@article{ott_sung_melvin_sheats_haugh_adler_jones_2013, title={Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein}, volume={8}, ISSN={["1932-6203"]}, url={http://europepmc.org/abstract/med/23840497}, DOI={10.1371/journal.pone.0066512}, abstractNote={Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.}, number={6}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Ott, Laura E. and Sung, Eui Jae and Melvin, Adam T. and Sheats, Mary K. and Haugh, Jason M. and Adler, Kenneth B. and Jones, Samuel L.}, editor={Aspenstrom, PontusEditor}, year={2013}, month={Jun} }
 @article{welf_ahmed_johnson_melvin_haugh_2012, title={Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism}, volume={197}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201108152}, abstractNote={Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.}, number={1}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Welf, Erik S. and Ahmed, Shoeb and Johnson, Heath E. and Melvin, Adam T. and Haugh, Jason M.}, year={2012}, month={Apr}, pages={105–114} }
 @article{melvin_welf_wang_irvine_haugh_2011, title={In Chemotaxing Fibroblasts, Both High-Fidelity and Weakly Biased Cell Movements Track the Localization of PI3K Signaling}, volume={100}, ISSN={["0006-3495"]}, DOI={10.1016/j.bpj.2011.02.047}, abstractNote={Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.}, number={8}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Melvin, Adam T. and Welf, Erik S. and Wang, Yana and Irvine, Darrell J. and Haugh, Jason M.}, year={2011}, month={Apr}, pages={1893–1901} }
 @article{weiger_wang_krajcovic_melvin_rhoden_haugh_2009, title={Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts}, volume={122}, ISSN={["1477-9137"]}, DOI={10.1242/jcs.037564}, abstractNote={During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3′-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3′-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts.}, number={3}, journal={JOURNAL OF CELL SCIENCE}, publisher={The Company of Biologists}, author={Weiger, Michael C. and Wang, Chun-Chao and Krajcovic, Matej and Melvin, Adam T. and Rhoden, John J. and Haugh, Jason M.}, year={2009}, month={Feb}, pages={313–323} }