Lipid signaling affects primary fibroblast collective migration and anchorage in response to stiffness and microtopography

Abstract

Cell migration is regulated by several mechanotransduction pathways, which consist of sensing and converting mechanical microenvironmental cues to internal biochemical cellular signals, such as protein phosphorylation and lipid signaling. While there has been significant progress in understanding protein changes in the context of mechanotransduction, lipid signaling is more difficult to investigate. In this study, physical cues of stiffness (10, 100, 400 kPa, and glass), and microrod or micropost topography were manipulated in order to reprogram primary fibroblasts and assess the effects of lipid signaling on the actin cytoskeleton. In an in vitro wound closure assay, primary cardiac fibroblast migration velocity was significantly higher on soft polymeric substrata. Modulation of PIP2 availability through neomycin treatment nearly doubled migration velocity on 10 kPa substrata, with significant increases on all stiffnesses. The distance between focal adhesions and the lamellar membrane (using wortmannin treatment to increase PIP2 via PI3K inhibition) was significantly shortest compared to untreated fibroblasts grown on the same surface. PIP2 localized to the leading edge of migrating fibroblasts more prominently in neomycin-treated cells. The membrane-bound protein, lamellipodin, did not vary under any condition. Additionally, fifteen micron-high micropost topography, which blocks migration, concentrates PIP2 near to the post. Actin dynamics within stress fibers, measured by fluorescence recovery after photobleaching, was not significantly different with stiffness, microtopography, nor with drug treatment. PIP2-modulating drugs delivered from microrod structures also affected migration velocity. Thus, manipulation of the microenvironment and lipid signaling regulatory drugs might be beneficial in improving therapeutics geared toward wound healing.

Examination of lipid profiles in abdominal fascial healing using MALDI-TOF to identify potential therapeutic targets

“…Fibroblasts contribute significantly to wound healing, creating extracellular matrix components and contracting the wound [48] . Lipid signalling can affect fibroblast proliferation [49] , [50] . The plasma membrane of fibroblasts contains micrometer-scale patches enriched with sphingolipids that can metabolise to signalling molecules regulating cell survival and proliferation [51] , [52] .…”

Hang on tight: reprogramming the cell with microstructural cues

“…Lipid signaling at the cell membrane has also been shown to rapidly affect focal adhesion assembly, facilitating conversion of mechanical cues to signaling events. Phosphatidylinositol 4,5-biphosphate (PIP2) signaling has been shown to modulate organization of the actin cytoskeleton and lamellar architecture (Mkrtschjan et al 2018a). In the context of focal adhesions and the actin cytoskeleton, PIP2 plays key roles in the recruitment of adaptor proteins, such as vinculin, talin and paxillin, to the focal adhesions enabling propagation of forces to the cytoskeleton (Zhang et al 2012).…”

“…Loading of a drug into microrods may also be useful for wound healing applications. PEGDMA microrods were loaded with high concentrations of neomycin, a small molecule drug capable promoting cell migration in vitro through modulation of lipid signaling, and delivery was maintained for over 12 h. When cultured with primary rat primary fibroblasts, the drug released to the media significantly increased the migration velocity of cells into the gap in a wound closure assay (Mkrtschjan et al 2018a). Hence, development of these neomycin microrod devices for a therapeutic application might improve wound healing.…”

Extracellular matrix mineralization in the mouse osteoblast-like cell line MC3T3-E1 is regulated by actin cytoskeleton reorganization and non-protein molecules secreted from the cells themselves

“…Alexa Fluor fluorescent antibody staining [32]. PIP 2 is a phospholipid that is located in the cell membrane and is a known substrate for a number of important signaling proteins and is related to actin fiber assembly [33][34][35][36][37].…”