3  Researches

Paramecium cells with endosymbionts

Left, P. caudatum with symbiotic bacteria Holospora obtusa in the macronucleus (Photograph, M. Fujishima).

Right, P. bursaria with symbiotic algae Chlorella variabilis in the cytoplasm (Kodama and Fujishima, Endosymbionts in Paramecium, pp.31-55, Springer, 2009).

Both endosymbionts can be isolated from homogenates of their host cells by Percoll continuous gradient centrifugation for Holospora and sucrose discontinuous centrifugation for Chlorella cells, and can induce reestablishment of endosymbiosis by mixing the isolated symbionts with the symbiont-free host cells. We clarified infection processes of Holospora and Chlorella cells to their host Paramecium cells as shown in schematic diagrams described below.

Infection process and life cycle of Holospora species

The spherical digestive vacuole (DV)-I differentiates to a condensed and acidified DV-II vacuole by fusion of acidosomes and evagination of the DV membrane to the cytoplasm. Then the vacuole differentiates to a swollen DV-III vacuole by fusion of primary lysosomes. Undigested materials remain in the acid phosphatase-less DV-IV vacuole. The DV-IV vacuole fuses to a cytoproct and discharges the content. Some infectious-form (IF) cells of Holospora species escape from the acidified DV without wrapping with the DV membrane. Immediately after escaping from the host DV, the bacteria differentiate to activated forms in the host cytoplasm, and migrate toward the target nucleus with the help of the host actin. The bacteria distinguish their target nucleus by specific binding between the outer membrane substance and the unknown nuclear envelope substance. Then, the bacterium penetrates the target nuclear envelope with an invasion tip leading. After the invasion, the bacterial cytoplasmic region increases and the large periplasmic region decreases to form constrictions for differentiation to the reproductive forms (RFs). During this infection process, the bacterium decreases its buoyant density from 1.16 to 1.09 g/ml. The RF continues to divide by binary fission when the host cell is also growing by binary fission, but the RF halts the binary fission, elongates itself, and differentiates to the IF when the host cell starves or the host's protein synthesis is inhibited. During this differentiation, the bacterium increases the buoyant density, and forms a large periplasmic region, an invasion tip, and two nucleoids. The infectious forms are freed from the cells. Acridine orange stains the DV-II vacuole orange, the DV-III vacuole yellow, and other DVs yellow-green. 

Infection process of symbiotic Chlorella variabilis to Paramecium bursaria

Using pulse label and chase method, four important cytological events necessary to establish endosymbiosis were clarified. First, 3 min after mixing, some algae acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs), even when the digested ones coexist. Second, 30 min after mixing, the algae start to escape from DVs as the result of budding of the membrane into the cytoplasm. Third, within 15 min after the escaping, the DV membrane enclosing a single green Chlorella differentiated to the perialgal vacuole (PV) membrane, which provides protection from lysosomal fusion. Finally, the alga localizes beneath the host cell cortex. At about 24 h after mixing, the alga increases by cell division and establishes endosymbiosis. (from Fujishima M. and Kodama Y. Endosymbionts in Paramecium. Eur. J. Protistol., 48, 124?137, 2012.)

Recent Fundings  from 2010)

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