Cytoskeleton Protein’s New Role Magnified by Novel Imaging Method

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Actin is a cytoskeletal protein involved in multiple cellular processes. Actin provides cellular structure and aids in cell migration, cell division, and organelle trafficking. Researchers at the Salk Institute have recently developed a new imaging technique, which has allowed them to observe how actin aids mitochondria divide in two.

Their work, “Actin chromobody imaging reveals sub-organellar actin dynamics,” is published in the journal Nature Methods and led by Uri Manor, PhD, director of Salk’s Biophotonics Core facility.

Mitochondria govern many metabolic processes. In addition, mitochondria sense the status of metabolism and change their functions to regulate energy production, cell death, and thermogenesis. Mitochondrial structural remodeling through division and fusion is critical to the organelle’s function. Abnormalities in mitochondrial division and fusion are linked to the pathophysiology of metabolic diseases such as diabetes and obesity. However, the exact way in which one mitochondrion pinches off into two mitochondria has been poorly understood, particularly how the initial constriction happens. Studies have found that removing actin from a cell entirely, among many other effects, leads to less mitochondrial fission, suggesting a role for actin in the process. But destroying all the actin causes so many cellular defects that it is difficult to study the protein’s exact role in any one process.

“Actin is the most abundant protein in the cell, so when you image it, it’s all over the cell,” stated Manor.

From left: Uri Manor, PhD, Cara Schiavon, PhD, and Tong Zhang, PhD. [Salk Institute]
“The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed ‘actin chromobodies’ (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics,” the researchers wrote.

The researchers created an actin probe targeted to the outer membrane of mitochondria. Only when actin is within 10 nm of the mitochondria does it attach to the sensor, causing the fluorescence signal to increase. Rather than finding actin scattered around all mitochondrial membranes, the team saw bright hotspots of actin. When they took a closer look at the hotspots, they observed they were located at the same locations where the endoplasmic reticulum crosses the mitochondria, previously found to be fission sites.

The team observed actin hotspots light up and disappear over time, and they discovered that 97% of mitochondrial fission sites had actin fluorescing around them.

“This is the clearest evidence I’ve ever seen that actin is accumulating at fission sites,” added Cara Schiavon, PhD, a joint postdoctoral fellow in the labs of Manor and Salk professor Gerald Shadel, PhD. “It’s much easier to see than when you use any other actin marker.”

The researchers were able to piece together the order in which different components join the mitochondrial fission process, by altering the actin probe so that it attached to the endoplasmic reticulum membrane rather than the mitochondria. Their results suggest that the actin attaches to the mitochondria before it reaches the endoplasmic reticulum. This lends important insight towards how the endoplasmic reticulum and mitochondria work together to coordinate mitochondrial fission.

The team also reported 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.

Looking forward, the researchers are planning to look at how genetic mutations known to alter mitochondrial dynamics might also affect actin’s interactions with the mitochondria. They also plan to adapt the actin probes to visualize actin that’s close to other cellular membranes.

“This is a universal tool that can now be used for many different applications,” explained Tong Zhang, PhD, a light microscopy specialist at Salk and co-first author of the paper. “By switching out 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 instruments with ever higher resolution are always being invented; but in spite of that there are still major limitations to what you can see,” said 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.”

Their work sets a new stage for microscopy and may one day offer insight on mitochondrial dysfunction, which has been linked to cancer, aging, and neurodegenerative diseases.

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