstrating the 3D tilt of the MP. The yellow arrow represents the MP rotation. Cells are stained with actin cortex (magenta), microtubules (cyan), KTs (green), and CHs (red). Scale bars represent 10 µm. d) Representative fixed images showing the measurement of inter-KT separation and the quantitation showing an increase in the average inter-KT distance with confinement. Scale bars represent 5 µm. e) Confinement causes a drop in the mitosis duration, N = 5, n = 47.

    Article Snippet: Nanofiber networks were sterilized with 70% ethanol and functionalized with 4 μg/mL of rhodamine-conjugated fibronectin (Cytoskeleton Inc.) in PBS for 1 h to enable cell–fiber attachment.

    Techniques: Microscopy, Staining, Quantitation Assay

    Computational modeling recapitulate experimentally observed MP tilt with increasing confinement. a) (i, ii) Representative images of fixed unconfined and confined cells with top ( xy ), side ( yz ), and front ( xz ) views shown, actin, histone H2B, and fibronectin are shown in red, cyan, and green to identify the cell cortex, MP, and the fibers, respectively. Scale bars are 10 µm. (iii) MP tilt as a function of the extent of confinement ( h / H ). Inset images show front and side views of fixed unconfined ( h / H ∼0.1) and confined ( h / H ∼0.5) cells. The angle is measured from a normal drawn to the fiber axis (green dashed lines shown for confined and unconfined cases) ( N = 10, n = 73). Scale bars are 10 µm. b) (i) Schematic showing the quantification of the RF coverage. (ii) RF coverage decreases with h / H . Inset images showing RF organization in representative front views of fixed, confined and unconfined cells. Scale bars are 10 µm ( N = 5, n = 44). c) (i) Intensity heat map demonstrating the average projection of yz side views in actin-stained cells. Scale bars are 10 µm. Band-like arrangement emerges for RF organization in confined cells, while unconfined cells have triangular forms of RF regions that extends downwards from the mid-cortical level, corresponding adopted RF regions for the computational simulations. (ii) Computational model of the mitotic cell between the two external fibers (marked in green). CSs attract each other ( f CS - CS ) or KTs ( f CS - KT ) and are attracted to the cortex region devoid of RFs ( f CS − CRTX ) or coupled to RFs ( f CS − RF ). CSs repel CH arms ( f CS − CH ). All relevant components of the mitotic machinery are marked and indexed below. f CS − RF attraction is considered several times stronger than f CS − CRTX . (iii) Computational data for MP tilt as a function of h / H without cortex deformation, representative snapshots showing the front and side views of spherical unconfined and confined cells. Average MP tilt is estimated from ∼500 simulations with different random initializations for each condition. Error bars represent the SEM measured with respect to the corresponding mean values of the data. Note that crosses in a(iii) and c(iii) show the low and high MP tilt angles observed at high confinement along with representative images. The average of these two angles is plotted in the main figure, and the origin of two configurations is explained in Fig. .

    Journal: PNAS Nexus

    Article Title: Confinement in fibrous environments positions and orients mitotic spindles

    doi: 10.1093/pnasnexus/pgaf201

    Figure Lengend Snippet: Computational modeling recapitulate experimentally observed MP tilt with increasing confinement. a) (i, ii) Representative images of fixed unconfined and confined cells with top ( xy ), side ( yz ), and front ( xz ) views shown, actin, histone H2B, and fibronectin are shown in red, cyan, and green to identify the cell cortex, MP, and the fibers, respectively. Scale bars are 10 µm. (iii) MP tilt as a function of the extent of confinement ( h / H ). Inset images show front and side views of fixed unconfined ( h / H ∼0.1) and confined ( h / H ∼0.5) cells. The angle is measured from a normal drawn to the fiber axis (green dashed lines shown for confined and unconfined cases) ( N = 10, n = 73). Scale bars are 10 µm. b) (i) Schematic showing the quantification of the RF coverage. (ii) RF coverage decreases with h / H . Inset images showing RF organization in representative front views of fixed, confined and unconfined cells. Scale bars are 10 µm ( N = 5, n = 44). c) (i) Intensity heat map demonstrating the average projection of yz side views in actin-stained cells. Scale bars are 10 µm. Band-like arrangement emerges for RF organization in confined cells, while unconfined cells have triangular forms of RF regions that extends downwards from the mid-cortical level, corresponding adopted RF regions for the computational simulations. (ii) Computational model of the mitotic cell between the two external fibers (marked in green). CSs attract each other ( f CS - CS ) or KTs ( f CS - KT ) and are attracted to the cortex region devoid of RFs ( f CS − CRTX ) or coupled to RFs ( f CS − RF ). CSs repel CH arms ( f CS − CH ). All relevant components of the mitotic machinery are marked and indexed below. f CS − RF attraction is considered several times stronger than f CS − CRTX . (iii) Computational data for MP tilt as a function of h / H without cortex deformation, representative snapshots showing the front and side views of spherical unconfined and confined cells. Average MP tilt is estimated from ∼500 simulations with different random initializations for each condition. Error bars represent the SEM measured with respect to the corresponding mean values of the data. Note that crosses in a(iii) and c(iii) show the low and high MP tilt angles observed at high confinement along with representative images. The average of these two angles is plotted in the main figure, and the origin of two configurations is explained in Fig. .

    Article Snippet: Nanofiber networks were sterilized with 70% ethanol and functionalized with 4 μg/mL of rhodamine-conjugated fibronectin (Cytoskeleton Inc.) in PBS for 1 h to enable cell–fiber attachment.

    Techniques: Staining