Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Dec;5(12):1990-2000.
doi: 10.1128/EC.00195-06. Epub 2006 Sep 29.

The Tetrahymena Thermophila Phagosome Proteome

Affiliations
Free PMC article

The Tetrahymena Thermophila Phagosome Proteome

Mary Ellen Jacobs et al. Eukaryot Cell. .
Free PMC article

Abstract

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.

Figures

FIG. 1.
FIG. 1.
Summary of Tetrahymena phagosome proteome analysis. Tetrahymena micrographs are combined Nomarski and fluorescent images. Dark intracellular inclusions are bead-filled phagosomes (PGSMs), and the arrow indicates a single extracellular latex bead. See the text for additional details.
FIG. 2.
FIG. 2.
(A) Western blot analysis with antibodies to Grl8p, histone H1, and α-tubulin. Blots contain 104 cell equivalents of total Tetrahymena proteins (lanes 1) or 104 (lanes 2) or 106 (lanes 3) cell equivalents of phagosomes. In the Grl8p blot, the arrowhead denotes the position of the 45-kDa Grl8p proprotein, and the arrow indicates the 22-kDa mature form. (B) Ethidium bromide-stained agarose gels containing nucleic acids (2 μl or 20 μl) isolated from fractions of a sucrose gradient (10 to 62% sucrose) used to purify phagosomes. The positions of the 26S and 17S rRNAs are indicated.
FIG. 3.
FIG. 3.
Localization of GFP-tagged constructs in live Tetrahymena cells. Pairs of differential interference contrast (DIC) and fluorescence (FLUOR) confocal microscopy images are shown. (A) Control parental CU522 cell line. (B) GFP-tagged cathepsin B transformant cell line. (C and D) GFP-tagged Tpp2p transformant cell line (different confocal image sections of the same cell are shown in panels C and D). (E and F) GFP-tagged Tpp9p transformant cell line fed beads for 15 min and 2 h, respectively. (G) GFP-tagged Tpp5p transformant cell line. Images are oriented with the cell anterior directed toward the upper left corner of the image. Long arrows, bead-containing phagosomes; short arrows, phagosomes without beads; large arrowheads, contractile vacuoles (CV). MA, macronucleus. All images were obtained under the same microscopy conditions and were uniformly processed.

Similar articles

See all similar articles

Cited by 26 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback