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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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Inhibition of Ad2  DNA import in nocodazole-,  but not taxol-treated HeLa  cells. HeLa cells were pretreated with either nocodazole (20 μM) or taxol (25 nM)  as described in Fig. 2. Wt Ad2  (20 μg/ml) was bound in the  cold in the presence or absence of drugs and internalized in DME-BSA medium in  the absence (a) or presence of  taxol (b) or nocodazole (d)  for 180 min. A parallel sample of nocodazole-treated  cells was washed several  times in drug-free medium  and incubated for an additional 180 min in DME-BSA  (e). Alternatively, nocodazole was added to cells from  90 up to 180 min p.i. (f). All  samples including noninfected cells (c) were then analyzed by in situ hybridization  using TR-labeled genomic  Ad2 DNA followed by anti-lamin immunolabeling of nuclear envelopes and confocal  laser scanning microscopy.
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Figure 3: Inhibition of Ad2 DNA import in nocodazole-, but not taxol-treated HeLa cells. HeLa cells were pretreated with either nocodazole (20 μM) or taxol (25 nM) as described in Fig. 2. Wt Ad2 (20 μg/ml) was bound in the cold in the presence or absence of drugs and internalized in DME-BSA medium in the absence (a) or presence of taxol (b) or nocodazole (d) for 180 min. A parallel sample of nocodazole-treated cells was washed several times in drug-free medium and incubated for an additional 180 min in DME-BSA (e). Alternatively, nocodazole was added to cells from 90 up to 180 min p.i. (f). All samples including noninfected cells (c) were then analyzed by in situ hybridization using TR-labeled genomic Ad2 DNA followed by anti-lamin immunolabeling of nuclear envelopes and confocal laser scanning microscopy.

Mentions: We next analyzed if intact MTs were required for virus targeting to the nucleus. HeLa cells were incubated with 20 μM nocodazole for 30 min and virus was bound to the cell surface for 60 min in the cold to synchronize infection and to further depolymerize MTs. Cells were then shifted to 37°C in drug-containing medium for 75 min. Under these conditions, MTs were completely depolymerized as shown with mAb 1A2 and anti–β-tubulin immunostainings (data not shown). Nocodazole strongly, but not completely inhibited localization of TR-labeled virus to the nuclear envelope as indicated by confocal microscopy (Fig. 2 B, panel c). Similar results were obtained if the internalization time was extended to 135 min (data not shown), thus suggesting that the low level of nuclear targeting was most likely due to particles entering from a plasma membrane area near the nucleus. If MTs were restored by washing out nocodazole for 75 min, significant virus targeting to the nucleus was observed, similar to cells not treated with the drug (Fig. 2 B, panels a and e). These results were confirmed by analyzing viral DNA import into the nucleus in nocodazole-treated cells using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy at 180 min p.i. Control cells (no drug) had a strong signal of viral DNA inside the nucleus as concluded from an anti-lamin immunostaining of the nuclear envelope (Fig. 3 a). Noninfected cells had no viral DNA signal, indicating that the FISH conditions were specifically detecting viral genomes (Fig. 3 c) (see also Greber et al., 1997). In nocodazole-treated cells most of the DNA signal was present in the cytoplasm, sometimes in an aggregated form (Fig. 3 d). Note that the combined cytoplasmic signal was somewhat lower than the nuclear signal in the control (no drug)-treated cells, partly because some cytoplasmic virus had been artificially released during the FISH treatments. The absence of viral DNA in the nucleus was not due to a lack of detection or rapid nuclear export, since viral genomes were readily found in the nucleus of cells that were treated with nocodazole beginning at 90 min p.i., when the majority of virus particles had already arrived at the nuclear envelope (Fig. 3 f). The cytoplasmic DNA signals in the nocodazole-treated cells decreased after washing out the drug and a strong nuclear DNA signal appeared (Fig. 3 e). Taken together, these results suggested that intact MTs are needed for targeting of incoming virus particles to and for import of viral DNA into the nucleus.


