Cells with endogenously labeled Arp2/3 complex reveal striking substrate-dependent actin network architecture at the leading edge.

(A) Frames from time lapse movies of endogenously-labeled ARPC2-mScarlet expressed by Fibroblasts plated on glass coverslips coated with either fibronectin or poly-L-Lysine alongside associated kymographs of protrusive regions marked with a dashed pink line in the left panels. Scale bars span 10 microns. (B) Plot of mean ARPC2-mScarlet intensities along line scans starting near the middle of the largest cellular protrusion, aligned perpendicular to the cell edge, and directed inwards for 5 microns while avoiding regions of obvious ruffles and folds in cells plated as described in (A). n = 16 cells from 3 experiments (C) Plot of mean ARPC2-mScarlet intensities at and within roughly 1 micron of the cell edge along the largest protrusion in fibroblasts plated as described in (A). n = 16 cells from 3 experiments (D) Plot of width measurements in microns of ARPC2-mScarlet signal that is above 50% the maximum intensity recorded, referred to as “Full Width Half Max” and abbreviated as F.W.H.M, in the line scans shown and described in (B). (E) Plot of retrograde flow rates visualized by bleaching GFP-β-Actin signal at the edge of protrusions in cells plated as described in (A) and measured by calculating the slope of recovering signal in kymographs generated from bleached regions. n = 33 and 32 cells for PLL and Fn conditions, respectively, from 3 experiments. Note that the data from cells on poly-L-Lysine coated glass in panels (B-E) were collected at the same time as other experimental conditions for a separate study, and values for fibronectin here have been published as the 10 ug/ml control condition previously ( Chandra, Butler et al. 2022).

Increased Rac activation is not sufficient to induce dense branched actin organization on PLL.

(A) Frames from time lapse movies of endogenously-labeled ARPC2-mScarlet and lentiviral-transduced mEmerald-WAVE1 expressed by Fibroblasts plated on glass coverslips coated with either fibronectin or poly-L-Lysine and associated kymographs of protrusive regions marked with a dashed red line. Scale bars span 10 microns. (B) Frames from time lapse movies of endogenously-labeled ARPC2-mScarlet and lentiviral-transduced Cortactin-mEmerald expressed by Fibroblasts plated on glass coverslips coated with either fibronectin or poly-L-Lysine and associated kymographs of protrusive regions marked with a dashed red line. Scale bars span 10 microns. (C) Images of control fibroblasts with Arp2/3 intact plated on poly-L-Lysine coated surfaces and visualized using fluorescently-labelled phalloidin. Scale bar spans 10 microns. (D) Images of 4HT-treated Arpc2 knockout fibroblasts plated on poly-L-Lysine coated surfaces and visualized using fluorescently-labelled phalloidin. Scale bar spans 10 microns. (E) Images from timelapse movies of Tiam1-DH/PH-TagRFPt-SspBmicro and ARPC2-HaloTag (labeled with JF646 Halo Ligand) stably expressed by 4HT-treated Arpc2 knockout fibroblasts via lentiviral transduction that have been plated on a glass coverslip coated with poly-L-Lysine (top panels) or fibronectin (bottom panels) and regularly stimulated with 405 nm light inside the region of interest (ROI) labeled with a yellow dashed oval starting at t = 0. These cells additionally express Venus-iLid-caax (not shown). The associated kymograph was generated from a line (not shown) bisecting the yellow dashed oval ROI along the long axis, and the associated plot is a measure of the intensity of the associated marker within ∼1 micron of the cell edge down the length of the accompanying kymograph. Scale bars span 10 microns.

Enrichment of Arp2/3-branched actin is linked to cell spreading.

(A) Representative images of ARPC2-mScarlet endogenously expressed by fibroblasts plated on glass coverslips coated with either poly-L-Lysine or fibronectin as they reached a projected cell spread area of 66%, 75%, and 99% of the maximum spread area recorded. Graphs below the image panels show the distribution of intensity around the cell periphery at a given distance from the cell edge for each of the two plating conditions shown above. Scale bar spans 10 microns. (B) Plot of Full Width Half Max ARPC2-mScarlet mean intensity around the cell periphery at various stages of spreading relative to the maximum recorded when plated on surfaces coated with poly-L-Lysine or fibronectin. (C) Plot of the maximum value from a range of mean Arpc2-mScarlet intensities measured in 1-pixel intervals around the cell spanning from the cell edge to 5 microns inward, which is divided by the mean value of all intensities throughout this same range,, shown as an alternative measure for the values shown in (B) and graphically detailed in Figure 3 Supplement 1. (D) Plot of the maximum/mean ARPC2-mScarlet intensity within 5 microns of the cell edge around the entirety of the cell periphery against cell spread area as a percentage of the maximum recorded, showing a significantly steeper slope for these values as a group when measured in cells plated on fibronectin compared to those plated on poly-L-Lysine. (E) Minimum and maximum projected spread areas of the cells measured. n = 12 for PLL and 16 for Fn from 3 experiments for (B-E).

