Methodological Approaches to Assessing the Size and Morphology of Microvesicles of Cell Lines.

Morphological properties and the size of microvesicles were assessed using atomic force microscopy, electron microscopy, and granulometric analysis. As these methods require significant numbers of microvesicles, we chose microvesicles derived from cell lines for our research.

Host and symbiont intraspecific variability: The case of Paramecium calkinsi and "Candidatus Trichorickettsia mobilis".

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species "Candidatus Trichorickettsia mobilis" (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split "Candidatus Trichorickettsia" into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed "Candidatus Trichorickettsia" hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.

[Fibrillar actin in the nuclear apparatus of the ciliate Paramecium caudatum].

Due to their nuclear dualism, ciliates provide a good model for studying the role of actin in spatial organization and transcription activity of the nucleus. The actin in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloiodin and indirect immunocytochemistry. Fibrillar actin was demonstrated in both of the nuclei. Actin was revealed in the chromatin areas, and was often associated with the periphery of the amplified nucleoli in the macronucleus. Redistribution of actin was observed depending on different physiological state of the cells. Stable infection of the macronulear with the intranuclear endobionts Holospora obtuse led to the loss of nuclear actin accompanied by significant nuclear fragility and redistribution of the phosphorylated form of the actin-binding protein cofilin. Spherical bodies resembling karyosphere were found in the macronuclear anlagen.

Actin-based mechanism of holospora obtusa trafficking in Paramecium caudatum.

Holospora obtusa, an alpha-proteobacterium, is an obligate endonuclear pathogen of the ciliate Paramecium caudatum. It is engulfed by the host cell in the course of phagocytosis but soon escapes from the phagosome and is transported across the host cell cytoplasm to the paramecium macronucleus. Electron microscopy reveals a comet-like tail resembling that of Listeria trailing after H. obtusa in the host cytoplasm. In this study we investigated the role of the host cell actin and Arp3 in the process of infection with Holospora. Cytochalasin D treatment significantly reduced the rate of nuclear infection. Using immunocytochemistry and experimental infection of GFP-actin-transfected paramecia we demonstrated that the Paramecium actin1-1 took part in the bacterial escape from the phagosome, its trafficking in the cytoplasm and entry into the host macronucleus. Rapid assembly/disassembly of actin filaments in P. caudatum led to quick loss of actin1-1 from the trails left by H. obtusa. Immunocytochemistry using anti-bovine Arp3 antibodies demonstrated the presence of Arp3 in these trails. Our data indicate that details of H. obtusa infection are rather similar to those of Listeria and Rickettsia.