Trichinella spiralis-infected muscle cells: abundant RNA polymerase II in nuclear speckle domains colocalizes with nuclear antigens

Abstract

Infection of mammalian skeletal muscle cells by Trichinella spiralis causes host nuclei to become polyploid (ca. 4N) and abnormally enlarged. It has been postulated that this enlargement reflects an infection-induced elevation of host transcription. Anthelmintic treatment of T. spiralis-infected rodents with mebendazole (MBZ) causes a reduction in the size of infected cell nuclei and a significant reduction in the total RNA content of individual infected muscle cells. A monoclonal antibody to the large subunit of RNA polymerase II (Pol II) was used here to assess the effects of infection on Pol II levels in isolated infected cell nuclei. Pol II was localized to speckle domains in isolated infected cell nuclei. Similar domains have been previously localized to sites of RNA synthesis or processing. When compared to the levels in nuclei from other, uninfected host cells, speckle-localized Pol II (SL-Pol II) levels were significantly elevated in infected cell nuclei by a mean of 3.9- to 6.8-fold. Nuclear antigens (NA) recognized by antibodies against T. spiralis localized to infected cell nuclei. By use of confocal microscopy, a subpopulation of NA was found colocalized with most speckle domains defined by Pol II. MBZ treatment of chronically infected mice, which depletes NA from infected cell nuclei, caused a significant depletion of SL-Pol II from infected cell nuclei. Control nuclei had a mean of 70% more SL-Pol II than MBZ-treated nuclei. The mean residual level of Pol II in these polyploid nuclei remained elevated by 120% over the level in 2N control nuclei. These observations may indicate two distinct effects of infection on Pol II levels in host cells.

FIG. 1

Immunoblot using MAb 8WG16. Extracts of infected cell nuclei (5 × 10⁵), isolated as described in Materials and Methods, were probed by immunoblotting using MAb 8WG16 (lane 1) or an isotype control MAb (IgG2a) (lane 2), each at 10 μg ml⁻¹. The label on the right indicates bands at estimated 210 and 240 kDa which coincide with major hypo- and hyperphosphorylated forms of the large subunit of Pol II. The dashes on the left indicate size standards in the descending order of 220, 97.4, 66, 46, 30, 21.7, and 14.3 kDa.

FIG. 2

Detection of Pol II in infected cell nuclei. (A) IFA of isolated infected cell nuclei was done with MAb 8WG16 (anti-Pol II) (20 μg ml⁻¹) alone (a), mixed with a heptapeptide repeat (21-mer) containing the MAb epitope (d), or mixed with a control competitor peptide (24-mer) for a 44-kDa B. bovis protein (e). (b) Lack of binding by an isotype control antibody (IgG2a). (c) Nomarski image of the nucleus evaluated in panel b. Bar, 10 μm. (B) Inhibition of MAb 8WG16 binding to infected cell nuclei. The IFA results shown in panel A were quantitated by microfluorimetry. Columns: 1, MAb 8WG16 alone (20 μg/ml); 2, MAb plus peptide for a 44-kDa B. bovis protein; 3, MAb plus peptide for a 225-kDa B. bovis protein; 4, MAb plus a Pol II heptapeptide repeat (21-mer). A 1:200 molar ratio was used for all peptides. Error bars indicate standard deviations. The asterisk indicates a significant reduction (P < 0.001), compared to the data shown in column 1, which was observed only for the Pol II peptide. The percent reduction (55%) obtained with this heptapeptide is similar to that achieved by others using purified Pol II in an enzyme-linked immunosorbent assay (22).

FIG. 3

Pol II and NA distributions in infected cell nuclei. (a) Confocal microscopy of Pol II in speckle domains using MAb 8WG16 (2 μg ml⁻¹) in an IFA as described in the legend to Fig. 2. Green localizes MAb binding at one level, red localizes it at another level 1 μm away, and yellow shows the coincidence of binding at the two levels. (b to d) Dual labeling to determine the localizations of Pol II and NA in infected cell nuclei. (b) Pol II localization using MAb 8WG16. (c) NA localization using anti-NA antibody (α-DG p43) at the same level as that shown in panel b. (d) Merger of panels b and c. The yellow color shows colocalization. These results were obtained with 0.5-μm sections. Similar results were obtained with 0.1-μm sections and at different levels within nuclei. The results show that NA localize to speckle domains characterized by Pol II. Bar, 10 μm.

FIG. 4

Comparison of Pol II in infected cell nuclei and inflammatory cell nuclei. The confocal picture shows infected cell nuclei (large arrows) and two infiltrating cell nuclei (small arrows). Nuclei were coisolated from chronically infected muscle cells and analyzed in an IFA using MAb 8WG16 as described in the legend to Fig. 2. Bar, 10 μm. Quantitation of immunofluorescence for the two classes of nuclei is shown in Table 1.

FIG. 5

MBZ treatment of chronic T. spiralis infections depletes NA and Pol II from infected cell nuclei. (A) Immunoblot of infected cell nuclei (n = 62,500) isolated from animals treated with vehicle alone (lane C) or MBZ (lane M) for 4 days. Proteins separated on 7.5 to 17.5% polyacrylamide gels were detected using anti-NA antibodies. The results demonstrate that NA were depleted from infected cell nuclei by the MBZ treatment. Size standards on the left are in kilodaltons. (B) Pol II levels in infected cell nuclei (NC) and infiltrating cell nuclei (IC) from mice treated as described for panel A. Measurements were made after 10 days of treatment. IFAs were conducted with MAb 8WG16, and Pol II levels were quantitated by microfluorimetry. Error bars indicate standard deviations. The asterisk indicates significant (P < 0.001) differences between the control and MBZ-treated infected cell nuclei. Results are representative of two experiments.