Inducing cell spreading on poly-L-Lysine with myosin inhibition results in enrichment of branched actin in cell protrusions.

(A) Frames from time lapse movies of endogenously-labeled ARPC2-mScarlet and lentiviral transduced GFP-Paxillin expressed by Fibroblasts plated on glass coverslips coated with poly-L-Lysine and treated with either 50 μM para-amino-Blebbistatin (Bleb.) or DMSO control at t = 10 minutes showing the change in projected cell spread area over time. Scale bar spans 10 microns (B) Plot of projected cell spread area over time measured among cells shown and described in (A), with the red arrow at t = 10 minutes marking when wash-ins were performed. n = 25 from 2 experiments for Bleb treatment and 14 for DMSO-treated cells. (C) Images of cells similarly treated as described in (A) and fixed either 10 minutes prior to wash-ins or 50 minutes after for accurate measurements of ARPC2-mScarlet distribution. Scale bar spans 10 microns (D) Line scans of ARPC2-mScarlet intensity along lines 5 microns long and running inward from the cell edge at the largest protrusion under the indicated conditions. n = 30 cells from 2 experiments for all three groups. (E) Plot of mean ARPC2-mScarlet intensities at and within roughly 1 micron of the cell edge along the largest protrusion from the same cells measured for (D). (F) Plot of Full Width Half Max values calculated from the line scans shown in (D).

Increased extracellular viscosity can induce cell spreading in the absence of dense ECM and promotes enriched actin network branching in lamellipodial protrusions.

(A) Frames from time lapse movies of endogenously-labeled ARPC2-mScarlet and lentiviral transduced GFP-Paxillin expressed by Fibroblasts plated on glass coverslips coated with poly-L-Lysine and either treated with 0.6% methylcellulose at t = 10 minutes or not, showing the change in projected cell spread area over time. Scale bar spans 10 microns (B) Plot of projected cell spread area over time measured among cells shown and described in (A), with the red arrow at t = 10 minutes marking when wash-ins were performed. n = 44 from 2 experiments for each condition. (C) Images of cells similarly treated as described in (A) and fixed either 10 minutes prior to wash-ins or 50 minutes after for accurate measurements of ARPC2-mScarlet distribution. Scale bar spans 10 microns (D) Line scans of ARPC2-mScarlet intensity along lines 5 microns long and running inward from the cell edge at the largest protrusion under the indicated conditions. n = 30 cells from 2 experiments for all three groups. (E) Plot of mean ARPC2-mScarlet intensities at and within roughly 1 micron of the cell edge along the largest protrusion from the same cells measured for (D). (F) Plot of Full Width Half Max values calculated from the line scans shown in (D).

Robust spreading in response to increased extracellular viscosity requires Arp2/3-branched actin.

(A) Frames from time lapse movies of lentiviral transduced LifeAct-miRFP670 expressed by control and 4-HT-treated Arpc2 knockout Fibroblasts plated on glass coverslips coated with poly-L-Lysine and treated with 50 μM para-amino-Blebbistatin at t = 10 minutes. Scale bars span 10 microns. (B) Plot of projected cell spread area over time measured among cells shown and described in (A), with the red arrow at t = 10 minutes marking when wash-ins were performed. n = 27 for control and 28 for Arp2/3 Null cells from 2 experiments. (C) Frames from time lapse movies of lentiviral transduced LifeAct-miRFP670 expressed by control and 4-HT-treated Arpc2 knockout Fibroblasts plated on glass coverslips coated with poly-L-Lysine and treated with 0.6% methylcellulose at t = 10 minutes. Scale bars span 10 microns. (D) Plot of projected cell spread area over time measured among cells shown and described in (A), with the red arrow at t = 10 minutes marking when wash-ins were performed. n = 33 for control and 32 for Arp2/3 Null cells from 2 experiments for each condition.

Viscosity-induced spreading on PLL-coated soft substrates generates smaller traction forces than spreading via robust Integrin-ECM engagement.

