Adhesion-mediated mechanosignaling forces mitohormesis

Abstract

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programmed cell death. The biochemical milieu regulates mitochondrial function and contributes to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of ageing. Extracellular matrix stiffness and cytoskeletal tension are also altered in cancer and in aged tissues. We 5 determined that cytoskeletal tension elicits a mitochondrial stress response that modifies mitochondrial function via SLC9A1-dependent ion exchange and HSF1-dependent transcription. Our data indicate that this cytoskeletal tension-induced mitochondrial stress response, termed mitohormesis, adaptively tunes mitochondrial metabolism and facilitates oxidative stress resilience. These findings demonstrate that cytoskeletal 10 tension regulates mitochondrial function and suggests that mechanical forces influence tissue behavior by modulating mitochondrial metabolism.One Sentence Summary: Adhesion-mediated mechanotransduction programs mitochondrial metabolism through an adaptive stress response.Main Text: Nutrient availability, oxygen content, and pH regulate cellular metabolism. 15Extracellular matrix (ECM) stiffness can change cellular metabolism by regulating the levels and/or activity of cytoplasmic enzymes responsive to cytoskeletal polymerization (1, 2). Many aspects of cellular metabolism depend upon the mitochondria, a key metabolic organelle that is structurally altered by physical forces and the cytoskeleton (3-5). Since mitochondrial structure influences mitochondrial function, we sought to 20 determine if ECM stiffness and cytoskeletal tension influence cellular metabolism by regulating mitochondrial structure and function. Cytoskeletal tension alters mitochondrial structure and functionTo explore associations between ECM stiffness and mitochondrial structure, we examined the mitochondrial morphology of nonmalignant human mammary epithelial 25 cells (MECs; MCF10A) cultured for 24 hours on polyacrylamide hydrogel gels (PA-gel) ranging in elasticity (stiffness) between normal breast stroma (400 Pa) and breast tumors (6k -60k Pa) (6, 7) (tissue culture polystyrene ~3G Pa). MECs cultured on this range of PA-gel elasticities displayed a variety of mitochondrial morphologies, ranging from thin interconnected filaments (400 Pa), to thickened filaments (6k Pa), and then ~300 nM 30 diameter fragments with toroidal shapes (60k Pa) ( Fig.1A and S1A-B). Cells respond to ECM stiffness by ligating ECM adhesion receptors that induce Rho-GTPase-dependent cytoskeletal remodeling and increase actomyosin tension through type-II myosins (8). Pharmacological inhibition of Rho-associated protein kinase (ROCK) with Y27632 or type-II myosins with blebbistatin, reduced the prevalence of the thick toroidal 35 mitochondrial fragments in MECs plated on the stiffest ECM PA-gels (Fig.1A). These data indicate that mitochondrial structure is sensitive to cytoskeletal tension generated in response to stiffness of the ext...

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization

“…A recent study found that hyperactivation of integrin signaling through several paradigms (growth on increased stiffness, constitutive active 1 integrin, hyperglycemia, etc.) results in mitochondrial fragmentation and robust UPR MT activation through cytoskeletal remodeling (14). Consistent with these findings, we find that overexpression of 1 integrin in mammalian cells is sufficient to cause mitochondrial fragmentation and induce canonical UPR MT targets (fig.…”

“…Consistent with previous findings (39), we find that knockdown of a select few genes that induce UPR MT and therefore likely cause mitochondrial stress also affects the cytosolic HSR. It is likely that under conditions of mitochondrial stress, HSF-1 (heat-shock factor 1) can be mobilized to affect targets of the cytosolic HSR to improve organismal homeostasis (14,39,40). RNAi knockdown of most of our candidate genes failed to induce the HSR, and all failed to induce the UPR ER , suggesting that our screening paradigm was highly specific in identifying genes involved in mitochondrial homeostasis.…”

“…S5C). These studies were performed in MCF10A mammary epithelial cells grown on soft surfaces because a previous study showed that growth on plastic substrates were sufficient to drive integrin-mediated changes to mitochondria (14). Through these data, we hypothesized that a loss of eps-8 can induce mitochondrial fragmentation and UPR MT activation via an increase in integrin signaling.…”

“…However, a large-scale screening study showed that the primary response to mitochondrial dysfunction caused by drugs targeting mitochondrial translation, respiration, and import was the integrated stress response (ISR) mediated by ATF4 (13). It is possible that the lack of understanding can be due to the inherent nature of an in vitro cell culture system, as growth on stiff surfaces caused major changes to mitochondrial metabolism (14). Overall, it is still unclear how UPR MT is regulated in human cells, and it is possible that the discrepancy between studies is due to the many differences that exist between cell types and their optimal culturing conditions.…”

“…The in vivo model system allows for functional studies of mitochondrial stress response without the potential pitfalls that exist under standard culturing conditions. For example, artifacts of mitochondrial form and function have been seen in high glucose, differences in oxygen levels, or the high mechanical tension that comes from growth on plastic petri dishes (14,15). Thus, cross-examination across two distinct systems, each with their unique strengths, would create a robust and highly confident candidate gene list important for mediating mitochondrial quality control.…”