Ultrastructural analysis of human leukemia U-937 cells after apoptosis induction: Localization of proteasomes and perichromatin fibers.

We studied the ultrastructure of human histiocytic lymphoma U-937cells after apoptosis induction with two external agents, hypertonic shock and etoposide. Appearance of aggregates of particles of nuclear origin within the nuclei and cytoplasm of the induced cells was the first and the most prominent morphological sign of apoptosis. These aggregates were not coated by a membrane, had variable shape, density and size. Two types of particles dominated in the aggregates: perichromatin fibers (PFs) and proteasomes (PRs). PFs represent a morphological expression of transcriptional and co-transcriptional processing of pre-mRNA (Biggiogera et al., 2008), PRs are involved in hydrolysis of proteins and nucleoproteins, and participate in regulation of apoptosis (Ciechanover, 1998; Liu et al., 2007). We examined the ultrastructure and localization of PFs and PRs, and confirmed the proteasome nature of the latter by immunoelectron microscopy. We traced the formation and migration of the aggregates along the nucleus and their exit into the cytoplasm across the nuclear pores. Finally, we demonstrated degradation of the aggregates and relocating their content into exosomes at the terminal stages of apoptosis with aid of exosomes. We suggest that proteasomes function as morphologically definite and independent intracellular organelles. Alongside with proteasomes, autophagic vacuoles were revealed in apoptotic cells. Occurrence of autophagic vacuoles in apoptotic cells may suggest that both proteolytic pathways, autophagy and proteasome degradation, are coordinated with each other along the programmed cell death pathway.

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

α-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.

Immunoelectron microscopic study of BASP1 and MARCKS location in the early and late rat spermatids.

Immunoelectron microscopy was used to locate the proteins BASP1 and MARCKS in the post-meiotic spermatids of male rat testis. It was shown that in early spermatids, BASP1 and MARCKS accumulate in chromatoid bodies, which are characteristic organelles for these cells. During spermatogenesis, while the spermatid nucleus is still active, the chromatoid body periodically moves to the cell nucleus and absorbs the precursors of definite mRNAs and small RNAs. mRNAs are preserved in the chromatoid body until the corresponding proteins are needed, but their "fresh" mRNA cannot be formed due to the nucleus inactivation. The chromatoid body (0.5-1.5µm in diameter) has a cloud-like fibrous appearance with many fairly round cavities. In the chromatoid body, BASP1 and MARCKS are distributed mainly around the cavities and at periphery. Based on the known functions of BASP1 and MARCKS in neurons, it is conceivable that these proteins participate in non-random movements of the chromatoid body to the nucleus and in Ca(2+)-calmodulin enrichment. In late spermatids, BASP1 and MARCKS are located in the outer dense fiber layer belonging to a metabolically active spermatozoon region, the tail mid-piece. In spermatozoa, as in chromatoid body, BASP1 and MARCKS may bind Ca(2+)-calmodulin and therefore contribute to the activation of calcium-dependent biochemical processes.

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.

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, gammaCOP 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.