Figure 6.
RUSH-α5 is delivered to the tip of adhesions and mediates adhesion growth. (A and B) Representative immunofluorescence image of U2OS cells expressing RUSH-α5 and pmKate2-Paxillin (white, colocalization) plated on FN (10 µg/ml) and anti-GFP (2.5 µg/ml; to trap cell surface RUSH-α5 at the point of delivery). Insets represent ROIs that are magnified. ROI2 is a FA demarcated into four equal areas for analysis and is further magnified in B. Scale bars 20 µm (whole cell image), 5 µm (ROI1), and 0.5 µm (ROI2 and B). (C) Representative image of an already established FA where RUSH-α5 delivery was quantified. Scale bar 0.5 µm. (D) Cartoon showing clutch model elements. Myosin motors pull on actin filaments with a speed v. This applies force to a substrate via integrins and adapter proteins (talin). The effect of force regulates the unbinding rates from integrins to the substrate (koff) and the folding/unfolding rates of talin (kfold/kunfold). When talin unfolds, adhesion reinforcement is assumed to happen, which is modeled by an increase in integrin density with value dadd. Changes in integrin availability are modeled by changing the parameter dadd. (E) Model prediction of adhesion growth with time for conditions in which integrin availability is low (dadd = 0.005 integrins/μm2) or high (dadd = 0.01 integrins/μm2). Adhesion growth (y-axis) is modeled through integrin density, which is plotted normalized to the starting value. (F) Quantification of adhesion growth in U2OS cells expressing RUSH-α5 and plated on FN or collagen ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (G) Quantification of adhesion growth in WT and ITGA5 KO U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (H) Quantification of cell spreading in WT and ITGA5 KO cells expressing RUSH-α5 ± biotin treatment for the indicated times. Data are mean ± SEM. (I) Quantification of the length of the longest protrusion (extending furthest from the initial plasma membrane localization during imaging) formed per cell after 45 min of biotin. Data are mean ± SEM. (J) Schematic depiction of the regulation of cell dynamics by transport of integrins through the biosynthetic pathway. Adhesion and cell spreading-dependent delivery of integrin from the ER is detected rapidly after release in cell protrusions. Canonical Golgi-dependent delivery is also polarized to cell protruding areas in an ECM-specific manner and contributes to FA growth and cell protrusion. (F–H) One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal, but this was not formally tested. (F)N = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments. (G) 57 WT cells and 52 ITGA5 KO cells, (H) 59 WT cells and 55 ITGA5 KO cells, pooled from three independent experiments. (I) Mann–Whitney test, N = 55 cells on FN, 66 cells on collagen, pooled from three independent experiments.

RUSH-α5 is delivered to the tip of adhesions and mediates adhesion growth. (A and B) Representative immunofluorescence image of U2OS cells expressing RUSH-α5 and pmKate2-Paxillin (white, colocalization) plated on FN (10 µg/ml) and anti-GFP (2.5 µg/ml; to trap cell surface RUSH-α5 at the point of delivery). Insets represent ROIs that are magnified. ROI2 is a FA demarcated into four equal areas for analysis and is further magnified in B. Scale bars 20 µm (whole cell image), 5 µm (ROI1), and 0.5 µm (ROI2 and B). (C) Representative image of an already established FA where RUSH-α5 delivery was quantified. Scale bar 0.5 µm. (D) Cartoon showing clutch model elements. Myosin motors pull on actin filaments with a speed v. This applies force to a substrate via integrins and adapter proteins (talin). The effect of force regulates the unbinding rates from integrins to the substrate (koff) and the folding/unfolding rates of talin (kfold/kunfold). When talin unfolds, adhesion reinforcement is assumed to happen, which is modeled by an increase in integrin density with value dadd. Changes in integrin availability are modeled by changing the parameter dadd. (E) Model prediction of adhesion growth with time for conditions in which integrin availability is low (dadd = 0.005 integrins/μm2) or high (dadd = 0.01 integrins/μm2). Adhesion growth (y-axis) is modeled through integrin density, which is plotted normalized to the starting value. (F) Quantification of adhesion growth in U2OS cells expressing RUSH-α5 and plated on FN or collagen ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (G) Quantification of adhesion growth in WT and ITGA5 KO U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (H) Quantification of cell spreading in WT and ITGA5 KO cells expressing RUSH-α5 ± biotin treatment for the indicated times. Data are mean ± SEM. (I) Quantification of the length of the longest protrusion (extending furthest from the initial plasma membrane localization during imaging) formed per cell after 45 min of biotin. Data are mean ± SEM. (J) Schematic depiction of the regulation of cell dynamics by transport of integrins through the biosynthetic pathway. Adhesion and cell spreading-dependent delivery of integrin from the ER is detected rapidly after release in cell protrusions. Canonical Golgi-dependent delivery is also polarized to cell protruding areas in an ECM-specific manner and contributes to FA growth and cell protrusion. (F–H) One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal, but this was not formally tested. (F)N = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments. (G) 57 WT cells and 52 ITGA5 KO cells, (H) 59 WT cells and 55 ITGA5 KO cells, pooled from three independent experiments. (I) Mann–Whitney test, N = 55 cells on FN, 66 cells on collagen, pooled from three independent experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal