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Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions.

Fraley SI, Wu PH, He L, Feng Y, Krisnamurthy R, Longmore GD, Wirtz D - Sci Rep (2015)

Bottom Line: However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment.The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified.Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA.

ABSTRACT
Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

No MeSH data available.


Related in: MedlinePlus

Influence of matrix microstructure on cell motility.(A) Reflection confocal micrographs of 3D collagen matrices of increasing collagen density, scale bar is 5 μm. (B) Average extent of collagen fiber alignment measured by FFT analysis in matrices of varying concentrations of collagen. (C) Average pore size of collagen fibers in matrices of varying density. (D) Correlation plot of normalized average pore size vs. alignment of fibers. (E) Global shear elastic modulus of collagen matrices of varying densities. (F) Correlation plot of normalized 3D cell speed vs. alignment of fibers. (G) Correlation plot of normalized 3D cell speed vs. average pore size. (H) Correlation plot of normalized 3D cell speed vs. shear elastic modulus. N = 3 independent gelation repeats for each graph of matrix characteristics; images of at least 5 different positions within the central region of the matrix, far from the container walls, were used from each repeat. Error bars represent s.e.m. ***p < 0.001; **p < 0.01; *p < 0.05.
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f2: Influence of matrix microstructure on cell motility.(A) Reflection confocal micrographs of 3D collagen matrices of increasing collagen density, scale bar is 5 μm. (B) Average extent of collagen fiber alignment measured by FFT analysis in matrices of varying concentrations of collagen. (C) Average pore size of collagen fibers in matrices of varying density. (D) Correlation plot of normalized average pore size vs. alignment of fibers. (E) Global shear elastic modulus of collagen matrices of varying densities. (F) Correlation plot of normalized 3D cell speed vs. alignment of fibers. (G) Correlation plot of normalized 3D cell speed vs. average pore size. (H) Correlation plot of normalized 3D cell speed vs. shear elastic modulus. N = 3 independent gelation repeats for each graph of matrix characteristics; images of at least 5 different positions within the central region of the matrix, far from the container walls, were used from each repeat. Error bars represent s.e.m. ***p < 0.001; **p < 0.01; *p < 0.05.

Mentions: Since several physical properties of the matrix (pore size, stiffness, fibrilar structure, etc.) can change concurrently with changes in collagen density, we aimed to characterize these finer physical details and ask how these features varied with each other and with observed motility responses. Analysis of reflection confocal images of 1–6 mg/ml matrices (Fig. 2A) showed that fiber alignment (measured at length scales of the cell) varied biphasically with collagen concentration (Fig. 2B). The average pore size varied somewhat irregularly, but overall was reduced with increasing collagen concentration (Fig. 2C). These two matrix parameters correlated significantly with one another across all collagen densities (Fig. 2D). Quantitative shear rheometry was used to measure the bulk matrix elasticity, which varied somewhat irregularly with increasing collagen concentration (Fig. 2E). Although previous studies suggest that the elastic modulus should scale positively with increasing collagen concentration2122, it is critical to note that the process by which collagen is extracted23, the gelation conditions2224, and even the thickness of the matrix22 can all significantly alter its mechanical properties and related microstructure25. Collagen density alone, which is often the only information provided in studies, is not sufficiently descriptive to enable direct comparisons. In our collagen matrices, the alignment of fibers decreased significantly when collagen density was increased from 1 mg/ml to 1.5 mg/ml, but the average pore size did not change. That the mesh is not tighter, but is significantly less aligned likely explains the decrease in the elastic modulus from 1–1.5 mg/ml. Then from 1.5 to 2 mg/ml pore size decreases dramatically while fiber alignment drops only slightly. This means that 2 mg/ml represents a tighter mesh, which would be expected to increase the elastic modulus compared to 1.5 mg/ml. Comparing the measured changes in matrix microstructure and mechanics to changes in cell motility revealed a significant correlation of cell speed and invasion distance with both fiber alignment and pore size (Fig. 2F,G) across the varying densities of collagen. Cell speed and invasiveness did not correlate with matrix stiffness, at least within the range of collagen concentrations tested (Fig. 2H).


Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions.

Fraley SI, Wu PH, He L, Feng Y, Krisnamurthy R, Longmore GD, Wirtz D - Sci Rep (2015)

Influence of matrix microstructure on cell motility.(A) Reflection confocal micrographs of 3D collagen matrices of increasing collagen density, scale bar is 5 μm. (B) Average extent of collagen fiber alignment measured by FFT analysis in matrices of varying concentrations of collagen. (C) Average pore size of collagen fibers in matrices of varying density. (D) Correlation plot of normalized average pore size vs. alignment of fibers. (E) Global shear elastic modulus of collagen matrices of varying densities. (F) Correlation plot of normalized 3D cell speed vs. alignment of fibers. (G) Correlation plot of normalized 3D cell speed vs. average pore size. (H) Correlation plot of normalized 3D cell speed vs. shear elastic modulus. N = 3 independent gelation repeats for each graph of matrix characteristics; images of at least 5 different positions within the central region of the matrix, far from the container walls, were used from each repeat. Error bars represent s.e.m. ***p < 0.001; **p < 0.01; *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589685&req=5

f2: Influence of matrix microstructure on cell motility.(A) Reflection confocal micrographs of 3D collagen matrices of increasing collagen density, scale bar is 5 μm. (B) Average extent of collagen fiber alignment measured by FFT analysis in matrices of varying concentrations of collagen. (C) Average pore size of collagen fibers in matrices of varying density. (D) Correlation plot of normalized average pore size vs. alignment of fibers. (E) Global shear elastic modulus of collagen matrices of varying densities. (F) Correlation plot of normalized 3D cell speed vs. alignment of fibers. (G) Correlation plot of normalized 3D cell speed vs. average pore size. (H) Correlation plot of normalized 3D cell speed vs. shear elastic modulus. N = 3 independent gelation repeats for each graph of matrix characteristics; images of at least 5 different positions within the central region of the matrix, far from the container walls, were used from each repeat. Error bars represent s.e.m. ***p < 0.001; **p < 0.01; *p < 0.05.
Mentions: Since several physical properties of the matrix (pore size, stiffness, fibrilar structure, etc.) can change concurrently with changes in collagen density, we aimed to characterize these finer physical details and ask how these features varied with each other and with observed motility responses. Analysis of reflection confocal images of 1–6 mg/ml matrices (Fig. 2A) showed that fiber alignment (measured at length scales of the cell) varied biphasically with collagen concentration (Fig. 2B). The average pore size varied somewhat irregularly, but overall was reduced with increasing collagen concentration (Fig. 2C). These two matrix parameters correlated significantly with one another across all collagen densities (Fig. 2D). Quantitative shear rheometry was used to measure the bulk matrix elasticity, which varied somewhat irregularly with increasing collagen concentration (Fig. 2E). Although previous studies suggest that the elastic modulus should scale positively with increasing collagen concentration2122, it is critical to note that the process by which collagen is extracted23, the gelation conditions2224, and even the thickness of the matrix22 can all significantly alter its mechanical properties and related microstructure25. Collagen density alone, which is often the only information provided in studies, is not sufficiently descriptive to enable direct comparisons. In our collagen matrices, the alignment of fibers decreased significantly when collagen density was increased from 1 mg/ml to 1.5 mg/ml, but the average pore size did not change. That the mesh is not tighter, but is significantly less aligned likely explains the decrease in the elastic modulus from 1–1.5 mg/ml. Then from 1.5 to 2 mg/ml pore size decreases dramatically while fiber alignment drops only slightly. This means that 2 mg/ml represents a tighter mesh, which would be expected to increase the elastic modulus compared to 1.5 mg/ml. Comparing the measured changes in matrix microstructure and mechanics to changes in cell motility revealed a significant correlation of cell speed and invasion distance with both fiber alignment and pore size (Fig. 2F,G) across the varying densities of collagen. Cell speed and invasiveness did not correlate with matrix stiffness, at least within the range of collagen concentrations tested (Fig. 2H).

Bottom Line: However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment.The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified.Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA.

ABSTRACT
Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

No MeSH data available.


Related in: MedlinePlus