Stress‐dependent macromolecular crowding in the mitochondrial matrix

Abstract

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water‐accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein‐rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox‐to‐condensed transition or a mitochondrial unfolded protein response. CAP‐treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons

“…7), which could conceivably reflect an adaptation to metabolic activity 53,54 that becomes altered with age. Alternatively, decreased molecular crowding within the mitochondria 55 or changes in their membrane fluidity as a result of oxidative damage 23,25 could explain reduced viscosity. Interestingly, protein diffusion was shown to be enhanced in mitochondria of human fibroblasts that are deficient for complex I activity 56 , likely a sign of reduced viscosity, while mitochondria hyperpolarisation correlates with increased viscosity in HepG2 cells 55 .…”

“…Alternatively, decreased molecular crowding within the mitochondria 55 or changes in their membrane fluidity as a result of oxidative damage 23,25 could explain reduced viscosity. Interestingly, protein diffusion was shown to be enhanced in mitochondria of human fibroblasts that are deficient for complex I activity 56 , likely a sign of reduced viscosity, while mitochondria hyperpolarisation correlates with increased viscosity in HepG2 cells 55 . Thus, our data support the idea that the reduced ΔΨ m and mitochondrial viscosity in aged DRGs are mechanistically linked.…”

“…1-2) could explain reduced viscosity. Interestingly, mitochondria of human fibroblasts that are deficient for complex I activity display enhanced protein diffusion (Koopman et al, 2008), likely a sign of reduced viscosity, while mitochondria hyperpolarisation correlates with increased viscosity in HepG2 cells (Bulthuis et al, 2023). Thus, our data support the idea that the reduced ΔΨ m and mitochondrial viscosity in aged DRGs are mechanistically linked.…”

The biophysics of water in cell biology: perspectives on a keystone for both marine sciences and cancer research

“…This revealed fascinating images of cellular water subpopulations within neuroblastoma cells ( Ramos and Lee, 2023 ). The consequence of the crowded space on water activity was also studied in the mitochondrial matrix, revealing the impact of its changes on biochemical reactions ( Bulthuis et al, 2023 ). Another case showed the impact of crowding and cellular interactions on in vivo folding of the three-helix bundle protein B in the cytoplasm ( Russell et al, 2023 ).…”

Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects

“…Thus, in general, crowding refers to the effects of volume exclusion due to the presence of molecular crowders, while confinement represents the restricted motions of the protein being confined within a cage-like, impenetrable boundary formed by the crowder molecules. , Recent studies seek a more comprehensive understanding of how molecular crowding impacts protein folding, stability, aggregation, diffusion, interaction with other biomolecules, and catalysis. These studies on crowding effects vary with regard to the model system, the type of molecular crowders used, as well as their size and concentration. − …”