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Developmental Biology

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Published: May 19th, 2016

DOI:

10.3791/53973

1Division of Pulmonary Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, 2Department of Physiology, University of Tennessee Health Science Center
* These authors contributed equally

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

Cell migration is a complicated multi-step process. Studies have shown that during migration cells are polarized into leading and trailing edges. By adhering to the extracellular matrix, the leading edge provides the traction necessary for the cell body to move forward. Finally, the trailing edge releases rear attachments and completes the migration cycle1,2.

Cell polarization for efficient cell migration is regulated by spatial segregation of intracellular signaling. Cellular second messengers, such as cAMP, mediate the compartmentalization of the signaling events required for fine-tuned directional cell migration3,4.....

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1. Ingenuity Pathway Analysis

  1. Uploading Protein-interactome Dataset
    1. Insert the proteins/genes of interest in a spreadsheet with their unique gene identifiers (preferably gene symbols and gene identifier numbers as obtained with mass spectrometric data).
    2. Assign one column in the spreadsheet for Gene identifier number and one column for observational value (e.g., Fold-change or p-value). Select the 'contains column header' option to view the column headings.
    3. Up.......

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To study the effect of MRP4 on fibroblast migration, we used a wound-healing assay utilizing high-content microscopy14. Precise wounds were made on confluent monolayers of MEFs isolated from either Mrp4-/- or Mrp4+/+ mice, and images were taken at 1 hr intervals for 24 hr. We observed a higher migration rate for Mrp4-/- MEFs compared to Mrp4+/+ MEFs (Figur.......

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Cell migration is an intricate process that plays indispensable roles in many important physiological events including wound healing1,2. Aberrant cell migrations may cause catastrophic events, such as tumor metastasis and angiogenesis24,25. Therefore, fine-tuned regulation of cell migration is required to maintain normal body function.

Using high-content microscopy18, we demonstrated that MRP4-deficient MEFs migrate faster compared to wild-type fibroblasts.......

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This work was supported by National Institutes of Health grants R01-DK080834 and R01-DK093045. We thank J. Denise Wetzel, CCHMC Medical Writer, for editing of the manuscript.

....

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Name Company Catalog Number Comments
Lipofectamine 2000 Invitrogen(Carlsbad, CA)  11668-027
DMEM Invitrogen (Carlsbad, CA)  11965-092
IncuCyte Zoom Essen BioScience
96-well IncuCyte Image-Lock microplates  Essen BioScience 4493
Latrunculin B Sigma-Aldrich (St. Louis, MO). L5288 Stock in DMSO
H-89 Enzo Life Sciences (Farmingdale, NY) BML-EI196 Stock in DMSO
35-mm glass-bottomed dishes  (MatTek Corporation; Ashland, MA) P35G-1.5-20-C 
Fibronectin Sigma-Aldrich (St. Louis, MO). F1141
Opti-MEM Reduced Serum Media Invitrogen (Carlsbad, CA)  31985-088
FRET microscopy system Olympus inverted microscope (IX51)
CCD camera  Hamamatsu, Japan ORCA285
SlideBook software 5.5 Intelligent Imaging Innovation ( Denver, CO)
Ingenuity Pathway Analysis software IPA, QIAGEN Redwood City,
Forskolin Tocris (Ellisville, MO).  1099 Stock in100% EtOH
DMEM F-12   Invitrogen (Carlsbad, CA)  11330-057
HBSS Invitrogen (Carlsbad, CA)  14025-134
Excel Microsoft
PBS Invitrogen(Carlsbad, CA)  10010-023
Trypsin/EDTA Solution (TE) Invitrogen(Carlsbad, CA)  R-001-100
Penicillin-Streptomycin Invitrogen(Carlsbad, CA)  15140-122

