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.

1. Ingenuity Pathway Analysis
<ol>
	<li>Uploading Protein-interactome Dataset
	<ol>
		<li>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).</li>
		<li>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 &#39;contains column header&#39; option to view the column headings.</li>
		<li>Up...

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

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...

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. Preferential accumulations of cAMP and cAMP-dependent kinase PKA activity at the leading edge play key roles in directional cell migration5,6. By phosphorylating small GTPases such as Ras-related C3 botulinum toxin substrate (Rac) and cell division control protein 42 homolog or Cdc42, PKA activates actin-related protein 2/3 (Arp 2/3) at the leading edge and induces the formation of lamellipodia7-9. PKA also phosphorylates an anti-capping agent, vasodilator stimulated phosphoprotein (VASP), thereby regulates the oscillatory cycles of membrane extension and retraction10,11.
In cells, cAMP levels are regulated by three major processes: i) synthesis by adenylate cyclase, ii) degradation by phosphodiesterases, and iii) transportation by membrane-bound efflux transporters3. Multidrug resistance protein 4 (MRP4), a member of ATP-binding cassette (ABC) family of membrane transporters, functions as an endogenous efflux transporter of cyclic nucleotides. Therefore, MRP4 can regulate intracellular cAMP levels and cAMP-dependent cellular signaling11-13. We have previously shown that in Mrp4-/-, fibroblasts contain relatively higher levels of cyclic nucleotides and migrate faster compared to Mrp4+/+ fibroblasts14. We also reported a biphasic effect of cyclic nucleotides on fibroblast migration. Based on previous studies and our finding that Mrp4-/- fibroblasts contain more polarized cAMP during the course of migration, we hypothesized that this MRP4-mediated regulation of fibroblast migration is cAMP dependent. In order to understand the downstream mechanism, we took a direct yet multifaceted approach.
To identify the proteins associated and in interplay with MRP4, we immunoprecipitated MRP4-containing macromolecular complexes from HEK293 cells that over express MRP4. Using mass-spectrometry, we identified multiple MRP4-interacting proteins and analyzed their interconnectivity using Ingenuity Pathway Analysis (IPA). IPA is a useful tool to analyze protein-protein interactions (both structural and functional) and explore their contributions in particular physiological and pathological events based on the literature and experimental evidences15,16. IPA indicated that F-actin is a major downstream target of MRP4 in the context of cell migration where cAMP and cGMP are the key signaling molecules17. These data were further confirmed by high-content microscopy. High-content microscopy can capture and analyze cell behaviors such as cell migration in a more convenient, accurate and high-throughput manner18. The high-content microscopy data demonstrated that the effect of MRP4 on fibroblast migration is completely abolished upon disruption of the actin cytoskeleton or inhibition of PKA17.
Additionally, we used a F&#246;rster resonance energy transfer (FRET)-based PKA sensor to monitor the PKA dynamics in migrating cells in real time. FRET-based kinase sensors usually consist of specific phosphorylation substrate peptides flanked by CFP and YFP fluorophores19-21. pmAKAR3 is an improved and membrane targeted FRET-based PKA sensor that contains forkhead-associated domain 1 (FHA1) and the PKA substrate sequence LRRATLVD5,22. Phosphorylation of pmAKAR3 by the PKA catalytic subunit increases FRET signal between CFP and YFP19. Insertion of a lipid modification domain into the sensor targets it to the plasma membrane for monitoring PKA dynamics, specifically at the membrane compartment23.
Using pmAKAR3, we demonstrated that the leading edge of migrating Mrp4-/- fibroblasts exhibited more polarized PKA activity than Mrp4+/+ fibroblasts, which in turn increased the cortical actin formation at the cell&#39;s leading edge17. Together, these events resulted in better cellular polarization and faster directional cell migration in the absence of MRP4. Our specific and direct approach identified key downstream targets for MRP4 and provides an important, but as of yet unexplored mechanism for MRP4-dependent regulation of fibroblast migration.

