A nuclear protein involved in apoptotic-like DNA degradation in Stylonychia: implications for similar mechanisms in differentiating and starved cells

Abstract

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.

Figure 1

Detection of a protein homologous to Tetrahymena Pdd1p (Spdd1p) in Stylonychia nuclear extracts by immunoblot analyses. Nuclear proteins with the same concentrations were separated by SDS-PAGE, stained with Coomassie Blue (a, c, e), blotted, and incubated with the Pdd1p antiserum (b, d, f). (a) Whole nuclear extracts from exconjugants (days 1–5 after mating). (b) Anti-Pdd1p Western blot of the proteins shown in a. (c) Nuclear extracts of exconjugant nuclei (day 2 after mating), which were separated by size. Lane 1, 30–40 μm; lane 2, 25–30 μm; lane 3, 10 -25 μm; lane 4, <10 μm. (d) Anti-Pdd1p Western blot analysis of the extracts shown in c. (e) Whole nuclear extracts from vegetative growing cells (+, lane 1 and lane 5 with the double concentration), from starved cells (3 d without feeding, −, lane 2), from isolated macronuclei (diameter 10–25 μm, lane 3), and from isolated micronuclei (<10 μm, lane 4). (f) Anti-Pdd1p (dilution of antiserum 1:1000) Western analysis of the extracts shown in e. Spdd1p is labeled by an arrow. Sizes are given in kilodaltons (Sigma molecular weight marker).

Figure 2

Tetrahymena Pdd1p inhibits binding of Pdd1p antiserum to Spdd1p. (a) Whole Tetrahymena macronuclear anlagen (MaA) nuclear lysates (lane 1, 10 μl; lane 2, 30 μl) and HPLC-purified Tetrahymena Pdd1p (lane 3, 10 μl; lane 4, 30 μl), separated by SDS-PAGE. (b) Anti-Pdd1p Western analysis (dilution of antiserum 1:2000) of the extracts shown in a (control for d). (c) Stylonychia exconjugants nuclear extracts (day 4) after pairing, separated by SDS-PAGE (lane 1). (d) The same amount of protein as in c, lane 1, was separated by SDS-PAGE, blotted, and incubated with the Pdd1p antiserum (dilution 1:2000) after preincubation without Tetrahymena Pdd1p (lane 1) or after preincubation with 1 μl (lane 2), 5 μl (lane 3), or 15 μl (lane 4) Tetrahymena Pdd1p (purified protein fraction as shown in a and b, lanes 3 and 4). The staining reaction was performed for 4 h with each membrane strip. Sizes are given in kilodaltons (Sigma molecular weight marker). Arrowhead, Spdd1p; double arrowhead, Tetrahymena Pdd1p.

Figure 3

Immunofluorescence analysis of vegetatively growing, starved, and exconjugant cells. Vegetative growing cells (a–c), starved cells (2 d without feeding, d–f), and exconjugant cells during polytene chromosome stage (g–i) and at a later stage (k–m) were fixed and incubated with the Pdd1p antiserum (secondary anti-rabbit Cy2-labeled antibody), and the DNA was stained with DAPI. (a, d, g, k) Phase-contrast microscopy, (b, e, h, l) DAPI stain (UV light), (c, f, i, m) anti-Pdd1p stain (blue light). Ma, macronucleus; MaA, macronuclear anlagen; mi, micronucleus. The replication band is labeled by an arrow. Bar, 10 μm.

Figure 4

Spdd1p expression in late Stylonychia macronuclear anlagen. Macronuclear anlagen during polytene chromosome stage (a, b), at the beginning of DNA fragmentation (c–f), and in the vesicle stage during DNA degradation (g, h) were fixed, squashed, and stained with the Pdd1p antiserum (Cy2-stained anti-rabbit secondary antibody). (i, k) Macronucleus of nonconjugating cells (negative control). (a, c, e, g, i) Phase-contrast microscopy. (b, d, f, h, k) Anti-Pdd1p immunofluorescence (blue light). mi, micronucleus. Bar, 10 μm.

Figure 5

Localization of Spdd1p to the bands of polytene chromosomes. Macronuclear anlagen were fixed, squashed, incubated with the Pdd1p antiserum, and analyzed by immunofluorescence microscopy. (a–f, h) Anti-Pdd1p immunofluorescence stain (blue light); (g) phase-contrast image of h. Bar, 10 μm.

Figure 6

TUNEL assay with starved and exconjugant cells. Vegetatively growing cells (a–c), starved cells (d–f), exconjugants during polytene chromosome stage (g–i), exconjugants during beginning of DNA fragmentation (k–m), and cells with very late macronuclear anlagen and a newly synthesized macronucleus (n–p) were fixed for the TUNEL assay. In addition, nuclear DNA was stained with DAPI. (a, d, g, k, n) Phase contrast microscopy; (b, e, h, l, o) DAPI stain; (c, f, i, m, p) TUNEL assay (FITC-labeled secondary anti-mouse antibody). Ma, macronucleus; MaA, macronuclear anlagen; mi, micronucleus. Arrows, replication band. Bar, 10 μm.