  1. Ridley, A. J., et al. Cell migration: integrating signals from front to back. Science. 302 (5651), 1704-1709 (2003).
  2. Lauffenburger, D. A., Horwitz, A. F. Cell migration: a physically integrated molecular process. Cell. 84 (3), 359-369 (1996).
  3. Arora, K., et al. Compartmentalization of cyclic nucleotide signaling: a question of when, where, and why?. Pflugers Arch. 465 (10), 1397-1407 (2013).
  4. Howe, A. K., Baldor, L. C., Hogan, B. P. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration. Proc Natl Acad Sci U S A. 102 (40), 14320-14325 (2005).
  5. Lim, C. J., et al. Integrin-mediated protein kinase A activation at the leading edge of migrating cells. Mol Biol Cell. 19 (11), 4930-4941 (2008).
  6. Paulucci-Holthauzen, A. A., et al. Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc. J Biol Chem. 284 (9), 5956-5967 (2009).
  7. Weaver, A. M., Young, M. E., Lee, W. L., Cooper, J. A. Integration of signals to the Arp2/3 complex. Curr Opin Cell Biol. 15 (1), 23-30 (2003).
  8. Le Clainche, C., Carlier, M. F. Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev. 88 (2), 489-513 (2008).
  9. Raftopoulou, M., Hall, A. Cell migration: Rho GTPases lead the way. Dev Biol. 265 (1), 23-32 (2004).
  10. Krause, M., Dent, E. W., Bear, J. E., Loureiro, J. J., Gertler, F. B. Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol. 19, 541-564 (2003).
  11. Hara, Y., et al. Inhibition of MRP4 prevents and reverses pulmonary hypertension in mice. J Clin Invest. 121 (7), (2011).
  12. Russel, F. G., Koenderink, J. B., Masereeuw, R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux tra (7), 2888-289nsporter for drugs and signalling molecules. Trends Pharmacol Sci. 29 (4), 200-207 (2008).
  13. Cheepala, S., et al. Cyclic nucleotide compartmentalization: contributions of phosphodiesterases and ATP-binding cassette transporters. Annu Rev Pharmacol Toxicol. 53, 231-253 (2013).
  14. Sinha, C., et al. Multi-drug resistance protein 4 (MRP4)-mediated regulation of fibroblast cell migration reflects a dichotomous role of intracellular cyclic nucleotides. J Biol Chem. 288 (6), 3786-3794 (2013).
  15. Popovici, C., et al. Direct and heterologous approaches to identify the LET-756/FGF interactome. BMC Genomics. 7 (105), (2006).
  16. Soler-Lopez, M., Zanzoni, A., Lluis, R., Stelzl, U., Aloy, P. Interactome mapping suggests new mechanistic details underlying Alzheimer's disease. Genome Res. 21 (3), 364-376 (2011).
  17. Sinha, C., et al. PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration. Cell Signal. 27 (7), 1345-1355 (2015).
  18. Liu, L., Wang, Y. D., Wu, J., Cui, J., Chen, T. Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR-dependent motility in alveolar rhabdomyosarcoma cells. BMC Cancer. 12 (154), (2012).
  19. Zhang, J., Ma, Y., Taylor, S. S., Tsien, R. Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A. 98 (26), 14997-15002 (2001).
  20. Sinha, C., et al. Forster resonance energy transfer - an approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling. Biochim Biophys Acta. 1840 (10), 3067-3072 (2014).
  21. Sato, M., Ozawa, T., Inukai, K., Asano, T., Umezawa, Y. Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat Biotechnol. 20 (3), 287-294 (2002).
  22. Allen, M. D., Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun. 348 (2), 716-721 (2006).
  23. Ananthanarayanan, B., Ni, Q., Zhang, J. Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity. Proc Natl Acad Sci U S A. 102 (42), 15081-15086 (2005).
  24. Yamaguchi, H., Condeelis, J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta. 1773 (5), 642-652 (2007).
  25. Lamalice, L., Le Boeuf, F., Huot, J. Endothelial cell migration during angiogenesis. Circ Res. 100 (6), 782-794 (2007).
  26. Ghosh, M. C., Makena, P. S., Gorantla, V., Sinclair, S. E., Waters, C. M. CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2. Am J Physiol Lung Cell Mol Physiol. 302 (9), L846-L856 (2012).
  27. Vicente-Manzanares, M., Webb, D. J., Horwitz, A. R. Cell migration at a glance. J Cell Sci. 118 (Pt 21), 4917-4919 (2005).
  28. Zaccolo, M., et al. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol. 2 (1), 25-29 (2000).

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