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			<td height="21" style="height:21px;width:272px;">Lipofectamine 2000</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
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			<td>Invitrogen (Carlsbad, CA)&#160;</td>
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			<td height="21" style="height:21px;">96-well IncuCyte Image-Lock microplates&#160;</td>
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			<td style="width:187px;">4493</td>
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			<td height="21" style="height:21px;">Latrunculin B</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>L5288</td>
			<td>Stock in DMSO</td>
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			<td height="21" style="height:21px;">H-89</td>
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			<td>BML-EI196</td>
			<td>Stock in DMSO</td>
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			<td height="21" style="height:21px;">35-mm glass-bottomed dishes&#160;</td>
			<td style="width:324px;">(MatTek Corporation; Ashland, MA)</td>
			<td style="width:187px;">P35G-1.5-20-C&#160;</td>
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			<td height="21" style="height:21px;width:272px;">Fibronectin</td>
			<td style="width:324px;">Sigma-Aldrich (St. Louis, MO).</td>
			<td>F1141</td>
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			<td height="21" style="height:21px;width:272px;">Opti-MEM Reduced Serum Media</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">31985-088</td>
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			<td height="21" style="height:21px;width:272px;">FRET microscopy system</td>
			<td>Olympus inverted microscope (IX51)</td>
			<td style="width:187px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:272px;">CCD camera&#160;</td>
			<td style="width:324px;">Hamamatsu, Japan</td>
			<td style="width:187px;">ORCA285</td>
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			<td height="21" style="height:21px;width:272px;">SlideBook software 5.5</td>
			<td style="width:324px;">Intelligent Imaging Innovation ( Denver, CO)</td>
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			<td height="21" style="height:21px;">Ingenuity Pathway Analysis software</td>
			<td>IPA, QIAGEN Redwood City,</td>
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			<td height="21" style="height:21px;">Forskolin</td>
			<td>Tocris (Ellisville, MO).&#160;</td>
			<td style="width:187px;">1099</td>
			<td>Stock in100% EtOH</td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM F-12&#160;&#160;</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">11330-057</td>
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			<td height="21" style="height:21px;width:272px;">HBSS</td>
			<td>Invitrogen (Carlsbad, CA)&#160;</td>
			<td style="width:187px;">14025-134</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Excel</td>
			<td style="width:324px;">Microsoft</td>
			<td style="width:187px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:272px;">PBS</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">10010-023</td>
			<td></td>
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			<td height="21" style="height:21px;width:272px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penicillin-Streptomycin</td>
			<td style="width:324px;">Invitrogen(Carlsbad, CA)&#160;</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
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methods to study mrp4-containing macromolecular complexes in the 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.

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...

Watch this Scientific Journal Video about Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration at JoVE.com

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

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 ...

The overall goal of this experimental procedure is to observe the cyclic AMP dependent regulation of fibroglast migration by MRP4.High contact microscopy can help answer key questions in the field of cell biology.For example, in addition to conferring germ resistance, MRP4 can also regulate physiological process such as cell migration by modulating intracellular cyclic nucleotide concentration.The main advantage of FRET imaging that we are going to attempt today is to allow monitoring of the intracellular dynamics of cyclic AMP and cyclic AMP dependent kinase in a real-time fashion.The implication of high contact microscopy is it can be used in drug discovery.Once the system is set up, all the instrument is set up, there is no human intervention involved in this plate-based task.Though this method can provide insight into cell migration, it can be applied to other systems, such as lipe cell nonparturbing, high contact imaging of immune cell killing, cooler dilution, angiogenesis, cell proliferation, chemotaxis, and treatese phyllite.To prepare cells for high-contact microscopy under a cell culture hood, use a one percent fibronectin solution in PBS to coat the wells of a 96 well plate.Incubate the plate in a CO2 incubator at 37

methods-to-study-mrp4-containing-macromolecular-complexes-regulation

Research

JoVE Journal

Developmental Biology

6.6K vistas.  Cincinnati Children’s Hospital Medical Center. The overall goal of this experimental procedure is to observe the cyclic AMP dependent regulation of fibroglast migration by MRP4.High contact microscopy can help answer key questions in the field of cell biology.For example, in addition to conferring germ resistance, MRP4 can also regulate physiological process such as cell migration by modulating intracellular cyclic nucleotide concentration.The main advantage of FRET imaging that we are going to attempt today is to allow monitoring of the ...