[Show abstract][Hide abstract] ABSTRACT: Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, alpha-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix.
Full-text · Article · Jun 2010 · Nature Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Figure S1. shRNA depletion of FA proteins in HT0180 and E006AA
cells. Western blots of fibrosarcoma HT1080 (A-H) or
prostate E006AA (I-K) cells infected with lentivirus expressing
the indicated shRNA (see methods for a list of shRNAs) directed against
indicated FA proteins, or mock infected, or infected with lentivirus
expressing a nonspecific shRNA against luciferase. Loading controls are
either α-tubulin or α-tubulin. A) Talin 1,2.
B) Vinculin. C) Paxillin. D) FAK.
E) α-actinin 1,4. In lane 3 is sh822 that did not
result in efficient knockdown and was not used further. Only sh1299 and
sh2287 were used to knockdown α-actinin in experiments.
F) P130Cas. G) VASP. In lane 5 cells were
transduced with a lentivirus that expressed both VASP sh444 and an
RNAi-resistant isoform of VASP (rrhVASP-FH), containing a Flag and His
epitope tag. Knockdown of endogenous VASP was rescued by co-transduction
with RNAi-resistant VASP. H) Zyxin and concurrent endogenous
Zyxin knockdown with sh756 and rescue with RNAi-resistant rrhZyxin-FH (right
panel). I) E006AA cells and Zyxin. J) E006AA cells
[Show abstract][Hide abstract] ABSTRACT: Figure S5. Correlation between cell motility in a 3-D matrix
and cell-matrix interaction parameters. A-E. Correlations between
(A) 3-D HT-1080 cell speed and total bead movement in the matrix (total
matrix deformation), (B) 3-D cell speed and % permanent matrix deformation
(matrix remodeling), (C) growth rate of protrusions and maximum bead
displacement (traction), (D) number of protrusions per 90 min per cell and
maximum bead displacement (traction), (E) 3-D cell speed and maximum bead
displacement per cell (traction per pseudopodia).
[Show abstract][Hide abstract] ABSTRACT: Figure S4. Regulation of length and lifetime of protrusions by
FA proteins. A and B. Maximum length and average protrusion
length of protrusions for various HT-1080 cells depleted of FA proteins.
C. Correlation function between 3-D cell speed and the
lifetime of protrusions. D-F. Correlations between (D) 3-D cell
speed and averaged protrusion length, (E) averaged protrusion length and 3-D
persistence distance, and (F) lifetime of protrusions and 3-D persistence
distance for HT-1080 cells inside a 3-D collagen matrix. Bar graphs show
mean and SEM values of three independent experiments.
[Show abstract][Hide abstract] ABSTRACT: Figure S2. Dependence of 3-D cell speed on β
1 integrin, and correlations between 2-D and 3-D persistent time
and protrusion number and growth rate. A. 3-D cell speed of
control HT-1080 cells, HT-1080 cells in the presence of an anti-β1
integrin function-blocking antibody, and HT-1080 cells in the presence of a
non-specific mouse IgG antibody. NS: P > 0.05. ***: P < 0.001.
Bar graphs show mean and SEM values of three independent experiments.
B. Absence of correlation between the persistence time of
HT-1080 cells placed on conventional 2-D collagen I-coated substrates and
cells placed inside 3-D collagen matrix. C and D. Correlation
function between the number of actively growing protrusions per 90 min per
cell and persistence distance during random cell motility (C) and between
the growth rate of protrusions and the persistence distance for HT-1080
cells inside a 3-D collagen matrix (D).
[Show abstract][Hide abstract] ABSTRACT: Table ST1. Table of correlations among cell motility
parameters, protrusion dynamics parameters, and cell-matrix interaction
parameters. The three numbers in each box represent the slope of
a linear fit between the two parameters considered, the R squared value of
the fit, and the p value. Boxes in red show high statistical significance.
Cell motility parameters include: speed, persistence time, and persistence
distance of cells both on 2-D substrates and inside a 3-D matrix. Protrusion
dynamics parameters include: lifetime, length, maximum length, number of
protrusions per 90 min, and growth rate of individual protrusions.
Cell-matrix interaction parameters include: traction (averaged maximum bead
displacements), total matrix deformation (averaged total bead movement),
matrix remodeling (percent permanent matrix deformation), and traction per
pseudopodia (averaged maximum bead displacements per cell). A strong
correlation is defined as having slope ≥ 0.80 and R2
≥ 0.70 and p ≤ 0.009.
[Show abstract][Hide abstract] ABSTRACT: Figure S3. Regulation of cell speed on a 2-D substrate by FA
proteins in E006AA cells. 2-D cell speed of WT E006AA human
prostate cancer cells and E006AA cells depleted of either p130Cas or zyxin
on collagen coated glass. Bar graphs show mean and SEM values of three
[Show abstract][Hide abstract] ABSTRACT: The enzyme Transglutaminase is used as a novel method of increasing the stiffness of collagen gel without affecting other characteristics. This discovery has allowed for a more accurate analysis of cancer cell motility in a stiffer ECM leading to a potential understanding of the mechanism behind tumor metastasis. Johns Hopkins University, Office of the Provost
[Show abstract][Hide abstract] ABSTRACT: The progression of several human cancers correlates with the loss of cytoplasmic protein alpha-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of alpha-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking alpha-catenin and these cells where alpha-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of alpha-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of alpha-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of alpha-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.
No preview · Article · Jun 2009 · Journal of Biological Chemistry