Mechanometabolism of Cell Adhesion: Vinculin regulates cell bioenergetics in a ROCK dependent manner

Introduction: Physiological processes, such as wound healing, cell proliferation, and metastasis, require energy. The energetic demands needed to support these processes are due in part to cytoskeletal remodeling, an energy-intensive process. The actin cytoskeleton is directly linked to focal adhesions (FAs), where vinculin (Vcl), a primary constituent of FAs, plays a role in regulating its organization and dynamics. Vcl expression levels have been shown to alter the migratory behavior of cells. Additionally, bioenergetics is a defining determinant of cell migration since actin cytoskeleton remodeling requires ATP to fuel the actomyosin complex. However, our understanding of cellular bioenergetics in response to actin cytoskeleton disruption and how this may affect downstream events, such as cell migration and proliferation, remains poorly understood. Our prior work indicates that knocking out Vcl in metastatic MDA-MB-231 (MDA) breast cancer cells results in the cells losing their ability to polarize active mitochondria to the front of the cell during confined migration [1]. This suggests that Vcl may play a prominent role in regulating energetic machinery during cell migration and has raised the question of “Does Vcl regulate energy levels in a cell?” Since actin cytoskeleton dynamics require energy, and Vcl aids in modulating connections between F-actin and FAs, we hypothesized that Vcl alters bioenergetics of cancer cells via regulating dynamics of the actin cytoskeleton.

Materials and

Methods: A Vcl knockout (KO) cell line was generated using electroporation to transfect MDA cells with CRISPR/Cas9 targeting Vcl. A Vcl rescue cell line was produced by transducing Vcl KO cells with Vcl tagged with mVenus (Vcl-mVenus), in addition to an empty vector control cell line (Vcl KO + pRRL). ATP:ADP was quantified in live cells using the ratiometric signal from PercevalHR. Glucose uptake was assessed using 2-NBDG, and mitochondrial membrane potential was studied using tetramethylrhodamine, methyl ester (TMRM) dye. Cell proliferation was quantified using the percentage of EdU incorporation in thymidine bases during S-phase. Cell migration was tracked using time-lapse microscopy, and centroid displacement was used to determine cell speed in Fiji. Color-coded time lapses were made by manually outlining cells in Fiji. For Rho kinase (ROCK) inhibition experiments, cells were incubated in media containing 20 µM Y27632 (Y27) or the vehicle control (deionized water) for 20 hours. Statistical analysis was performed using GraphPad Prism 10. For comparing two groups, data were compared using either the unpaired t test or Mann-Whitney test, where appropriate. For comparing more than two groups, Kruskal-Wallis analysis of variance testing was used followed by a post hoc Tukey’s test.

Results, Conclusions, and Discussions:

Results: Since Vcl stabilizes the linkage of the actin cytoskeleton to FAs, facilitating the regulation of actin dynamics, and actin cytoskeletal dynamics are energetically demanding, we investigated the effect of Vcl depletion on bioenergetics. We found that Vcl deficiency led to higher ATP:ADP, glucose uptake, and mitochondrial membrane potential (Fig. 1A-B), suggesting that Vcl-deficient cells exhibit increased energy production and utilization. To investigate the impact of Vcl-deficiency induced increases in energetics, we assessed migration and proliferation, energetically demanding processes, and interestingly, did not observe enhanced proliferation or faster migration (Fig. 1C-D). Since actin cytoskeletal dynamics are energy intensive, we next more closely examined cell behavior over time, finding that Vcl KO cells experience greater changes in cell shape and dynamic bleb-like protrusions (Fig. 1E). To determine if the dynamic protrusion behavior contributes to the increased energetics in the Vcl KO cells, we treated cells with Y27 to inhibit ROCK, disrupting myosin functionality, since myosin activity can drive blebbing. Y27 caused the Vcl KO cells to suppress both dynamic protrusions and energetics (Fig. 1E-G), suggesting that without Vcl, cells undergo more frequent changes in cell shape, supported by actin reorganization, requiring more energy.

Discussion: We demonstrated that Vcl participates in determining the bioenergetic phenotype of cells. Vcl KO cells exhibit increased energetics, but a subsequent reduction in energetics following Y27 treatment, suggesting Vcl contributes to changes in energetics through its role in mediating actomyosin activity. Without Vcl, cell blebbing increases concomitant with energy, implying that energetics drive cell shape changes. Since Vcl links the actin cytoskeleton to matrix-bound integrins via FAs, our data suggests that without Vcl, cells may be over-activating RhoA, which further activates its effector ROCK, to drive blebbing and a potential switch in migration mode when this actin-matrix linkage is interfered with.

Conclusions: We showed that Vcl-deficient cells exhibit energy production dependent on ROCK driven changes in cell shape. Since altered energetics often lead to changes in fundamental cell behaviors, understanding the link between cell-matrix adhesion and bioenergetics, and the mechanisms facilitating these changes, may aid in elucidating therapeutic avenues.

References: [1] Mosier et al. Nanoscale Adv. 2023

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