Abstract
Cell migration is the directed movement of cells from one location to another often in response to chemical or physical signals in their environment and is essential in various biological phenomena such as wound healing, immune responses, cancer metastasis, and morphogenesis. Furthermore, it is an energetically costly process requiring significant stores of cellular energy in the form of adenosine triphosphate (ATP) to fuel processes such as actin cycling, actomyosin contractility, and phosphorylation of cell adhesion signaling proteins. The major forms of cell migration are single and collective. In single cell migration, individual cells move independently to position themselves within tissues and interstitial spaces. In contrast, collective migration involves groups of cells that remain interconnected, coordinating their movements as a unit. Twenty-first century advancements in tools for manipulating and assaying bioenergetics in live samples support initial investigations into the regulation of metabolism in single cell migration, but studies in collective cell migration remain limited. The mechanical coordination of cell-extracellular matrix (ECM) and cell-cell adhesions underlie efficient collective migration. However, the mechanistic links between cell biomechanics and bioenergetics in the energetically costly process of collective cell migration remain elusive.
In Chapter 2 we report an increase in mitochondrial activity along the leading row of motile Xenopus laevis gastrula-stage mesendoderm cells at sites where fibronectin-𝛂5ꞵ1 integrin substrate traction stresses are greatest. Real-time metabolic analyses demonstrate that 𝛂5ꞵ1 integrin dependent increases in oxidative phosphorylation occur in mesendoderm cells on fibronectin substrates. This elevation in metabolic activity is reduced following pharmacologic inhibition of focal adhesion kinase (FAK) signaling. The integrin-linked mitochondrial oxidative phosphorylation via FAK signaling is conserved in cells from various human tissues. Attachment of mesendoderm cells to fibronectin-Glutathione-S-Transferase (GST) fusion protein fragments that support differing 𝛂5ꞵ1 integrin conformational and ligand-binding affinity states, leads to an increase in mitochondrial activity when both the Arg-Gly-Asp (RGD) and Pro-Pro-Ser-Arg-Asn (PPSRN) synergy sites of fibronectin are engaged by the receptor. Stretching single mesendoderm cells on deformable fibronectin substrates also results in a FAK-dependent increase in mitochondrial membrane potential. Inhibition of mitochondrial membrane potential or mitochondrial ATP synthase activity slows collective cell migration velocity in vivo, further suggesting that integrin-dependent adhesion and signaling contribute to ATP production.
In Chapter 3, I delved into the connections between mitochondrial metabolism and cell adhesion. I demonstrated that aconitase, a citric acid cycle enzyme, function is critical for collective mesendoderm migration in the intact embryo. Based on comparisons of mesendoderm cells on fibronectin-GST fusion proteins, we found that integrin activation fails to affect rates of mitochondrial biogenesis or degradation within the timescale of gastrulation. I observed that mesendoderm treated with manganese to produce integrin activation presented with high mitochondrial membrane potential. Examinations of mesendoderm cell clusters presented mitochondria with a high degree of clustering and high membrane potential near protrusion regions. This is in addition to clustering of mitochondria near the leading edges of mesendoderm cells in the migratory tissue. This was in addition into our observation of accumulations of mitochondria close to C-Cadherin adhesions in dissociated cells.
Finally, in Chapter 4 I aimed to further examine the role of metabolism in adhesive processes during gastrulation. I presented the preliminary metabolomics studies on Xenopus laevis gastrulae in the presence or absence of a fibronectin morpholino. Notably, metabolites related to pyrimidine and aromatic amino acid processes showed the greatest changes due to the knockdown. The potential significance of these processes in the developmental processes of gastrulation and future studies are discussed. In sum, this dissertation highlights an underexplored link between ECM-integrin adhesion and mitochondrial metabolism in early embryonic cell migration. We propose that fibronectin-integrin engagement provides an important signal for shaping the metabolic landscape of collectively migrating cells. Continued study of the interplay between adhesive processes and metabolism will inform our approaches for targeting dysregulated cell migration in pathological contexts.
