The biological significance of nuclear actin

Abstract

Background and purpose: One of the most abundant proteins of eukaryotic organisms is actin. This globular protein shows extremely high degree of evolutionary conservation, and the earliest form of actin evidently appeared in the common ancestor of bacteria, archaea and eukarya. In eukaryotic cells, actin fulfils innumerous cytoplasmic functions both in monomeric and polymeric forms, such as the building of the cytoskeleton, taking part in muscle contraction, intracellular transport, and cytokinesis, just to mention a few. Today it is clear, that actin is not an exclusively cytoplasmic protein; its nuclear presence is undoubted, and many experimentally verified nuclear functions have been revealed. The manifold cytoplasmic and nuclear functions of actin argue for the existence of a tightly regulated active transport for the protein through the nuclear pore complexes. Actin is exported out from the nucleus in complex with Exportin 6 and profilin, and Importin 9 carries cofilin-associated actin into the nucleus from the cytoplasm. The nuclear functions of actin include the regulation of transcription and nuclear size, modulation of chromatin organization and DNA damage repair, to name a few. It is important to note that the detailed mechanisms through which actin functions in these nuclear processes are still poorly understood, and actin is likely to be involved in many yet undescribed nuclear mechanisms. These facts indirectly suggest the essentiality of nuclear actin. In our work, we tested the robustness of the nuclear localization of actin, and investigated whether the perturbations of its nuclear presence affect the viability of the whole organism. Thereby, we investigated the biological significance of nuclear actin. Our model organism, Drosophila melanogaster, has 6 actin coding genes, from which Act5C and Act42A encode less specialized, cytoplasmic isoforms. The structure and regulation of the Act5C gene is well known, and its absence is lethal, therefore it was the perfect subject of the experiments. Experimental approaches: To achieve our aims, we generated a modular genetic system that enables the observation of changes in nuclear actin levels and thus, it allows to indirectly investigate the essentiality and functions of nuclear actin. The experimental setup consists of the Act5C null mutation and transgenes expressing several modified Act5C protein variants, including a Nuclear Export Signal (NES)-tagged isoform which ensures forced nuclear export of actin. To investigate the effect of sumoylation of actin, implicated earlier to serve as a nuclear retention signal, the Act5C gene was modified with mutagenic PCR. In addition, the known importin of actin (RanBP9 in Drosophila) was knocked out with P-element remobilization. The effects of these genetic modifications on nuclear actin levels were quantitated by immunostaining cultured Drosophila cells or larval salivary glands. In the rescue experiments we investigated the combined effect of various genetic modifications through monitoring the viability of male progeny. Screening for new importin of actin was carried out in vitro, by using pull-down assay. The obtained results were verified with in vivo, in BiFC system on S2R+ cultured cells. Key results: The validation of modified (NES-, V5-, and FLAG-tagged) Act5C expressing stocks, and the rescue of the Act5C null-mutant lethal phenotype with them confirmed, that NES-tagging of Act5C significantly reduces the nuclear level of actin. However, this decrease neither affected the viability of flies nor did lead to any other discernable phenotype. Because the disruption of actin’s sumoylation motif failed to enhance its nuclear export, we concluded that sumoylation does not act as a major nuclear retention signal of actin in Drosophila. In contrast, the complete elimination of the RanBP9 importin significantly lowered nuclear actin levels, however viability decreased only if the rescue was carried out together with NES-tagging of Act5C. The reduction of viability was still not 100% in this case, which implies the existence of nuclear import mechanisms acting in parallel to the RanBP9 pathway. With the help of in vitro and in vivo assays we identified four β-importins that interact with actin. Conclusion: We successfully developed a genetic system that is suitable for the investigation of nuclear actin functions at the level of the organism. We found that the nuclear actin pool is maintained not exclusively by RanBP9, but multiple parallel acting import mechanisms ensure the nuclear localization of the protein. Our results provide direct evidence that the nuclear presence of actin is greatly secured, which in turn argues for essential biological functions for nuclear actin.