An engineered protein assembles into a complex in mouse cells only where they touch a partner cell, and can recruit an intercellular channel and a protein that directs cell division (preprint)
An engineered protein assembles into a complex in mouse cells only where they touch a partner cell, and can recruit an intercellular channel and a protein that directs cell division (preprint)
On October 29, Nick Shin, Wendell Lim (University of California, San Francisco), Rohit Pappu (Washington University in St. Louis), and colleagues posted a preprint on bioRxiv, a paper that has yet to undergo peer review. One of their engineered proteins remains dispersed inside an isolated mouse cell but forms dense patches at the cell boundary after two hours of contact with a partner cell.
At neuronal synapses and other cell contacts, a scaffold protein recruits other proteins through multiple binding sites and flexible, disordered regions that interact weakly with one another. Cell receptors bind proteins on a neighboring cell and accumulate at the contact site, recruiting scaffold molecules through their intracellular ends. The authors test the following hypothesis: when receptors are sparse, the scaffold remains dispersed, but receptor accumulation at a contact recruits enough scaffold to the membrane to push its concentration above a threshold, triggering abrupt assembly into a dense complex.
A computational model from Pappu's group identified three regimes. When scaffold molecules attract one another strongly, they assemble everywhere; when attraction is weak, they assemble nowhere. At an intermediate strength of attraction, just below the threshold for assembly without receptors, the scaffold assembles when more receptors accumulate at a contact. In the experiments, the receptor carried the fluorescent protein GFP on its extracellular side, while partner cells carried an antibody fragment that binds GFP. The authors built the scaffold from three PDZ modules that bind the receptor inside the cell, with two disordered regions between them. This is the most common arrangement in human PDZ-containing proteins at cell contacts.
The disordered regions came from proteins whose tendency to assemble has been well studied. In most cells, the scaffold containing a region from hnRNPA1 assembled only at contacts. The scaffold containing a region from FUS assembled even without a partner cell, while the scaffold containing a region from DDX4 assembled almost nowhere, matching the model's three regimes. The authors tuned the strength of attraction by substituting amino acids in the hnRNPA1 region: substitutions that strengthened attraction enabled assembly without a partner, while those that weakened attraction reduced assembly. A second control was the number of functional PDZ modules: assembly at contacts requires all three.
The authors tested the principle with another set of proteins, replacing both the extracellular pair through which cells recognize one another and the intracellular link between receptor and scaffold. They estimate that the new link is at least 700 times stronger than the original. With this new set, even the scaffold containing the DDX4 region assembled at contacts, although it had assembled almost nowhere with the original set. The authors conclude that assembly depends on the full set of interactions between receptor and scaffold.
In 2018, Lim's laboratory used synNotch, a receptor that converts cell contact into an instruction to genes, to make cells self-assemble into multilayered structures. The present scaffold builds a complex at the contact site. In a mixture containing two types of partner cells, a cell carrying both sets of proteins assembled both complexes at once, each next to its corresponding partner.
The authors coupled several proteins to the scaffold. In another cell line, the scaffold and modified connexin-43, a protein that forms intercellular channels, accumulated together at contacts, and dye transfer between cells over one hour increased by a factor of 1,9 compared with cells without the scaffold. The division axis was randomly oriented in cells carrying the scaffold alone. When the scaffold was coupled to Par6, a protein that directs cell division, the axis acquired a preferred orientation relative to the contact. Activators of Cdc42 and Rac, proteins that regulate the actin cytoskeleton, reshaped the contact into thin protrusions and a broad sheet, respectively.