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Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.

Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, Greber UF - J. Cell Biol. (1999)

Bottom Line: No directed movement was observed in nocodazole-treated cells.Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment.The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, University of Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

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Ad2 arrives in the cytosol in the absence of intact microtubules. (A) HeLa cells were either kept in drug-free medium  (lanes 1–5) or pretreated with 20 μM nocodazole at 37°C for 15  min (lanes 6–11). [35S]methionine Ad2 was bound in the cold, followed by warming in the presence or absence of nocodazole for  indicated times. Surface-bound virus was digested with trypsin  for 1 h in the cold followed by analysis of cleaved and trypsin-resistant hexon by SDS-PAGE and phosphorimaging as described earlier (Greber et al., 1993). (B) HeLa cells were either  treated with 20 μM nocodazole (panel b) or left in drug-free medium (panels a, c, and d) as described in A. 50 μg/ml wt Ad2  (panels a and b) or 50 μg/ml ts1 virus (panel c) or no virus (panel  d) was bound at 4°C for 90 min. Cells were warmed to 37°C in  growth medium containing 20 mg/ml HRP with or without nocodazole for 15 min and then incubated in HRP-free medium for  15 min, fixed, and then prepared for thin section EM. HRP activity was visualized using diaminobenzidine. Bar, 0.5 μm.
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Figure 4: Ad2 arrives in the cytosol in the absence of intact microtubules. (A) HeLa cells were either kept in drug-free medium (lanes 1–5) or pretreated with 20 μM nocodazole at 37°C for 15 min (lanes 6–11). [35S]methionine Ad2 was bound in the cold, followed by warming in the presence or absence of nocodazole for indicated times. Surface-bound virus was digested with trypsin for 1 h in the cold followed by analysis of cleaved and trypsin-resistant hexon by SDS-PAGE and phosphorimaging as described earlier (Greber et al., 1993). (B) HeLa cells were either treated with 20 μM nocodazole (panel b) or left in drug-free medium (panels a, c, and d) as described in A. 50 μg/ml wt Ad2 (panels a and b) or 50 μg/ml ts1 virus (panel c) or no virus (panel d) was bound at 4°C for 90 min. Cells were warmed to 37°C in growth medium containing 20 mg/ml HRP with or without nocodazole for 15 min and then incubated in HRP-free medium for 15 min, fixed, and then prepared for thin section EM. HRP activity was visualized using diaminobenzidine. Bar, 0.5 μm.

Mentions: It was possible that nocodazole affected endocytosis and/or virus escape from the endosome. We therefore measured the internalization of [35S]methionine virus into drug-treated cells using a cell surface trypsinization assay, which measures virus uptake by the emergence of a trypsin-resistant form of the major capsid protein hexon (Greber et al., 1993). The results indicate that in the presence of nocodazole half of the virus particles were endocytosed at 15 min postwarming, whereas in control cells the half-time of internalization was ∼9 min (Fig. 4 A). The efficiency of entry at 30 min postwarming was close to 75% under both conditions. The remaining viruses were most likely shed from the cells into the medium (Greber et al., 1993). The data demonstrated that virus uptake into cells lacking intact MTs was slightly slower, but no less efficient than into control cells, in agreement with earlier observations showing that nocodazole had no effect on the formation of endocytic vesicles (Gruenberg et al., 1989).


Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.

Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, Greber UF - J. Cell Biol. (1999)

Ad2 arrives in the cytosol in the absence of intact microtubules. (A) HeLa cells were either kept in drug-free medium  (lanes 1–5) or pretreated with 20 μM nocodazole at 37°C for 15  min (lanes 6–11). [35S]methionine Ad2 was bound in the cold, followed by warming in the presence or absence of nocodazole for  indicated times. Surface-bound virus was digested with trypsin  for 1 h in the cold followed by analysis of cleaved and trypsin-resistant hexon by SDS-PAGE and phosphorimaging as described earlier (Greber et al., 1993). (B) HeLa cells were either  treated with 20 μM nocodazole (panel b) or left in drug-free medium (panels a, c, and d) as described in A. 50 μg/ml wt Ad2  (panels a and b) or 50 μg/ml ts1 virus (panel c) or no virus (panel  d) was bound at 4°C for 90 min. Cells were warmed to 37°C in  growth medium containing 20 mg/ml HRP with or without nocodazole for 15 min and then incubated in HRP-free medium for  15 min, fixed, and then prepared for thin section EM. HRP activity was visualized using diaminobenzidine. Bar, 0.5 μm.
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Related In: Results  -  Collection

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Figure 4: Ad2 arrives in the cytosol in the absence of intact microtubules. (A) HeLa cells were either kept in drug-free medium (lanes 1–5) or pretreated with 20 μM nocodazole at 37°C for 15 min (lanes 6–11). [35S]methionine Ad2 was bound in the cold, followed by warming in the presence or absence of nocodazole for indicated times. Surface-bound virus was digested with trypsin for 1 h in the cold followed by analysis of cleaved and trypsin-resistant hexon by SDS-PAGE and phosphorimaging as described earlier (Greber et al., 1993). (B) HeLa cells were either treated with 20 μM nocodazole (panel b) or left in drug-free medium (panels a, c, and d) as described in A. 50 μg/ml wt Ad2 (panels a and b) or 50 μg/ml ts1 virus (panel c) or no virus (panel d) was bound at 4°C for 90 min. Cells were warmed to 37°C in growth medium containing 20 mg/ml HRP with or without nocodazole for 15 min and then incubated in HRP-free medium for 15 min, fixed, and then prepared for thin section EM. HRP activity was visualized using diaminobenzidine. Bar, 0.5 μm.
Mentions: It was possible that nocodazole affected endocytosis and/or virus escape from the endosome. We therefore measured the internalization of [35S]methionine virus into drug-treated cells using a cell surface trypsinization assay, which measures virus uptake by the emergence of a trypsin-resistant form of the major capsid protein hexon (Greber et al., 1993). The results indicate that in the presence of nocodazole half of the virus particles were endocytosed at 15 min postwarming, whereas in control cells the half-time of internalization was ∼9 min (Fig. 4 A). The efficiency of entry at 30 min postwarming was close to 75% under both conditions. The remaining viruses were most likely shed from the cells into the medium (Greber et al., 1993). The data demonstrated that virus uptake into cells lacking intact MTs was slightly slower, but no less efficient than into control cells, in agreement with earlier observations showing that nocodazole had no effect on the formation of endocytic vesicles (Gruenberg et al., 1989).

Bottom Line: No directed movement was observed in nocodazole-treated cells.Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment.The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, University of Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

Show MeSH
Related in: MedlinePlus