Mitochondria, or how our textbooks call them, ​“the powerhouses of the cell”, help generate energy required to fuel eukaryotic life. For this, they rely on the inner mitochondrial membrane (IMM). This membrane is packed with enzymes that form the electron transport chain (ETC), generating ATP — the cell’s energy currency — through respiration. ATP generation is made possible with the help of a proton gradient that is established across the IMM. To establish this gradient, the IMM requires charge carriers to move around freely. 

Fluidity of the IMM should be crucial in this scenario; more fluid the IMM, more mobile would the charge carriers be. And more mobile the charge carriers, more efficient would be the respiration. Researchers are interested in this link between respiration and IMM fluidity; how do the properties of the IMM affect respiration?

“For a membrane that is so densely packed with proteins, you also need movement of molecules,” said Akash Gulyani, Associate Professor, University of Hyderabad. ​“You have to balance the movement of mobile electron carriers in this densely organised membrane with invaginations and cristae. The more we thought about it, the more we got intrigued by the properties of the IMM.” However, studying IMM fluidity in living cells has proven challenging. Probing mitochondria with dyes can stress the organelles and damage cells.

To investigate IMM dynamics in living cells, Gulyani and his group at Institute of Stem Cell and Regenerative Biology (InStem), Bangalore, along with the University of Hyderabad, adopted a rather advanced technique to measure IMM fluidity. They used fluorescence lifetime imaging microscopy (FLIM) in living cells, measuring fluidity using a fluorescent molecular rotor localised to the mitochondrial membrane. 

When a fluorescent molecule gets excited, it spends time in the excited state before emitting a photon and returning to the ground state. This time is the fluorescence lifetime, and the researchers observed that as respiration increased, the fluorescence lifetime of the rotor decreased, indicating an increase in the fluidity of the IMM. The findings were recently published in the Proceedings of the National Academy of Sciences (PNAS).

In a previous study, researchers took a complementary approach to modulate the fluidity of the respiring membranes in bacteria and yeast, resulting in changes in respiration. In the current PNAS paper, Gulyani and his team focused on modifying respiration, and observed how cells adjusted their own IMM fluidity in response.

The fluorescent molecular rotor used by Gaurav Singh, a post-doc in the lab, is Mitorotor‑1. It is a harmless, voltage-sensitive dye that is drawn to the potential gradient across the IMM, thereby causing it to localise to the mitochondria. The fluorescence lifetime of a molecule depends on its environment and the viscosity surrounding it. For Mitorotor‑1, a more fluid environment enables faster rotation and photon emission, resulting in shorter fluorescence lifetimes. When the researchers stimulated respiration by starving the cells, the fluorescence lifetime of their probe reduced, indicating increased fluidity.

When a molecule deexcites quickly due to increased fluidity, its intensity also reduces. But intensity also depends on the number of fluorescent molecules localised to the mitochondria. ​“Lifetime, interestingly, is an intrinsic property of the molecule, so the signal is independent of how many molecules are at the membrane,” said Gulyani.

“This is a very exciting study that provides an approach to simultaneously measure IMM fluidity and mitochondrial metabolic activities using FLIM, opening up a route to study the correlation between mitochondrial structures and metabolic activities,” said Xingbo Yang, Research Group Leader, TU Dresden, Germany, who was not involved in the study. ​“The quantitative characterisation of the IMM fluidity sensor is very encouraging.”

The observation that intact cells can modulate the properties of the IMM by changing its fluidity is a significant finding, and it begs the question of the mechanism behind it. Some possibilities that Gulyani speculates are cardiolipin (an important phospholipid in the IMM), ions like calcium, or perhaps a mechanical force.