Imaging method marks new role for cellular ‘skeletal’ protein

While your skeleton helps your body move, fine skeletal filaments in your cells as well as cellular structures help to move. Now, Salk researchers have developed a new imaging method that allows them to control a small subset of these filaments, called actin.

“Actin is the most abundant protein in the cell, so if you picture it, it is the whole cell,” says Uri Manor, director of Salk’s Biophotonics Core facility and corresponding author of the paper. “Until now, it has been really difficult to tell where individual actin molecules are of interest because it is difficult to separate the relevant signal from any background.”

With the new imaging technology, the Salk team has been able to explain how actin mediates an important function: the cellular ‘power stations’, known as mitochondria, in two parts. The work, which appeared in the magazine Nature methods on August 10, 2020 could provide a better understanding of mitochondrial dysfunction, which is linked to cancer, aging, and neurodegenerative diseases.

Mitochondrial vision is the process by which these energy-generating structures, like organelles, divide and multiply as part of normal cellular maintenance; the organelles divide not only when a cell divides itself, but also when cells are under high levels of stress when mitochondria are damaged. The exact way in which one mitochondrion cleaves into two mitochondria is poorly understood, especially how the initial constriction occurs. Studies have found that removing actin completely from a cell, among many other effects, leads to less mitochondrial vision, suggesting a role for actin in the process. But the destruction of all actin causes so many cellular defects that it is difficult to study the exact role of the protein in one process, the researchers say.

That said, Manor and his colleagues are developing a new way to image actin. Instead of marking all the actin in the cell with fluorescence, they made an actin probe aimed at the outer membrane of mitochondria. Only when actin is within 10 nanometers of the mitochondria does it attach to the sensor, thereby increasing the fluorescent signal.

Rather than seeing actin spread randomly across all mitochondrial membranes, as they might if there were no discrete interactions between actin and the organelles, Manor’s team saw bright hotspots of actin. And when they looked well, the hotspots were located at the same locations where another organelle called the endoplasmic reticulum crosses the mitochondria, previously found to be fission sites. Indeed, when the team saw that actin hotspots light up and disappear over time, they discovered that 97 percent of mitochondrial fission sites had actin fluorescent around them. (They speculate that there was also actin in the other 3 percent of the fission sites, but that it was not visible).

“This is the clearest evidence I’ve ever seen that actin collects on fiction sites,” says Cara Schiavon, co-author of the paper and a joint postdoctoral fellow in the labs of Uri Manor and Salk Professor Gerald Shadel. “It’s much easier to see than using another actin marker.”

By modifying the actin probe so that it attaches to the endoplasmic reticulum membrane, instead of to the mitochondria, the researchers were able to share the sequence in which various components participate in the mitochondrial fish process. The team’s results suggest that actin attaches to the mitochondria before reaching the endoplasmic reticulum. This provides important insights into how the endoplasmic reticulum and mitochondria work together to coordinate mitochondrial vision.

In additional experiments described in a pre-print manuscript available on bioRxiv, Manor’s team also reports that the same accumulation of endoplasmic reticulum-associated actin is seen at the sites where other cellular organelles – including endosomes, lysosomes, and peroxisomes – divide. This suggests a broad new role for a subset of actin in organelle dynamics and homeostasis (physiological equilibrium).

In the future, the team hopes to look at how genetic mutations are known to alter mitochondrial dynamics, also affecting the interactions of actin with the mitochondria. They also plan to adapt the actin probes to visualize actin that is close to other cellular membranes.

“This is a universal tool that can now be used for many different applications,” says Tong Zhang, a light microscopy specialist at Salk and co-author of the paper. “By exchanging the targeting sequence or the nanobody, you can address other fundamental questions in cell biology.”

“We’re in a golden age of microscopy, where new, ever-resolving instruments are always being invented; but nonetheless, there are still great limitations to what you can see,” says Manor. “I think combining these powerful microscopes with new methods that select for exactly what you want to see is the next generation of imaging.”