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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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Rapid switching between minus- and plus end–directed  motions of Ad2 wt in control nondrug-treated cells, taxol-treated  TC7/MAP4/MTB–GFP cells, and also in cells reestablishing their  MTs after nocodazole wash out. Ad2-TR was internalized for 20– 40 min into TC7/MAP4-GFP cells not treated (A) or treated (B)  with taxol (100 nM). Alternatively, cells were pretreated with nocodazole (20 μM) for 15 min in growth medium, incubated with  Ad2-TR in cold binding medium in the presence of nocodazole  for 60 min, followed by virus internalization for 30 min in the  presence of nocodazole and 1–2 min after removal of the drug  (C). Time-lapse micrographs were recorded as described in Materials and Methods and directional ES plotted against time.  Representative traces of one particle for each condition are  shown. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-1.html
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Figure 7: Rapid switching between minus- and plus end–directed motions of Ad2 wt in control nondrug-treated cells, taxol-treated TC7/MAP4/MTB–GFP cells, and also in cells reestablishing their MTs after nocodazole wash out. Ad2-TR was internalized for 20– 40 min into TC7/MAP4-GFP cells not treated (A) or treated (B) with taxol (100 nM). Alternatively, cells were pretreated with nocodazole (20 μM) for 15 min in growth medium, incubated with Ad2-TR in cold binding medium in the presence of nocodazole for 60 min, followed by virus internalization for 30 min in the presence of nocodazole and 1–2 min after removal of the drug (C). Time-lapse micrographs were recorded as described in Materials and Methods and directional ES plotted against time. Representative traces of one particle for each condition are shown. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-1.html

Mentions: Dynamic turnover of MTs can be experimentally eliminated by incubating cells in low concentrations of taxol or nocodazole (Liao et al., 1995; Waterman-Storer and Salmon, 1997). To first test if dynamic MTs were needed for nuclear targeting of Ad2 we pretreated HeLa cells with 25 nM taxol for 30 min. Such treatment was sufficient to stabilize MTs against cold-induced depolymerization (Fig. 1, e and h). Ad2-TR was bound to the surface and then internalized in the presence of the drug for 75 min. Similar to control cells, the majority of virus particles were localized to the nuclear envelope (Fig. 2, g and h). Viral DNA was readily detected inside the nucleus at 180 min p.i. using the FISH assay described above (Fig. 3 b). Likewise, treatment of TC7/MAP4/MTB–GFP cells with low concentrations of nocodazole (1–10 μM for 15 min) in the absence of cold incubations neither affected the MT network nor virus targeting to the nucleus (data not shown). We next analyzed Ad2 motility in MAP4/MTB–GFP-expressing TC7 cells in the presence of taxol or low nocodazole. As expected from the static entry analysis (Fig. 2 B), the population mean speeds towards the MTOC and the periphery were not significantly different from the control cells (Table I). In cells treated with low concentrations of nocodazole, we also observed peak minus- and plus end– directed speeds of up to 1.7 μm/s (data not shown). Although the frequencies of either minus- or plus end– directed motions were significantly decreased in the presence of taxol and the no movement frequencies were increased from 12.8 to 40.8%, rapid switching between plus- and minus end–directed ES was readily observed, similar to control cells (Fig. 7, A and B). Within 2.6 s, single virus particles were frequently switching from +0.4 μm/s to −0.4 μm/s and vice versa, suggesting that stable MTs supported rapid bidirectional transport.


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)

Rapid switching between minus- and plus end–directed  motions of Ad2 wt in control nondrug-treated cells, taxol-treated  TC7/MAP4/MTB–GFP cells, and also in cells reestablishing their  MTs after nocodazole wash out. Ad2-TR was internalized for 20– 40 min into TC7/MAP4-GFP cells not treated (A) or treated (B)  with taxol (100 nM). Alternatively, cells were pretreated with nocodazole (20 μM) for 15 min in growth medium, incubated with  Ad2-TR in cold binding medium in the presence of nocodazole  for 60 min, followed by virus internalization for 30 min in the  presence of nocodazole and 1–2 min after removal of the drug  (C). Time-lapse micrographs were recorded as described in Materials and Methods and directional ES plotted against time.  Representative traces of one particle for each condition are  shown. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-1.html
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Related In: Results  -  Collection

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Figure 7: Rapid switching between minus- and plus end–directed motions of Ad2 wt in control nondrug-treated cells, taxol-treated TC7/MAP4/MTB–GFP cells, and also in cells reestablishing their MTs after nocodazole wash out. Ad2-TR was internalized for 20– 40 min into TC7/MAP4-GFP cells not treated (A) or treated (B) with taxol (100 nM). Alternatively, cells were pretreated with nocodazole (20 μM) for 15 min in growth medium, incubated with Ad2-TR in cold binding medium in the presence of nocodazole for 60 min, followed by virus internalization for 30 min in the presence of nocodazole and 1–2 min after removal of the drug (C). Time-lapse micrographs were recorded as described in Materials and Methods and directional ES plotted against time. Representative traces of one particle for each condition are shown. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-1.html
Mentions: Dynamic turnover of MTs can be experimentally eliminated by incubating cells in low concentrations of taxol or nocodazole (Liao et al., 1995; Waterman-Storer and Salmon, 1997). To first test if dynamic MTs were needed for nuclear targeting of Ad2 we pretreated HeLa cells with 25 nM taxol for 30 min. Such treatment was sufficient to stabilize MTs against cold-induced depolymerization (Fig. 1, e and h). Ad2-TR was bound to the surface and then internalized in the presence of the drug for 75 min. Similar to control cells, the majority of virus particles were localized to the nuclear envelope (Fig. 2, g and h). Viral DNA was readily detected inside the nucleus at 180 min p.i. using the FISH assay described above (Fig. 3 b). Likewise, treatment of TC7/MAP4/MTB–GFP cells with low concentrations of nocodazole (1–10 μM for 15 min) in the absence of cold incubations neither affected the MT network nor virus targeting to the nucleus (data not shown). We next analyzed Ad2 motility in MAP4/MTB–GFP-expressing TC7 cells in the presence of taxol or low nocodazole. As expected from the static entry analysis (Fig. 2 B), the population mean speeds towards the MTOC and the periphery were not significantly different from the control cells (Table I). In cells treated with low concentrations of nocodazole, we also observed peak minus- and plus end– directed speeds of up to 1.7 μm/s (data not shown). Although the frequencies of either minus- or plus end– directed motions were significantly decreased in the presence of taxol and the no movement frequencies were increased from 12.8 to 40.8%, rapid switching between plus- and minus end–directed ES was readily observed, similar to control cells (Fig. 7, A and B). Within 2.6 s, single virus particles were frequently switching from +0.4 μm/s to −0.4 μm/s and vice versa, suggesting that stable MTs supported rapid bidirectional transport.

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