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)

Inhibition of Ad2  DNA import in nocodazole-,  but not taxol-treated HeLa  cells. HeLa cells were pretreated with either nocodazole (20 μM) or taxol (25 nM)  as described in Fig. 2. Wt Ad2  (20 μg/ml) was bound in the  cold in the presence or absence of drugs and internalized in DME-BSA medium in  the absence (a) or presence of  taxol (b) or nocodazole (d)  for 180 min. A parallel sample of nocodazole-treated  cells was washed several  times in drug-free medium  and incubated for an additional 180 min in DME-BSA  (e). Alternatively, nocodazole was added to cells from  90 up to 180 min p.i. (f). All  samples including noninfected cells (c) were then analyzed by in situ hybridization  using TR-labeled genomic  Ad2 DNA followed by anti-lamin immunolabeling of nuclear envelopes and confocal  laser scanning microscopy.
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Related In: Results  -  Collection

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Figure 3: Inhibition of Ad2 DNA import in nocodazole-, but not taxol-treated HeLa cells. HeLa cells were pretreated with either nocodazole (20 μM) or taxol (25 nM) as described in Fig. 2. Wt Ad2 (20 μg/ml) was bound in the cold in the presence or absence of drugs and internalized in DME-BSA medium in the absence (a) or presence of taxol (b) or nocodazole (d) for 180 min. A parallel sample of nocodazole-treated cells was washed several times in drug-free medium and incubated for an additional 180 min in DME-BSA (e). Alternatively, nocodazole was added to cells from 90 up to 180 min p.i. (f). All samples including noninfected cells (c) were then analyzed by in situ hybridization using TR-labeled genomic Ad2 DNA followed by anti-lamin immunolabeling of nuclear envelopes and confocal laser scanning microscopy.
Mentions: We next analyzed if intact MTs were required for virus targeting to the nucleus. HeLa cells were incubated with 20 μM nocodazole for 30 min and virus was bound to the cell surface for 60 min in the cold to synchronize infection and to further depolymerize MTs. Cells were then shifted to 37°C in drug-containing medium for 75 min. Under these conditions, MTs were completely depolymerized as shown with mAb 1A2 and anti–β-tubulin immunostainings (data not shown). Nocodazole strongly, but not completely inhibited localization of TR-labeled virus to the nuclear envelope as indicated by confocal microscopy (Fig. 2 B, panel c). Similar results were obtained if the internalization time was extended to 135 min (data not shown), thus suggesting that the low level of nuclear targeting was most likely due to particles entering from a plasma membrane area near the nucleus. If MTs were restored by washing out nocodazole for 75 min, significant virus targeting to the nucleus was observed, similar to cells not treated with the drug (Fig. 2 B, panels a and e). These results were confirmed by analyzing viral DNA import into the nucleus in nocodazole-treated cells using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy at 180 min p.i. Control cells (no drug) had a strong signal of viral DNA inside the nucleus as concluded from an anti-lamin immunostaining of the nuclear envelope (Fig. 3 a). Noninfected cells had no viral DNA signal, indicating that the FISH conditions were specifically detecting viral genomes (Fig. 3 c) (see also Greber et al., 1997). In nocodazole-treated cells most of the DNA signal was present in the cytoplasm, sometimes in an aggregated form (Fig. 3 d). Note that the combined cytoplasmic signal was somewhat lower than the nuclear signal in the control (no drug)-treated cells, partly because some cytoplasmic virus had been artificially released during the FISH treatments. The absence of viral DNA in the nucleus was not due to a lack of detection or rapid nuclear export, since viral genomes were readily found in the nucleus of cells that were treated with nocodazole beginning at 90 min p.i., when the majority of virus particles had already arrived at the nuclear envelope (Fig. 3 f). The cytoplasmic DNA signals in the nocodazole-treated cells decreased after washing out the drug and a strong nuclear DNA signal appeared (Fig. 3 e). Taken together, these results suggested that intact MTs are needed for targeting of incoming virus particles to and for import of viral DNA into the nucleus.

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