Ganie, R. A.; Biswas, A.; Sasi, S.; Devarakonda, L. P.; Kale, I.; Mullick, S.; Siddappa, G.; Singh, N.; Gadadhar, S.; Sirajuddin, M.

Cytoskeletal protein expression and filament dynamics change significantly during cell state transitions. However, post-translational modifications of cytoskeletal proteins during these transitions have rarely been described. Here, using a synthetic glutamylation-binder (SB2B49) selected against a bi-glutamylated peptide epitope, we identify a distinct pool of glutamylated vimentin filaments. We demonstrate that vimentin glutamylation is enzymatically added by tubulin tyrosine ligase-like (TTLLs) and removed by cytosolic carboxypeptidases (CCPs). Mass spectrometry and mutagenesis reveal that glutamylation occurs on specific vimentin residues. We find that glutamylated vimentin levels are dynamically modulated during epithelial-mesenchymal plasticity. During collective migration in scratch-wound assays, glutamylated vimentin filaments are transiently depleted at the wound edge, a process controlled by canonical glutamylation writer-erasers. Our findings reveal a new layer of vimentin regulation via glutamylation and establish the glutamylation-binder as a valuable tool for exploring the diversity of vimentin proteoforms and glutamylation modifications in both physiological and pathological contexts.