Nanobody-based analysis of RhoGTPase stress response in Schizosaccharomyces pombe: Role of mtl2

Schizosaccharomyces pombe ( S.pombe ), commonly referred to as fission yeast, is a widely utilised model organism for studying cellular responses to stress. The stress response mechanisms are largely conserved, offering insights into similar processes in higher eukaryotes, including humans.We have developed an in vivo nanobody-library screening platform in Δ mtl 2 fission yeast subjected to lethal caspofungin stress, enabling the direct selection of nanobodies that improve survival. Through this method, we isolate a nanobody (Clone-C) that binds to and regulates the Rho1 GEF (Guanine nucleotide Exchange factor) Rgf1, enhancing cell survival and uncovering a hitherto unreported Mtl2–Rgf1–Rho1GTPase regulatory axis in the cell-wall stress response. This nanobody-based fluorescence-readout method offers a versatile tool for gene identification and antifungal target/ADC screening under certain stress conditions.