Nucleolar Division in the Promastigote Stage ofLeishmania majorParasite: A Nop56 Point of View

Abstract

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56) during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56 in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical, to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out by an analogous mechanism.

Nucleolar Structure and Function in Trypanosomatid Protozoa

“…Similar bipartite nucleoli are found in other organisms, including other protozoan, yeast, invertebrates, fish, and amphibians [7,26,33,34,35]. In contrast with higher eukaryotes, Trypanosoma and Leishmania undergo a closed mitosis in which the nuclear envelope remains intact, the chromatin does not condense, and the nucleolus persists as an intranuclear, ribonucleoproteic organelle during the whole cell division (Figure 2B) [30,36,37,38]. As mitosis progresses, the nucleolus lengthens and is pulled, via the spindle fibers, towards the opposite poles of the nucleus.…”

“…The T. brucei Snu13 protein shares 64% identity with the yeast orthologue and, as expected, it interacts with snoRNAs [109]. In L. major , in silico analyses showed that, despite sequence divergence, Nop56 contains the three structural and evolutionary conserved domains (NOP5NT, NOSIC and Nop) and that its predicted three-dimensional structure is very similar to that of the yeast orthologue [38].…”

“…Eventually, the nucleolar structure is divided into two large ribonucleoprotein complexes that are transmitted to each nascent nucleus without intervention of intermediary structures, such as the classical prenucleolar bodies (Figure 2B). Finally, late in mitosis, new nucleoli are clearly observed before partition of the cytoplasm by cytokinesis (Figure 2B) [36,37,38].…”

“…Under situations of nutritional starvation, such as metacyclogenesis, there is a pronounced decrease in transcriptional activity, mainly of Pol I, causing the dispersion of nucleolar material into the nucleoplasm and the disassembly of the bipartite nucleolus [165], suggesting that the T. cruzi nucleolus can act as a true stress sensor and a coordinator of cell response during the differentiation process. This hypothesis is reinforced by the fact that during the stationary phase of culture (which is a suitable in vitro condition that recreates the nutrient stress faced by trypanosomatids during the initiation of metacyclogenesis in vivo), the low rates of RNA synthesis are associated with both the subcellular relocation of several nucleolar antigens and the dramatic alterations of nucleolar ultrastructure [32,38,76,108,166]. In addition, the treatment of T. cruzi and L. mexicana with actinomycin D, a transcription inhibitor, provoke the nucleolar agglomeration of a select group of RNA binding proteins (RBPs) involved in mRNA metabolism [167,168].…”

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

“…The cellular localization of LmBdp1 labeled with a PTP tag was determined by indirect immunofluorescence, as previously described [26, 34]. For these assays, 1.5 × 10 7 mid-log promastigotes were incubated with a rabbit anti-Prot C polyclonal antibody (Delta Biolabs) and a secondary goat anti-rabbit antibody conjugated with Alexa-Fluor 488 (Life Technologies).…”