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.