Biophysicists unraveled a mystery of how muscles form at the molecular level and how they maintain their function. Nature Communications published the discovery, which may help in the design of treatments for muscle diseases such as dilated cardiomyopathy, one of the leading causes of heart failure.
"We've made a fundamental advance in understanding how the cellular cytoskeleton is assembled, especially in muscle cells," says Shashank Shekhar, assistant professor of physics at Emory University and senior author of the study.
The researchers upended a model relied on for more than four decades to explain how filaments of actin, a protein vital to cellular movement and other functions, form and maintain their length.
"Our work challenges a long-standing paradigm by uncovering a new mechanism, previously thought impossible, by which the protein leimodin builds actin filaments in muscle," Shekhar says.
"We also provide a molecular explanation for how defects in leimodin can disrupt the assembly of the contractile machinery of the heart," says Sudipta Biswas, an Emory PhD candidate and first author of the paper.
A genetic mutation in leimodin, for example, is linked to dilated cardiomyopathy, a condition which progressively weakens the heart muscles and impairs its ability to pump blood effectively.
An abundant, versatile protein
The Shekhar Lab studies the mechanical and chemical regulation of actin, one of the most abundant and versatile proteins in the body.
Actin assembles into filaments that are part of the skeleton of living cells, giving them shape. It is also essential to the mobility of cells. As actin filaments elongate inside a cell they push against its membrane, causing the cell to roll forward. These same forces generated by the dynamics of actin assemblies allow a cell to shape-shift and divide into two daughter cells. Or an immune cell to engulf and kill an invading bacterium.
In many of the cells in the body, actin filaments form into loose, mesh-like bundles. In muscle cells, however, actin and the protein myosin form straight, highly stable arrays known as sarcomeres — the smallest, organized units of muscle cells.
Actin in muscle cells is highly specialized to facilitate contraction. The actin filaments slide past the myosin filaments towards the middle of the sarcomere, gathering up the length of the sarcomere like a drawstring without any change in the length of the filaments.
"How well your muscles contract is controlled by the length of these actin filaments," Shekhar explains. "From birth, to death, the length of the actin filaments in muscle cells stays the same. It's very tightly controlled."
While the length of the actin filaments remains the same, however, actin filaments still need to grow and replenish themselves, so they can keep forming anew as they age, turn over and degrade.
How the filaments did this in muscles has been a mystery.
Sarcomeres grow differently
All actin filaments have two ends: one is called the pointed, or minus, end; the other is called the barbed, or plus, end. For decades, scientists have long known that the minus end of an actin filament is where the filaments depolymerize, or degrade and break off. And the plus end is where freshly energized actin subunits are added to the filament, like forming a new step in a staircase.
This process — losing an actin subunit from the minus end while gaining one at the plus end — is called "treadmilling," since the filament appears to move forward as new building blocks are added.
This fundamental process for actin filaments, however, does not work the same way in muscle cells. Actin filaments in sarcomeres strictly control their length. Their barbed, plus end is capped with a protein that prevents the addition of new molecules.
"And yet, the actin filaments in the sarcomeres you're born with are not the same ones in your sarcomeres today," Shekhar says. "No one knew how they keep assembling and remodeling themselves."
A clue to the mystery
In previous research, Shekhar and colleagues at Ohio State University found an interesting effect of a toxin from Vibrio cholerae bacteria on actin filaments in non-muscle cells. Vibrio, the pathogen that causes cholera, can hijack the machinery of actin filaments, reversing the treadmilling process so that growth occurs on the minus end.
Shekhar and Biswas wondered if something similar could be occurring in healthy actin filaments of muscle cells. Could some catalyst spark growth on the minus end?
"Sudipta did some really clever experiments to test this idea," Shekhar says.
Designing experiments
Biswas centered her investigation on leiomodin 2 — a particular type of leiomodin found near the pointed ends of actin filaments in cardiac muscle cells. "It has domains similar to the Vibrio toxin," she says.
"Previous laboratory dish experiments," she adds, "showed that if you delete leiomodin 2 from cardiac muscle cells, their actin filaments get shorter. And if you have too much leiomodin 2, the filaments grow longer than normal."
Biswas designed experiments using the highly specialized technique of microfluidic-assisted total internal reflection fluorescence microscopy (mf-TIRF) for studying the dynamics of how actin filaments remodel themselves.
The Shekhar Lab is one of a handful in the world using mf-TIRF to study how actin filaments grow and disassemble. The technique allows researchers to attach different colors of fluorescent dyes to single protein molecules then introduce them to a microfluidic system. The light from these dyes reveals movements of single molecules.
Biswas anchored molecules of leimodin 2 to the base of a microfluidic chamber. She then tagged single actin molecules with red fluorescence and introduced them to the chamber. Like tiny, red fluorescent worms growing, actin filaments began to assemble, remaining anchored to the base by the leimodin, which attaches to the pointed end of the filaments.
But were the filaments growing from their pointed, minus ends?
Seeing is believing
To find out, Biswas introduced more actin molecules into the system, this time tagged green and watched to see what would happen. The green molecules became anchored at the leimodin base. As each new green molecule got added to the base, the red molecules in the filaments gradually became displaced, moving away from the base in the direction of the flow.
"We provided the first direct molecular evidence that actin filaments grow from their pointed ends," Biswas says. "We proved those who thought this wasn't possible wrong."
The work provides a mechanistic explanation for why a mutation in Leiomodin2 leads to shorter, thin filaments or abnormally long, thin filaments in heart muscle cells.
"Understanding how a mutation causes a disease is often the first step towards finding ways to treat or prevent a disease," Biswas says.
Co-authors of the study include Tania Larrinaga (University of Arizona, Tucson, and Icahn School of Medicine in Mount Sinai, New York); Sandeep Choubey (Institute of Mathematical Sciences in Chennai, India, and Homi Bhabha National Institute in Mumbai); and Carol Gregorio (Icahn School of Medicine).
This research, reported in Nature Communications, July 2026, was supported by the National Institute of General Medical Sciences under grants (R35GM143050, R01GM120137) and National Heart, Lung, and Blood Institute under grant (R01HL123078). 100% of this project was financed with federal funds.
