A Protein’s Stiffness Determines How Easily It Enters the Nucleus

A gateway between the cytoplasm and the nucleus

Nuclear pore complexes (NPCs) offer a gateway between a cell’s nucleus – its control centre – and the cytoplasm.

These gateways enable the passage of biomolecules, such as proteins, that inform the internal control centre of what’s going on within the cell and beyond. In response, the nucleus can dictate whether certain genes must be “on” or “off” to serve a specific biological function. This mode of communication is selective, ironically, given that NPCs are somewhat larger than other biological pores.

What dictates how easily a protein can cross NPCs? Scientists have asked this question for many years. It’s well known that a protein’s size and composition are contributing factors, but a study from researchers at the Francis Crick Institute and King’s College London shows that the mechanical stability of a protein is also a key determinator in nuclear translocation. The research is published in Nature Physics.

“We’ve made a fundamental discovery that the mechanics of a protein – how soft or stiff it is in the region that leads translocation – control its entry into the cell’s nucleus,” said Dr. Sergi Garcia-Manyes, group leader of the Single Molecule Mechanobiology Laboratory at the Francis Crick Institute, professor of biophysics at King’s College London and the study’s lead author.

“Unanticipated” data show protein stiffness affects nuclear translocation

Garcia-Manyes and colleagues utilized single-molecule mechanics and single-cell optogenetic techniques in their experiments. Through creating proteins that contain a modified light-inducible nuclear export (LEXY) probe, they were able to control and track a protein’s movement. When blue light was applied, the LEXY probe exposes a nuclear export sequence, which triggers the protein’s movement out of the cell. When the light is turned off, the protein’s nuclear-localization sequence (NLS) acts as a signal that informs the cell to transport the protein – which is tagged with a fluorescent protein – back into the nucleus.

By engineering proteins to possess different properties, the researchers could directly analyze the factors that impact protein translocation, including mass, charge and mechanical stability.

Proteins that possessed a softer, more flexible leading protein domain – the domain nearest to the NLS – could move across the nucleus quicker than those that didn’t.

Hypothetically, manipulating the leading protein domain could assist more stubborn, stiff proteins in nuclear translocation. To test this, Garcia-Manyes and colleagues engineered a soft tag near to the NLS in the transcription factor MRTF, which assists cells in their movement around the body. Adding the tag enhanced MRTF’s ability to enter the nucleus, consequently increasing the movement of cells.

“Our findings were rather unanticipated,” said Dr. Rafael Tapia-Rojo, co-first author and former postdoc at the Crick. Tapia-Rojo is now a lecturer in biological physics at King’s College London. “It was striking to see how measurements at the single molecule level can be so directly linked to what happens at a cellular level, using a newly designed optomechanical approach.”

“Although we only looked at the nuclear pore, this mechanism could regulate entry into other parts of the cell, such as the mitochondria or proteasomes,” added Garcia-Manyes. As many proteins are drug targets, the study’s findings could create new possibilities in modern drug design, he said, “Knowing that a more flexible protein can enter the nucleus quicker could help us design more targeted drugs.”

Now, Garcia-Manyes and colleagues are exploring how certain transcription factors have evolved to possess flexible regions, allowing them to enter the nucleus quicker and with greater ease than other proteins.

Reference: Panagaki F, Tapia-Rojo R, Zhu T, et al. Structural anisotropy results in mechano-directional transport of proteins across nuclear pores. Nat Phys. 2024. doi: 10.1038/s41567-024-02438-8

This article is a rework of a press release issued by The Francis Crick Institute. Material has been edited for length and content.