Mitochondria are essential organelles involved in a variety of critical cellular processes, including energy production and intracellular signaling. They continuously sense and integrate both metabolic and mechanical cues to fine-tune their functions. Although their central role in energy metabolism has been extensively studied, the mechanical properties of mitochondria and their interactions with the cytoskeleton remain poorly characterized. Understanding how mitochondrial mechanical behavior relates to cytoskeletal dynamics is key to revealing the role of mitochondria in mechanotransduction.

To characterize in cellulo the mechanical properties of mitochondria and their interplay with the cytoskeleton, we employed two main techniques, optical tweezers and fluorescence lifetime imaging (FLIM), separately and in combination. Using an optical tweezers-based intracellular micromanipulation technique, we probed the mechanical properties of mitochondria and their microenvironment in RPE-1 cells. By applying controlled forces to deflect single mitochondria, we measured the effective stiffness of mitochondria while preserving their functional integrity. Additionally, we performed oscillatory rheology while selectively disrupting the actin and microtubule cytoskeletons to investigate their contributions to the mitochondrial mechanical environment. We further explored the mechanical interplay between mitochondria and the cytoskeletal components by quantifying force transmission within the mitochondrial network. Our findings demonstrate that the actin cytoskeleton is the main mediator of force transmission to mitochondria. Using FLIM measurements of Mito Flipper-TR to probe fluorescence lifetime at intermediate timescales, we found that mitochondrial membrane tension decreases upon microtubule depolymerization. However, when combining FLIM with optical tweezers-based deflection of mitochondria, mitochondrial membrane tension remains unchanged upon local force application, suggesting the existence of intrinsic tension-regulating mechanisms. Altogether, our results provide new insights into mitochondrial mechanics and uncover distinct roles of the cytoskeleton in shaping their microenvironment.

Legend: Confocal image of a fixed RPE-1 cell, revealing the complex organization of the mitochondrial network (magenta), as it closely associates with microtubules (green) and actin filaments (amber gold).