Cdc42, a Rho-family GTPase, plays a pivotal role in establishing polarity in Saccharomyces cerevisiae by accumulating on the membrane at the site of bud emergence. Cdc42’s ability to bind to membranes, mediated by prenylation, is essential for its function. Prenylation involves either the post-translational addition of a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group to Cdc42’s C-terminus. One of the major challenges in studying the physical and chemical interactions of Cdc42 at the polarity spot in vitro is obtaining prenylated Cdc42. Here, we present a streamlined, sortase A-based approach to farnesylate Cdc42 in vitro. This method uses Escherichia coli-expressed Cdc42 with a sortase A recognition motif, facilitating efficient farnesylation and purification using a size exclusion-based strategy. The farnesylated Cdc42 retains functionality, as evidenced by membrane binding and by its GEF-regulatory GTPase activity, making it suitable for further biophysical and biochemical investigations.

This study shows that coupling to designed plasmonic nanoparticles can modulate the electrophysiological function of proteins in living mammalian cells. Nanostar-shaped particles, that are robust to biological noise, are designed to enable near-field-coupling to plasma membrane-localized mutated Archaerhodopsin proteins in live cells. The coupled rhodopsins exhibit enhanced fluorescence and an increased response speed to membrane voltage. Incorporating this plasmonic enhancement into a Markov chain photocycle model of the Archaerhodopsin mutant QuasAr6a, shows an increased fluorescence emission rate and manipulation of the protein dynamics through a combination of photocycle transition rate enhancements. The results show an improvement in fluorescence and voltage-response dynamics of the functional QuasAr6a Archaerhodopsin mutant, beyond what has been achievable through genetic engineering. This opens up possibilities for engineering the biological functionality of proteins through plasmonics: manipulating protein photocycles could improve light sensitivity, change optogenetic applications, and lead to fluorescent biosensors with enhanced dynamics.

In this Perspective, Journal of Cell Science invited researchers working on cell and tissue polarity to share their thoughts on unique, emerging or open questions relating to their field. The goal of this article is to feature 'voices' from scientists around the world and at various career stages, to bring attention to innovative and thought-provoking topics of interest to the cell biology community. These voices discuss intriguing questions that consider polarity across scales, evolution, development and disease. What can yeast and protists tell us about the evolution of cell and tissue polarity in animals? How are cell fate and development influenced by emerging dynamics in cell polarity? What can we learn from atypical and extreme polarity systems? How can we arrive at a more unified biophysical understanding of polarity? Taken together, these pieces demonstrate the broad relevance of the fascinating phenomenon of cell polarization to diverse fundamental biological questions.