Evgeny Sagulenko

Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus

Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell.

Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death

High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.

Keys to eukaryality: planctomycetes and ancestral evolution of cellular complexity

Planctomycetes are known to display compartmentalization via internal membranes, thus resembling eukaryotes. Significantly, the planctomycete Gemmata obscuriglobus has not only a nuclear region surrounded by a double-membrane, but is also capable of protein uptake via endocytosis. In order to clearly analyze implications for homology of their characters with eukaryotes, a correct understanding of planctomycete structure is an essential starting point. Here we outline the major features of such structure necessary for assessing the case for or against homology with eukaryote cell complexity. We consider an evolutionary model for cell organization involving reductive evolution of Planctomycetes from a complex proto-eukaryote-like last universal common ancestor, and evaluate alternative models for origins of the unique planctomycete cell plan. Overall, the structural and molecular evidence is not consistent with convergent evolution of eukaryote-like features in a bacterium and favors a homologous relationship of Planctomycetes and eukaryotes.

Protein uptake by bacteria: An endocytosis-like process in the planctomycete Gemmata obscuriglobus

Endocytosis is a fundamental process of membrane-trafficking in eukaryotes, but has not been known to occur in bacteria or archaea. The origin of endocytosis is central to the understanding of evolution of the first eukaryotes and their endomembrane systems. In a recent study we have established that an endocytosis-like process for uptake of proteins into cells occurs in a bacterium, Gemmata obscuriglobus, a member of the distinctive phylum Planctomycetes of peptidoglycan-less budding bacteria. Members of this phylum characteristically possess cells divided into compartments separated by internal membranes, and in the case of G. obscuriglobus these compartments include one where a double membrane envelope surrounds its nucleoid DNA, as well as an outer ribosome-free region of cytoplasm. Proteins can be internalized by cells from the external milieu and collected into this ribosome-free compartment, and this process is energy-dependent and appears to be receptor-mediated. As in eukaryote endocytosis, internalized proteins are associated with vesicles, and can be subjected to proteolytic degradation. The discovery of this process in a bacterium has significant implications for our understanding of the origins of endocytosis in eukaryotes.

Structural studies of Planctomycete Gemmata obscuriglobus support cell compartmentalisation in a bacterium

Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.

Novel aphidicolin‐inducible common fragile site FRA9G maps to 9p22.2, within the C9orf39 gene

Common fragile sites represent a component of normal chromosome structure that form gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. In humans, cytogenetic locations of 89 common fragile sites are listed in the Genome Database; however, the exact number of fragile sites remains unknown. The application of high resolution mapping approaches continues to reveal new common fragile sites in the human genome. Here, we identified a novel aphidicolin‐inducible common fragile site FRA9G, which maps to chromosomal band 9p22.2. We have characterized the structure of the fragile DNA sequence that extends over a genomic region of ∼300 kb within the C9orf39 (chromosome 9 open reading frame 39) gene. Analysis of incidence in healthy individuals showed that FRA9G is commonly expressed in the population. Heterozygous BRCA2 mutation carriers exhibit an almost sevenfold increase of FRA9G expression compared to an unrelated control population group. Identification of a novel aphidicolin‐inducible common fragile site at 9p22 may have implications for understanding the mechanism of genetic instability in tumorigenesis and other genetic disorders.

Nested Bacterial Boxes: Nuclear and Other Intracellular Compartments in Planctomycetes

Bacteria in the phylum Planctomycetes and some related phyla challenge our concept of the typical bacterium as consisting of cells without internal compartments or membrane-bounded organelles. Cells of all species of planctomycetes examined consist of at least two major compartments, and there are two other types of compartmentation in which a third compartment is formed either by a double-membrane envelope around the nucleoid in the case of the aerobic Gemmata obscuriglobus or by a single but potentially energized membrane in the case of the anaerobic ammonium-oxidizing anammox planctomycetes. We examine here the nature of these planctomycete compartments in relation to function and their relationship to the endomembranes defining them, and discuss the implications of the remarkable compartment-confined process of protein uptake in Gemmata, which resembles receptor- and clathrin-mediated endocytosis of eukaryotes. Planctomycetes have implications for our understanding of the evolution of membrane-bounded organelles, of endomembranes, transport across endomembranes and membrane trafficking, and for how the complexity of a eukaryote style of cell organization could have originated.

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia.

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of ‘low microbial abundant’ (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges.

Electron tomography of the nucleoid of Gemmata obscuriglobus reveals complex liquid crystalline cholesteric structure

The nucleoid of the planctomycete Gemmata obscuriglobus is unique within the Bacteria in being both highly condensed and enclosed by a double-membrane nuclear envelope, seemingly analogous to the nucleus of eukaryotes. Here we have applied electron tomography to study high-pressure frozen, cryosubstituted cells of G. obscuriglobus and found multiple nested orders of DNA organization within the condensed nucleoid structure. Detailed examination of the nucleoid revealed a series of nested arcs characteristic of liquid crystalline cholesteric DNA structure. The finest fibers were arranged in parallel concentrically in a double-twist organization. At the highest order of nucleoid organization, several of these structures come together to form the core of the G. obscuriglobus nucleoid. The complex structure of DNA within this nucleoid may have implications for understanding the evolutionary significance of compartmentalized planctomycete cells.

Nuclear Pore-Like Structures in a Compartmentalized Bacterium.

Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a β-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence.