Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis

Abstract

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.

Figure 1. Establishment of a stable Arp2/3 complex depleted cell line

1A) Growth curve of wild-type (WT) and Arf −/− early passage MEFs infected with lentivirus expressing a non-specific sequence (NS) shRNA or Arp2 shRNA (Arp2-KD). Error bars: SEM 1B) IA32 cells were infected with lentivirus that expressed shRNAs against NS, p34Arc and Arp2 (2xKD), or shArp2 that also co-expressed human Arp2-GFP (Arp2-KDR). Lysates were blotted for p34Arc, Arp3, p21Arc, Arp2 and for GAPDH as a loading control 1C) Mixed NS (expressing GFP) and 2xKD cells (marked by red asterisks) were immunostained for p34Arc to verify knockdown of the endogenous gene and phalloidin to visualize F-actin. Also see Fig. 1D,E 1D) Growth curve of NS and 2xKD cells. Error bars: SEM 1E) DIC images of NS and 2xKD cells (Scale bar: 50 µm) 1F) 2xKD cells were microinjected with 5 mg/mL Arp2/3 complex. Representative time-lapse sequence before and after injection show the reappearance of lamellipodia. See movie 1 1G) Cryo-shadowing EM images showing the leading edge actin networks of NS and 2xKD cells. Left and middle panels are separate cells, right panel is magnified portion of middle panels indicated by black boxes See also Fig. S1 and Movie 1

Figure 2. Arp2/3-depleted cells show inefficient, filopodia-driven cell motility

2A) Time-lapse microscopy of NS, 2xKD, p34-KD Arp2-KD, p34-KDR and Arp2-KDR and 2KDR cell lines was used to determine single-cell speed, depicted in graph. Error bars represent 95% confidence intervals. ****P<0.0001 by Student's t-test 2B) Single-cell speed of NS and 2xKD cells treated with 100 µM Arp2/3 inhibitor CK-666 or the inactive control compound CK-689. Error bars represent 95% confidence intervals. ****P<0.0001 by Student's t-test 2C) Single-cell speed of NS and 2xKD cells treated with 1µM Latrunculin B (LatB) or DMSO as a control. Error bars represent 95% confidence intervals. ****P<0.0001, **P<0.01 by Student's t-test 2D) Single-cell speed of NS and 2xKD cells treated with 15 µM blebbistatin (BLB) or DMSO as a control. Error bars represent 95% confidence intervals. ****P<0.0001 by Student's t-test 2E) SEM images of NS and 2xKD cells treated with DMSO or BLB 2F) Number of filopodia/cell with DMSO or BLB treatment was calculated from >30 cells in each cell line from SEM images. ****P<0.0001 by Student's t-test 2G) Time-lapse images of 2xKD cells treated with DMSO or BLB. Scale bar: 5 µm See also Fig. S2 and Movie 2

Figure 3. Arp2/3 complex depletion does not affect chemotaxis

3A) Schematic of the microfluidic chamber for chemotaxis and haptotaxis experiments 3B) Left: fluorescent image of Cy5-dextran gradient formed inside the chamber as an indication of gradient formation and maintenance. Right: line-scan plot of the gradient along the yellow line depicted in the left panel, slope of the gradient is indicated under the curve 3C) Diagram showing FMI calculation method and representative end point scatter plots of NS and 2xKD cells in chemotaxis assays (Data from 120 ng/mL source PDGF concentration) 3D) Diagram showing Compass Parameter (CP) calculation and representative histograms showing angular turn per step of NS and 2xKD cells in the same PDGF chemotaxis assays as in 3C. P(α) is the probably distribution of angles measured relative to gradient, γ is a constant, g is the gradient steepness and δ is the angular step size (Arrieumerlou and Meyer, 2005) 3E) Table showing compass parameter (CP), forward migration index (FMI), velocity (V) and number of cells analyzed (N) in chemotaxis assays with indicated PDGF source concentrations. Numbers in parentheses for each entry are 95% confidence intervals 3F) Table showing Rat2 cell chemotaxis parameters in the presence of Arp2/3 inhibitor CK-666 or its inactive control CK-689 3G) Western blotting showing the change of phospho-Akt (pAkt) level upon PDGF stimulation. Cells were serum-starved overnight before stimulation with 40 ng/mL PDGF 3H) 2xKD cells before and after PDGF stimulation (images) and the number of filopodia before and after PDGF treatment of 2xKD cells was calculated from >30 2xKD cells. *P<0.05 by Student's t-test 3I) Example of the filopodia up and down the PDGF gradient in 2xKD cell chemotaxis was shown and the number of filopodia was calculated from >30 2xKD cells. *P<0.001 by Student's t-test See also Fig. S3 and Movies 3 and 4