(A) Traction maps generated from representative still images selected from time-lapse movies of fibroblasts plated on 20 kPa PDMS substrates with red fluorescent beads attached to the surface allowing for TFM measurements and coated with either poly-L-Lysine or fibronectin. Methylcellulose was added at a concentration of 0.6% to promote spreading on the poly-L-Lysine coated substrates with similar dynamics to the cells plated on fibronectin. (B) Plot of changes in strain energy as cells spread out under the two conditions shown and detailed in (A), with error bars representing the mean standard error of the mean. For spread areas of 250, 500, 750, 1,000, 1,250 and 1,500 square microns, n = 6, 23, 22, 15, 8 and 6 cells for PLL + 0.6% Methylcellulose and 18, 29, 32, 29, 21 and 17 cells for FN plating conditions, respectively, from 3 experiments. (C) Plot of the strain energy density of forces applied by the same cells measured in (B) as they reached 66%, 75%, and 99% of the maximum recorded projected spread area.

Cell protrusions generated via optogenetic Rac activation on substrates coated with poly-L-Lysine are facilitated by increased extracellular viscosity.

(A) Images from timelapse movies of Tiam1-DH/PH-TagRFPt-SspBmicro and ARPC2-HaloTag (labeled with JF646 Halo Ligand) stably expressed by a 4HT-treated Arpc2 knockout fibroblast via lentiviral transduction that has been plated on a glass coverslip coated with poly-L-Lysine and regularly stimulated with 405 nm light inside the region of interest (ROI) labeled with a yellow dashed oval starting at t = 0. These cells additionally express Venus-iLid-caax (not shown). The same cell is shown in all panels and was imaged during stimulation both before (top row) and after 0.6% methylcellulose addition (bottom row). Scale bar spans 10 microns. (B) Kymographs generated from a line (not shown) bisecting the long axis of the yellow dashed oval ROIs shown in (A). (C) Plot of the distances cells detailed in (A) protruded upon optogenetic activation of Rac either before or after addition of 0.6% methylcellulose. n = the same 9 cells across 3 experiments for both conditions.

Barbed end density increases with ARPC2-mScarlet enrichment in Fibroblasts on surfaces coated with dense fibronectin ECM.

(A) Images of Fibroblasts endogenously expressing ARPC2-mScarlet after simultaneously pulsing labeled Alexa Fluor 488 Actin and unlabeled phalloidin under conditions that make the cells slightly permeable to labeled the barbed ends of stabilized actin filaments. Scale bar spans 10 microns. (B) Plot of mean intensity of Alexa Fluor 488 Actin in cells shown and described in (A). n = 9 cells for each condition. (C) Plot of values for mean edge intensity of pulsed labeled Alexa Fluor 488 Actin shown in (B) against the similarly measured ARPC2-mScarlet in the same cell, with linear regression lines of similar slopes demonstrating a similar relationship between barbed end and ARPC2-mScarlet densities in cells plated on poly-L-Lysine (blue dots) when compared to fibronectin (grey dots).

Branched actin enrichment in fibroblasts plated on fibronectin depends on Integrin engagement.

(A) Images of Endogenous ARPC2-mScarlet and lentiviral transduced LifeAct-miRFP670 and GFP-Paxillin expressed by Fibroblasts plated on surfaces that have been coated with either poly-L-Lysine or fibronectin. Red arrows mark the edge of the largest cell protrusion where the presence or absence of small paxillin clusters can be seen. Scale bar spans 5 microns. (B) Plot of density of GFP-Paxillin clusters around the edge of the largest cell protrusion under the conditions shown in (A). n = 16 cells from 3 experiments. Note that the data from cells on poly-L-Lysine coated glass in panels (A & B) were collected at the same time as other experimental conditions for a separate study, and the images and values for the fibronectin condition here have been published as the 10 ug/ml control condition previously . (C) Images of Endogenous ARPC2-mScarlet and lentiviral transduced LifeAct-miRFP670 and GFP-Paxillin expressed by Fibroblasts plated on surfaces that have been coated with covalently-linked poly-L-Lysine, fibronectin, purified GRGDS peptides, or purified PHSRNGRGDNP. Scale bar spans 5 microns. (D) Plot of mean ARPC2-mScarlet intensities at and within roughly 1 micron of the cell edge along the largest protrusion in fibroblasts plated as described and shown in (C). n = 29, 27, 14 and 38 for pLL, Fn, GRGDS, and GRGDNP, respectively.

