Yan Yu. Komissarchik

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

Tubular structures of Mycoplasma gallisepticum and their possible participation in cell motility

In five strains of Mycoplasma gallisepticum, a protein with a molecular mass of about 40 kDa was detected by immunoblotting with anti-pig brain tubulin polyclonal and monoclonal antibodies. In eight other mycoplasma species similarly tested no reaction was observed. Thin serial sections of M. gallisepticum and Acholeplasma laidlawii cells examined by transmission electron microscopy revealed a submembrane system of tubules in M. gallisepticum but not in A. laidlawii. The intracellular spatial distribution of the tubular structures was reconstructed. Thin sections of M. gallisepticum treated with anti-tubulin antibodies and colloidal gold particles (immunogold labelling) revealed distinct labelling of the tubular system. Analysis of the tubular structures by high resolution electron microscopy and optical diffraction showed their helical organization to be: diameter 40 nm, helix pitch approximately 20 nm and electron-transparent core 10 nm in diameter. A possible involvement of the tubular system in mycoplasma motility is suggested.

Bacterial invasion of eukaryotic cells can be mediated by actin‐hydrolysing metalloproteases grimelysin and protealysin

Earlier, we have shown that spontaneously isolated non‐pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin‐like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep‐2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli‐carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep‐2 cells. Moreover, internalization of non‐invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

N-Acetylcysteine-induced changes in susceptibility of transformed eukaryotic cells to bacterial invasion

The effect of N‐acetylcysteine (NAC) on morphological and physiological properties of “normal” 3T3 and 3T3‐SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3‐SV40 fibroblasts demonstrated formation of well‐adhered cells with structured 3T3‐like stress‐fibers. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria‐susceptible 3T3‐SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC‐induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.

Visualization of early golgi compartments at proliferate and sporogenic stages of a microsporidian Nosema grylli.

Vesicular and tubular structures associated with Golgi complex (GC) are observed in sporoblasts during polar filament maturation. Staining with thiamin pyrophosphatase (TPP) proved them to be homologous to trans-Golgi cistemae of other eukaryotes in spite of the lack of the classic flattened organization [8]. At the same time we know very little about localization of the early secretory compartments (intermediate endoplasmic reticulum-GC and cis-GC) in the mirosporidian cells throughout the life cycle and actually nothing about the presence of GC at the proliferate stage of microsporidian development [9 for review 1.

Specific invasion of transformed cells by Escherichia coli A2 strain.

Bacteria of the spontaneously isolated non‐pathogenic strain Escherichia coli A2 producing actin‐specific protease ECP 32 (Usmanova and Khaitlina, 1989) were shown to be taken up by transformed cells, whereas finite and immortal cell lines were resistant to the infection.

The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma.

Abstractα-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

Decreased Sensitivity of Transformed 3T3-SV40 Cells Treated with N-Acetylcysteine to Bacterial Invasion

Long-term treatment of transformed 3T3-SV40 mouse fibroblasts with antioxidant N-acetylcysteine decreased cell level of ROS and increased the concentration of reduced glutathione. Removal of N-acetylcysteine from the medium led to the appearance of well-expressed stress fibrils, virtually absent in control cells. In contrast to control cells, these cells were not invaded by apathogenic Escherichia coli A2 strain producing ECP32 protease specifically cleaving actin. Antioxidant N-acetylcysteine can cause partial reversion of transformed phenotype at the expense of a shift of cell redox balance in favor of reduced glutathione.

Structural correlates of the transepithelial water transport.

Transepithelial permeability is one of the fundamental problems in cell biology. Epithelial cell layers protect the organism from its environment and form a selective barrier to the exchange of molecules between the lumen of an organ and an underlying tissue. This chapter discusses some problems and analyzes the participation of intercellular junctions in the paracellular transport of water, migration of intramembrane particles in the apical membrane during its permeability changes for isotonic fluid in cells of leaky epithelia, insertion of water channels into the apical membrane and their cytoplasmic sources in cells of tight epithelia under ADH (antidiuretic hormone)-induced water flows, the osmoregulating function of giant vacuoles in the transcellular fluxes of hypotonic fluid across tight epithelia, and the role of actin filaments and microtubules in the transcellular transport of water across epithelia.

Ultrastructural Study and X-Ray Microanalysis of Apoptotic Lymphoma Cells U-937

We studied the ultrastructure and ionic cell contents of human histiocytic lymphoma U-937 cells induced for apoptosis by two external agents, hypertonic shock and etoposide. Despite different mechanisms of action, the deterioration of cellular functions triggered by these agents followed similar patterns. The most dramatic changes–condensation of the chromatin and formation of “crescent-shaped nuclei”, nucleus fragmentation, disappearance of nucleoli, and appearance of aggregated particles in the interchromatin nuclear space were observed in the nucleus. The aggregated particles observed in the interchromatin nuclear space, were lacking limiting membrane and presumably derived from the aggregates of interchromatin granules (IGs), so called speckles. The speckles, were shown previously to be highly dynamic nuclear structures associated with: m-RNA-splicing, transcription, and processing of pre-mRNA [1]. We attribute the described here aggregates to the “HERDS” (heterogeneous ectopic RNP-derived structures) type described by Biggiogera and co-authors [2,3]. In contrast to other cytoplasmic aggregates such as aggresomes, processing bodies (Pbs) or stress bodies (SBs) HERDS were observed both in the nucleus, and in cytoplasm [4,5]. They emerge in response to impairment of transcription during apoptosis. In case of cellular physiological modifications, e.g. under spermiogenesis or animal hibernation, this process can be reversible [2,3]. Ultrastructural analysis and immune electron microscopy of the aggregates observed in U-937 cell line allowed identification of three types of nuclear particles dominating in these aggregates: interchromatin granules (IGs) of 10 x 40 nm, proteasomes (PRs) of 10-15 × 30 nm, and long perichromatin fibers (PFs) [4]. Both IGs and PRs were rod-like shaped particles of variable size. They demonstrated different immune properties: IGs recognized ABs against the splicing factor SC35 (Figure 1a), meanwhile PRs reacted with antibodies (ABs) against proteins of the proteasome protein complex (Figure 1b). Ultrastructurally PFs observed in this study essentially differed from the ones described earlier [3-6].