Failure to heal wounds and scarring (the opposite) constitute major medical challenges, afflicting African Americans to the greatest extent. These pathologies often arise from dysregulation of cell repopulation of the (missing) tissue. During tissue (re)generation and engineered repair stromal fibroblasts, endothelial cells, and stem cells migrate into the provisional matrix to form both supporting matrix and vasculature. The initial cel immigration is directional, driven by signals, `cues', that arise from within the wound bed. However, once within the tissue, the cells must distribute without evident stimuli gradients. Cells can locomote productively even in isotropic environments, suggesting that a major component is cell-intrinsic. Our long-term goal is to determine how cells establish and then maintain progressive motility to repopulate tissues in response to external stimuli. Adherent cells undertake two forms of motility based on both adhesive properties and tightness of weave (or pore size) of the matrix. The key controls that convert actin cytoskeletal effects to locomotion and provide directionality have been defined by others and us during current and prior grant periods. Less frequently, as these cells transiently shift to fit through small pores and when encountering low-adhesive matrices (such as the initial fibrin-rich clots), the cells compact and move forward by following blebs, in amoeboid motility. We propose that these two modes of motility are actually inter-related using a key, still to-be-defined regulator of cytoskeletal-membrane interactions. The findings during the current grant period found that alpha-actinin-4 (ACTN4) can bridge the actin cytoskeleton to the membrane and that its interaction with actin filament is regulated by both growth factor-triggered tyrosyl-phosphorylations and calpain-mediated cleavages, both of which modulate actin-binding capabilities. Further, ACTN4 can either bind actin filaments or phospho-inositides, suggesting a mechanism coupling the anti-parallel dimer of ACTN4 to bridge the cytoskeleton to the membrane. Thus, we hypothesize that the ACTN4 is a master regulator of cytoskeleton-membrane connectivity, and that it is asymmetrically regulated by calpain cleavages during both mesenchymal and amoeboidal motility. We will determine whether: I. ACTN4 is `locked' into an actin-bound conformation by calpain-2 to bring the rear of the cell forward. II. ACTN4 bridging of the membrane is loosened by a unique tandem but dependent phosphorylation motif. III. Amoeboid motility involves asymmetric signaling responses integrating PIPs and ACTN4 enabling blebbing. Accomplishment of these investigations will define molecular bases of the spatial restriction of receptor signaling pathways and resultant biophysical responses critical to migration into cell repopulation, providing missing information vital for understanding tissue regeneration. This knowledge will enable the design on a subcellular scale of `smart' scaffolds and materials for cell and tissue engineering of intrinsic and applied cells directing the synthesis of both the matrx and the vascular bed that is required to support tissue function.

Public Health Relevance

The orchestrated movement of many types of cells, controlled by signals from the external milieu or microenvironment, is crucial for organogenesis and wound repair. An outstanding question remains how does a cell determine how to organize its intracellular machinery for productive motility. Testing the proposed foundational model of phospho-inositide distribution dictating ACTN4 functioning and localizing the biochemical and biophysical processes would provide novel insights into the basic cell biology of wound repair and allow for future development of smart surfaces that could harness this information to direct reparative cells into regions of injury and control stem cells for regenerative therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069668-14
Application #
9204837
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
2004-01-15
Project End
2019-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
14
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Shao, Hanshuang; Lauffenburger, Douglas; Wells, Alan (2017) Tyro3 carboxyl terminal region confers stability and contains the autophosphorylation sites. Biochem Biophys Res Commun 490:1074-1079
Shao, Hanshuang; Wang, Anna; Lauffenburger, Douglas et al. (2017) Tyro3-mediated phosphorylation of ACTN4 at tyrosines is FAK-dependent and decreases susceptibility to cleavage by m-Calpain. Int J Biochem Cell Biol 95:73-84
Yates, Cecelia C; Rodrigues, Melanie; Nuschke, Austin et al. (2017) Multipotent stromal cells/mesenchymal stem cells and fibroblasts combine to minimize skin hypertrophic scarring. Stem Cell Res Ther 8:193
Wells, Alan; Nuschke, Austin; Yates, Cecelia C (2016) Skin tissue repair: Matrix microenvironmental influences. Matrix Biol 49:25-36
Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan et al. (2016) Time series modeling of live-cell shape dynamics for image-based phenotypic profiling. Integr Biol (Camb) 8:73-90
Nuschke, Austin; Rodrigues, Melanie; Wells, Albin W et al. (2016) Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation. Stem Cell Res Ther 7:179
Nuschke, Austin; Rodrigues, Melanie; Rivera, Jaime et al. (2016) Epidermal Growth Factor Tethered to ?-Tricalcium Phosphate Bone Scaffolds via a High-Affinity Binding Peptide Enhances Survival of Human Mesenchymal Stem Cells/Multipotent Stromal Cells in an Immune-Competent Parafascial Implantation Assay in Mice. Stem Cells Transl Med 5:1580-1586
Bodnar, Richard J; Satish, Latha; Yates, Cecelia C et al. (2016) Pericytes: A newly recognized player in wound healing. Wound Repair Regen 24:204-14
Shao, Hanshuang; Kirkwood, John M; Wells, Alan (2015) Tenascin-C Signaling in melanoma. Cell Adh Migr 9:125-30
Travers, Timothy; Shao, Hanshuang; Joughin, Brian A et al. (2015) Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function. Sci Signal 8:ra51

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