Fluorescent protein tags for human tropomyosin isoform comparison

Tropomyosin is an important actin cytoskeletal protein underpinning processes such as muscle contraction, cell shape and cell division. Defects in tropomyosin function can lead to diseases, including some myopathies and allergies. In cells, tropomyosin molecules form coiled-coil dimers, which then polymerise end-to-end with other dimers for actin association. Tropomyosin is challenging to tag for in vivo fluorescence microscopy without perturbing its polymerisation interfaces. We recently developed a fluorescent tag comprising a forty amino acid flexible linker capable of detecting tropomyosin in S. pombe actin cables and the actomyosin ring, and in patch-like structures that were previously unappreciated. We also used this strategy successfully to tag human TPM2.2, a prominent human muscle isoform. Here, we expand this tool to visualise eight other human tropomyosin isoforms, using mNeonGreen, mCherry and mStayGold tags. All show typical tropomyosin fluorescence, no signs of cytotoxicity and are compatible with super-resolution microscopy. These tools singly or in combination should aid detailed mechanistic investigations of tropomyosin isoforms. Summary Statement Green and red fluorescent fusion proteins with lengthy and flexible polypeptide linkers readily allow improved visualisation and comparison of tropomyosin isoform distribution in live human cells.