Ranjini Krishnamurthy

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (3)24.25 Total impact

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    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
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    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 and P130Cas.
    Preview · Dataset · May 2010
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    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).
    Preview · Dataset · May 2010
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    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.
    Preview · Dataset · May 2010
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    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).
    Preview · Dataset · May 2010
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    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.
    Preview · Dataset · May 2010
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    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 independent experiments.
    Preview · Dataset · May 2010
  • Denis Wirtz · Stephanie Fraley · Ranjini Krishnamurthy
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    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
    No preview · Article · Nov 2009
  • Saumendra Bajpai · Yunfeng Feng · Ranjini Krishnamurthy · Gregory D Longmore · Denis Wirtz
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    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

Publication Stats

282 Citations
24.25 Total Impact Points

Institutions

  • 2009-2010
    • Johns Hopkins University
      • Department of Chemical and Biomolecular Engineering
      Baltimore, Maryland, United States