Studying the function and behaviour of specific proteins frequently involves the use of genetically encoded epitope or fluorescent tags. When designing systems that use such tagged proteins, it is important to consider whether the size or location of the tag might affect important variables including proper localisation, protein–protein interactions and even the function of the protein itself. Many studies of the intermediate filament protein lamin A, which forms a structurally supportive network surrounding the nucleus, utilise expression of N-terminally tagged lamin because the C-terminus is crucial for processing of prelamin to mature lamin. In this Short Report (Odell and Lammerding, 2024), Jacob Odell and Jan Lammerding demonstrate that N-terminal FLAG and GFP tags can subtly but significantly interfere with lamin A function. In lamin A-knockout fibroblasts, FLAG-tagged and GFP-labelled lamin A did restore normal nuclear morphology but failed to completely rescue reduced nuclear stiffness compared to what was seen with untagged lamin A. Additionally, both tags disturbed interactions between lamin A and the nuclear envelope protein emerin based on localisation and immunoprecipitation studies. These data suggest that N-terminal tags might affect the assembly dynamics of lamin A networks and highlight that tagged proteins used for functional studies require rigorous validation to ensure that partial loss-of-function effects are not overlooked.
