The collective outcome of this C1-dependent proteolytic mechanism during apoptosis can play important roles in reducing the host response to self-antigens to reduce the development of autoimmunity, a good hypothesis to explain their genetic deficiencies becoming causal to SLE pathogenesis. surface to constructions resembling the filopodium type of cortical cytoskeleton (Fig. 2). In the cytoplasm, C1q binding sites were diffuse, appearing consistently networked. Because C1q has been reported previously to bind to monomeric actin (29), it is unclear whether the cortical and cytoplasmic binding by C1q were both contributed by actins. At this stage, binding to the nucleus was observed but remained low, and the bound constructions were particulate in gaps between condensed chromatin areas (Fig. 2). The particulate nature of Entecavir hydrate the C1q-bound nuclear constructions were apparent on the basis of both 0.38-m section images and reconstructed three-dimensional images. There was a definite lack of C1q binding to the large body of chromatins. By 3 h, related binding patterns were maintained. Treatment of these cells with detergent did not significantly alter the C1q binding pattern (data not demonstrated). Open in a separate window Number 2. Temporal C1q binding to early and advanced apoptotic cells. HeLa cells on glass coverslips were UV-irradiated and cultured in serum-free medium for 1, 3, 6, 12, and 24 h and, after fixation in 1% (w/w) PFA for 30 min, incubated with C1q. Bound C1q was recognized using a mouse anti-C1q antibody, followed by goat anti-mouse IgG (Cy3, = 5 m. C1q Binds Mainly to the Nucleolus in Advanced Apoptotic Cells Six hours after UV irradiation, both surface and cytoplasmic C1q Entecavir hydrate binding subsided, but binding to the nucleus was intensified (Fig. 2). Furthermore, the pattern of C1q binding in the nucleus also changed from small particulate constructions to one to two large congregated constructions (Fig. 2). The C1q-bound subnuclear region was surrounded by a rim of chromatins, with sparse chromatin fragments becoming also found within the C1q-bound region. This C1q binding pattern is definitely homogeneous among the cell human population at 6 h. By 12 h, some cells started to show nuclear fragmentation, and, in these cells, C1q binding was also found on the nuclear fragments (Fig. 2). More rigorous nuclear fragmentation was observed by 24 h, and C1q continued to bind to these fragments. In the mean time, the nuclear corpses remaining in the cell center began to shed C1q binding. Although C1q binding was generally near nuclear chromatins in advanced apoptotic cells, C1q probably bound to DNA-associated constructions rather than DNA itself. The size and morphology of the C1q-bound nuclear structure resembled that of the nucleolus, as demonstrated most clearly at 6 h (Fig. 2) (30, 31). In Advanced Apoptotic Cells, the Major C1q-bound Nuclear Region Is the Nucleolus To examine whether the C1q-bound subnuclear areas in the 6-h apoptotic cells were indeed nucleoli, cells were 1st incubated with C1q and then incubated with mouse anti-C1q and rabbit anti-NPM1 antibodies. NPM1 is definitely a nuclear protein found mainly in the granular component of the nucleolus (32). As demonstrated in Fig. 3, the C1q-bound nuclear areas were strongly labeled by anti-NPM1 antibody (Fig. 3). At this stage, NPM1 also diffused broadly in the cytoplasm, where C1q binding was absent. It suggests that, although C1q binds Rabbit Polyclonal to IGF1R to the nucleoli in late apoptotic cells, NPM1 is an unlikely C1q ligand. Actually in the nucleolar region, C1q binding showed an incomplete overlap with NPM1 (Fig. 3and and = 5 m. The Nuclear Lamina Shows Impairment in Advanced Apoptotic Cells At this point, the following two possibilities were regarded as: C1q ligands are Entecavir hydrate only induced late in Entecavir hydrate cell apoptosis, and the ligands exist constitutively in nucleoli, becoming inaccessible by C1q at earlier time points. In this part of the study, we evaluated the integrity of the nuclear lamina in apoptotic.
