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 40-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 expanded this tool to visualise eight other human tropomyosin isoforms, using mNeonGreen, mCherry, mStayGold(E138D) and mScarlet3-H tags. All showed 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.
Scott W, Polutranko V, Milczarek J, Hands-Portman I, Balasubramanian MK