A bipartite NLS motif mediates the nuclear import of Drosophila moesin.

The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.

Direct interaction of Su(var)2-10 via the SIM-binding site of the Piwi protein is required for transposon silencing in Drosophila melanogaster.

Nuclear Piwi/Piwi-interacting RNA complexes mediate co-transcriptional silencing of transposable elements by inducing local heterochromatin formation. In Drosophila, sumoylation plays an essential role in the assembly of the silencing complex; however, the molecular mechanism by which the sumoylation machinery is recruited to the transposon loci is poorly understood. Here, we show that the Drosophila E3 SUMO-ligase Su(var)2-10 directly binds to the Piwi protein. This interaction is mediated by the SUMO-interacting motif-like (SIM-like) structure in the C-terminal domain of Su(var)2-10. We demonstrated that the SIM-like structure binds to a special region found in the MID domain of the Piwi protein, the structure of which is highly similar to the SIM-binding pocket of SUMO proteins. Abrogation of the Su(var)2-10-binding surface of the Piwi protein resulted in transposon derepression in the ovary of adult flies. Based on our results, we propose a model in which the Piwi protein initiates local sumoylation in the silencing complex by recruiting Su(var)2-10 to the transposon loci.

FERM domain-containing proteins are active components of the cell nucleus.

The FERM domain is a conserved and widespread protein module that appeared in the common ancestor of amoebae, fungi, and animals, and is therefore now found in a wide variety of species. The primary function of the FERM domain is localizing to the plasma membrane through binding lipids and proteins of the membrane; thus, for a long time, FERM domain-containing proteins (FDCPs) were considered exclusively cytoskeletal. Although their role in the cytoplasm has been extensively studied, the recent discovery of the presence and importance of cytoskeletal proteins in the nucleus suggests that FDCPs might also play an important role in nuclear function. In this review, we collected data on their nuclear localization, transport, and possible functions, which are still scattered throughout the literature, with special regard to the role of the FERM domain in these processes. With this, we would like to draw attention to the exciting, new dimension of the role of FDCPs, their nuclear activity, which could be an interesting novel direction for future research.

Measuring Transposable Element Activity in Adult Drosophila Ovaries.

Transposons are genetic elements that use various mechanisms of transposition to move around the genome, thus posing a risk to genomic integrity. Repression of transposable elements (TEs) involves the complex PIWI pathway and several proteins associated with heterochromatinization. All players of TE repression are indispensable for proper reproductive fitness, as loss-of-function mutations in these genes result primarily in sterility and impaired reproductive development. When investigating the function of novel genes with similar phenotypes, elevated transposon expression in reproductive tissues can be a marker for involvement in the aforementioned processes. Here, we present a protocol for investigating TE levels in adult Drosophila ovaries, from dissection to data analysis.

Detection of Actin in Nuclear Protein Fraction Isolated from Adult Drosophila Ovary.

Much evidence supports the presence of cytoskeletal elements in the nucleus; however, the exact functions of these proteins in the nucleus are still uncertain. Of the cytoskeletal proteins, the activity and biological significance of nuclear actin has been the most extensively researched. It is now clear that actin performs essential tasks both in the cytoplasm and the nucleus, and that the dynamic balance between the large cytoplasmic and the significantly smaller nuclear actin pools is maintained by robust transport mechanisms. Therefore, the compartment-specific manipulation or investigation of actin has been an enormous challenge. Here, we present a protocol for the detection of actin in isolated nuclear protein fractions from Drosophila ovaries.

Parallel import mechanisms ensure the robust nuclear localization of actin in Drosophila.

