Mammalian cells in the service of plant science ; a different way to isolate new cytoskeletal proteins

Based on the high conservation of cytoskeleton building blocks between the plant and animal kingdoms, we have developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins in mammalian cells. A YFP-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automatic microscopic device. An initial set of candidate genes identified in the screen were isolated, sequenced, their full length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability to bind actin directly. Alternatively, indirect binding via the interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as a YFP chimera behind the 35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domain of Arabidopsis myosin VIII in a calcium-dependent manner. Interestingly, when the cDNAs of these two proteins were expressed in plants they were diffusely localized in the cytoplasm suggesting that a similar screen of a YFP-fused cDNA library in plant cells would not reliably identify these proteins as cytoskeleton-associated partners.