Figure 4. Arp2/3 depletion inhibits cell spreading

4A) NS and 2xKD cells expressing GFP-Pax were immunostained for phospho-tyrosin (pTyr) and F-actin at different time points during cell spreading 4B) Time-lapse images showing the spreading of NS and 2xKD cells 4C) Cell adhesion and spreading kinetics of NS and 2xKD cells were analyzed using an impedance based system and reported as arbitrary units (Cell Index). Error bars: SEM 4D) Western blotting showing the change of phosphorylated FAK (pFAK) level upon cell adhesion to fibronectin. Cells were serum-starved overnight and trypsinized and plated on fibronectin-coated surface. Lysates were blotted for pFAK, total FAK, Arp2 and GAPDH as control

Figure 5. Arp2/3 complex depleted cells cannot respond to concentration or gradient changes in extracellular matrix

5A) Single-cell speed of NS and 2xKD cells plated on different concentrations of fibronectin was plotted (N>30). Error bars represent 95% confidence intervals 5B) Speeds of Rat2 cells treated with CK-666 or CK-689 and plated on different concentrations of FN were plotted (N>30). Error bars represent 95% confidence intervals 5C) Left: fluorescent image of Cy5-fibronectin gradient formed on the glass surface inside the cell culture chamber. Right: line-scan plot of the gradient along the yellow line depicted in the left panel 5D) Representative end point scatter plots of NS and 2xKD cells in haptotaxis assays (250 µg/mL source fibronectin concentration) 5E) Representative histogram showing angular turn per step of NS and 2xKD cells in the same haptotaxis assays as in 5D 5F) Table showing compass parameter (CP), forward migration index (FMI), velocity (V) and number of cells analyzed (N) in haptotaxis assays with indicated FN source concentrations. Numbers in parentheses for each entry are 95% confidence intervals 5G) Table showing compass parameter (CP), forward migration index (FMI), velocity (V) and number of cells analyzed (N) in haptotaxis assays with indicated extracellular matrix 5H) Table showing compass parameter (CP), forward migration index (FMI), velocity (V) and number of cells analyzed (N) in Rat2 cell haptotaxis assays with 250 µg/mL source fibronectin concentration and treated with CK-666 or CK-689 See also Fig. S4 and Movie 5

Figure 6. Depletion of Arp2/3 complex leads to defective focal adhesion morphology and dynamics

6A) Mixed NS (expressing GFP) and 2xKD cells (marked by red asterisks) were immunostained for endogenous paxillin (Pax), focal adhesion kinase (FAK), vinculin (Vin) and F-actin (Scale bar: 10 µm). 6B) Representative time-lapse TIRF images of an NS cell expressing GFP-Pax and LifeAct-tagRFP 6C) Representative time-lapse TIRF images of a 2xKD cell expressing GFP-Pax and LifeAct-tagRFP 6D) Table of focal adhesion parameters: NS vs 2xKD plated on 1, 10 and 100 µg/mL FN; Rat2 fibroblasts treated with CK-666 or CK-689 on 100 µg/mL FN. Numbers after the +/− indicate 95% confidence intervals as determined by a t-distribution fit See also Fig. S5

Figure 7. Arp2/3 complex depletion leads to poor global alignment of focal adhesions

7A) Distribution of single focal adhesion mean deviations from their first orientation measurement starting point. The adhesions analyzed were from NS (17 cells and 3184 adhesions) or 2xKD (22 cells and 1132 adhesions) cells plated on 100 µg/mL fibronectin. Insets show single adhesions outlined in green through time, there are 6 minutes between each image 7B) Diagram showing adhesion filtering through a minimum axial ratio of 3 and two sample adhesion orientations 7C) Diagram showing the determination of dominant angle and focal adhesion alignment index (FAAI) 7D) Sample single cell cartoons showing representative high and low FAAI cells with corresponding adhesion orientation data sets 7E) FAAI of NS and 2xKD cells expressing GFP-Paxillin plated on different concentrations of fibronectin (number of cells same as in Fig. 6D, p-values for the difference between the means were calculated by bootstrapping with 10000 replicates) 7F) FAAI of NS and 2xKD cells with the adhesions grouped by size (p-values for each size range <0.0005) 7G) FAAI of NS and 2xKD cells expressing indicated focal adhesion markers. (n = number of cells analyzed, p-values calculated same as in 7E) 7H) FAAI of Rat2 cells with CK-666 or CK-689 7I) Conceptual model of cell motility events across length scales See also Fig. S6 and Movie 6