Actin, as an ancient and fundamental protein, participates in various cytoplasmic as well as nuclear functions in eukaryotic cells. Based on its manifold tasks in the nucleus, it is a reasonable assumption that the nuclear presence of actin is essential for the cell, and consequently, its nuclear localization is ensured by a robust system. However, today only a single nuclear import and a single nuclear export pathway is known which maintain the dynamic balance between cytoplasmic and nuclear actin pools. In our work, we tested the robustness of the nuclear import of actin, and investigated whether the perturbations of nuclear localization affect the viability of the whole organism. For this aim, we generated a genetic system in Drosophila, in which we rescued the lethal phenotype of the null mutation of the Actin5C gene with transgenes that express different derivatives of actin, including a Nuclear Export Signal (NES)-tagged isoform which ensures forced nuclear export of the protein. We also disrupted the SUMOylation site of actin, suggested earlier to be responsible for nuclear retention, and eliminated the activity of the single nuclear import factor dedicated to actin. We found that, individually, none of the above mentioned manipulations led to a notable reduction in nuclear actin levels and thus, fully rescued lethality. However, the NES tagging of actin, together with the knock out of its importin, significantly reduced the amount of nuclear actin and induced lethality, confirming that the presence of actin in the nucleus is essential, and thereby, over-secured. Supporting this, we identified novel nuclear importins specific to actin, which sheds light on the mechanism behind the robustness of nuclear localization of actin, and supports the idea of essentiality of its nuclear functions.

The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila.

Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.

Cellular Immune Response Involving Multinucleated Giant Hemocytes with Two-Step Genome Amplification in the Drosophilid Zaprionus indianus.

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.

Nuclear actin: ancient clue to evolution in eukaryotes?

Until recently it was widely accepted that the dynamic cytoskeletal matrix is exclusive to the cytoplasm of eukaryotes, evolving before the emergence of the cell nucleus to enable phagocytosis, cell motility and the sophisticated functioning of the endomembrane system within the cytosol. The discovery of the existence of a prokaryotic cytoskeleton has changed this picture significantly. As a result, the idea has taken shape that the appearance of actin occurred in the very first cell; therefore, the emergence of microfilaments precedes that of the eukaryotic cytoskeleton. The discovery of nuclear actin opened new perspective on the field, suggesting that the nuclear activities of actin reflect the functions of primordial actin-like proteins. In this paper, we review the recent literature to explore the evolutionary origin of nuclear actin. We conclude that both ancient and eukaryotic features of the actin world can be detected in the nucleus today, which supports the idea that the cytoskeleton attained significant eukaryotic innovations before the tandem evolution of the cytoskeleton and nucleus occurred.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.

The actin-binding ERM protein Moesin directly regulates spindle assembly and function during mitosis.

Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled.

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

Viability, longevity, and egg production of Drosophila melanogaster are regulated by the miR-282 microRNA.

The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5' cap and a 3'-poly(A) sequence and a mature microRNA of ∼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis.

Ecdysone induced gene expression is associated with acetylation of histone H3 lysine 23 in Drosophila melanogaster.

Posttranslational modification of histones regulates transcription but the exact role that acetylation of specific lysine residues plays in biological processes in vivo is still not clearly understood. To assess the contribution of different histone modifications to transcriptional activation in vivo, we determined the acetylation patterns on the ecdysone induced Eip74EF and Eip75B genes in Drosophila melanogaster larvae by chromatin immunoprecipitation. We found that acetylation of histone H3 lysine 23 is localized to promoters and correlates with endogenous ecdysone induced gene activation. In contrast, acetylation of lysines 8, 12 and 16 of histone H4 and lysine 9 of histone H3 showed minor differences in their distribution on the regulatory and transcribed regions tested, and had limited or no correlation with ecdysone induced transcriptional activity. We found that dCBP, which is encoded by the nejire gene, acetylates H3 lysine 23 in vivo, and silencing of nejire leads to reduced expression of the Eip74EF and Eip75B genes. Our results suggest that acetylation of specific lysine residues of histones contribute specifically to the dynamic regulation of transcription. Furthermore, along with previous studies identify CBP dependent H3 lysine 23 acetylation as an evolutionarily conserved chromatin modification involved in steroid induced gene activation.