Segmentation and quantification of Arp2/3 distribution near the cell periphery during cell spreading.

Representative images of ARPC2-mScarlet endogenously expressed by a fibroblast plated on a glass coverslip coated with poly-L-Lysine as it reached a projected cell spread area of 66% (top) and 99% (bottom) of the recorded maximum spread area. Yellow lines represent segmentation of the cell around the outer edge and 5 microns into the cell from the edge in 1-pixel steps, which was used to calculate the F.W.H.M. and maximum/mean intensities as measures of branched actin density and distribution around the cell periphery during spreading.

fibronectin promotes efficient spreading and flattening of Fibroblasts.

(A-B) Scanning electron microscopy (SEM) images of Fibroblasts plated on glass coverslips coated with poly-L-Lysine or fibronectin. Right panel is an enlarged view of the boxed region in the panel to the left. (C) Plot of projected cell spread area in fibroblasts plated on either fibronectin poly-L-Lysine over time. (D) Sample images and accompanying line scans measured from regions marked with yellow-shaded regions and arrows for cells plated on poly-L-Lysine. Note the maintained increase in spread area and Arp2/3-mScarlet density show in the panels for the top section i and both the increase and decrease of Arp2/3-density that is seen upon the increase and decrease of cell spread area shown in the panels for the bottom section ii.

Physically flattening cells or manipulating membrane tension with osmotic pressure leads to enriched protrusive branched actin.

(A) Frames from a time lapse movie of endogenously-labeled ARPC2-mScarlet expressed by Fibroblasts plated on glass coverslips coated with poly-L-Lysine and compressed under weighted agarose pucks at t = 5 minutes to physically force cell flattening and spreading. Scale bar spans 10 microns. (B) Plot of the fold change in projected cell spread area after physically compressing cells at t = 10 minutes (red arrow) as shown in (A). n = 25 cells from 2 experiments. (C) Plot of ARPC2-mScarlet intensities along line scans starting near the middle of the largest cellular protrusion, aligned perpendicular to the cell edge, and directed inwards while avoiding regions of obvious ruffles and folds in cells plated as detailed in (A-B) following bleach correction pre- (t = 0) and post-compression (t = 60 minutes). (D) Frames from a time lapse movie of endogenously-labeled ARPC2-mScarlet expressed by Fibroblasts plated on glass coverslips coated with poly-L-Lysine and treated with Sorbitol at a final concentration of 0.25M at t = 10 minutes. Scale bar spans 10 microns. (E) Plot of the fold change in projected cell spread area after treating with 0.25M Sorbitol at t = 10 minutes (red arrow) as shown in (D). n = 32 cells from 2 experiments. (F) Plot of ARPC2-mScarlet intensities along line scans starting near the middle of the largest cellular protrusion, aligned perpendicular to the cell edge, and directed inwards while avoiding regions of obvious ruffles and folds in cells plated as detailed in (D-E) before (t = 0) and after (t = 90 minutes) Sorbitol addition. (G) Plot of mean ARPC2-mScarlet intensity around and within ∼1 micron of the edge of the largest cell protrusion in cells shown and detailed in (D-F) before (t = 0) and after (t = 90 minutes) Sorbitol addition. (H) Plot of Full Width Half Max values calculated from the line scans shown in (F). (I) Scanning electron microscopy (SEM) images of Fibroblasts plated on glass coverslip coated with poly-L-Lysine and treated with 50 μM para-amino-Blebbistatin. Right panel is an enlarged view of the boxed region in the panel to the left. (J) Scanning electron microscopy (SEM) images of Fibroblasts plated on glass coverslip coated with poly-L-Lysine and treated with 0.25M Sorbitol. Right panel is an enlarged view of the boxed region in the panel to the left.

Neither control nor Arp2/3 null Fibroblasts plated on dense fibronectin ECM exhibit robust cells spreading in response to increased extracellular viscosity.

(A) Frames from time lapse movies of endogenous ARPC2-mScarlet expressed by control cells and lentiviral transduced GFP-Paxillin and LifeAct-miRFP670 expressed by both control and 4-HT-treated Arpc2 knockout Fibroblasts plated on glass coverslips coated with fibronectin and treated with 0.6% methylcellulose at t = 10 minutes. Scale bars span 10 microns. (B) Plot of projected cell spread area over time measured among cells shown and described in (A), with the red arrow at t = 10 minutes marking when wash-ins were performed. N = 27 for control and 15 for Arp2/3 Null cells from 2 experiments.