Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells

Abstract

Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is critical for muscle function. Myonuclei differ in transcriptional activity and protein accumulation but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism to spatially and temporally regulate transcription in individual myonuclei. The best characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS) but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using in vitro generated multinucleated muscle cells, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import vary during differentiation. Together, our results suggest that spatial and temporal regulation of nuclear import pathways may be important in muscle cell differentiation and protein regionalization in myofibers.