Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds

Abstract

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction

“…For this, we used the previously described non-electrospinning spinneret based tunable engineered parameters (STEP 35,36 ) method to fabricate anisotropic/aligned fibers. These could also serve as force sensing nanonets, as we described previously [32][33][34] . We followed, in realtime, the protrusions of different cell lines and proceeded to examine which behaviors, if any, were unique to fibroblastic cells (immortalized murine C2C12, immortalized embryonic fibroblast (MEF), and well-established NIH-3T3) and human mesenchymal stem cells (hMSCs) as well as Hela cells ( Supplementary Fig.…”

“…First, we quantitated the forces exerted by both twines and PLPs using nanonet force microscopy (NFM 32 – 34 , Fig. 6a(i) ) through the establishment of tension-bearing force vectors directed toward the cell body.…”

“…Thus, we inquired how forces developed in 3D-PLPs, and also investigated the resultant migratory and force response of cells as they spread from three to four and five fibers using 3D-PLPs. First, we quantitated the forces exerted by both twines and PLPs using nanonet force microscopy (NFM [32][33][34] , Fig. 6a(i)) through the establishment of tension-bearing force vectors directed toward the cell body.…”

“…We observed the protrusions formed in an integrin-dependent manner on small diameter fibers, and notably suspended flat ribbons of equivalent widths did not capture protrusive sensitivity to curvature 30 . Here, using aligned and suspended fiber nanonets that also act as force sensors 32 – 34 , we report the identification of force exerting side protrusions termed ‘3D perpendicular lateral protrusions (3D-PLPs)’ that do not require pre-existing fibers to extend upon. We quantitatively show that anisotropic ECMs, represented by suspended and aligned nanonets as well as CAF derived ECMs, promote production of PLPs to enable fibroblastic cell contraction, potentially explaining the single cell/ECM physical characteristics of desmoplastic expansion.…”

Recent advances in understanding the role of metabolic heterogeneities in cell migration

“…A Rex probe paired with yellow fluorescent protein reported that neural crest migration depends heavily on glycolysis and is abrogated in cells utilizing OXPHOS 62 . This has been echoed in recent studies, where increased glycolysis and its intermediates were linked to increased migration 29 , 63 – 68 . These probes allow visualization of the inherent metabolic plasticity of cells and how migratory phenotypes are altered when cells utilize different energy pathways.…”