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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 targeting to  the nucleus requires physiological temperature. TR-labeled Ad2 was cold bound  to HeLa cells in the absence  (a–f) or presence of taxol (25  nM) including a 15-min preincubation with drug in growth  medium at 37°C (g–i). Cells  were warmed for 29 min (d–i)  followed by a cold incubation  up to 31 min in the absence  (d–f) or presence (g–i) of  taxol or were warmed for 60  min in the absence of drug  (a–c). Samples were fixed  with pFA and analyzed by  epifluorescence microscopy.
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Figure 1: Ad2 targeting to the nucleus requires physiological temperature. TR-labeled Ad2 was cold bound to HeLa cells in the absence (a–f) or presence of taxol (25 nM) including a 15-min preincubation with drug in growth medium at 37°C (g–i). Cells were warmed for 29 min (d–i) followed by a cold incubation up to 31 min in the absence (d–f) or presence (g–i) of taxol or were warmed for 60 min in the absence of drug (a–c). Samples were fixed with pFA and analyzed by epifluorescence microscopy.

Mentions: The structural organization of the cytoplasm typically restricts the diffusional mobility of artificial particles of 50 nm in diameter to a few square micrometers per second (Luby-Phelps, 1994; Seksek et al., 1997). Particles larger than 50 nm, such as Ad2, are expected to have essentially no diffusion mobility under physiological conditions. To test if cytosolic virus was localized to the nucleus in the absence of energy metabolism at a reduced temperature, we bound TR-labeled Ad2 to the surface of HeLa cells in the cold and internalized for 29 min at 37°C. During this time virus is taken up into the cytoplasm but is not yet targeted to the nucleus (Greber et al., 1993, 1997). Cells were subsequently shifted to 4°C and incubated in the cold for up to 60 min postinternalization, whereas control cells were kept at 37°C. Since exposure of cells to reduced temperatures causes depolymerization of MTs, targeting was also analyzed in cells that had been pretreated with the MT-stabilizing drug taxol. In control cells the large majority of virus particles was directed to the nucleus during the 60-min internalization, in agreement with previous results (Fig. 1) (Greber et al., 1997). In contrast, at reduced temperatures, with or without taxol treatment, virus particles were found randomly distributed in the cytoplasm at 60 min. The same result was obtained if the cold incubation was extended up to 90 min (data not shown). Virus particles also remained cytoplasmic if cellular ATP levels were reduced after 30 min of internalization by shifting cells into a glucose-free medium containing 10 mM sodium azide and 20 mM 2-deoxy-d-glucose (data not shown). These results confirmed that incoming Ad2 does not move through the cytoplasm by diffusion, but uses some form of mediated 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)

Ad2 targeting to  the nucleus requires physiological temperature. TR-labeled Ad2 was cold bound  to HeLa cells in the absence  (a–f) or presence of taxol (25  nM) including a 15-min preincubation with drug in growth  medium at 37°C (g–i). Cells  were warmed for 29 min (d–i)  followed by a cold incubation  up to 31 min in the absence  (d–f) or presence (g–i) of  taxol or were warmed for 60  min in the absence of drug  (a–c). Samples were fixed  with pFA and analyzed by  epifluorescence microscopy.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132937&req=5

Figure 1: Ad2 targeting to the nucleus requires physiological temperature. TR-labeled Ad2 was cold bound to HeLa cells in the absence (a–f) or presence of taxol (25 nM) including a 15-min preincubation with drug in growth medium at 37°C (g–i). Cells were warmed for 29 min (d–i) followed by a cold incubation up to 31 min in the absence (d–f) or presence (g–i) of taxol or were warmed for 60 min in the absence of drug (a–c). Samples were fixed with pFA and analyzed by epifluorescence microscopy.
Mentions: The structural organization of the cytoplasm typically restricts the diffusional mobility of artificial particles of 50 nm in diameter to a few square micrometers per second (Luby-Phelps, 1994; Seksek et al., 1997). Particles larger than 50 nm, such as Ad2, are expected to have essentially no diffusion mobility under physiological conditions. To test if cytosolic virus was localized to the nucleus in the absence of energy metabolism at a reduced temperature, we bound TR-labeled Ad2 to the surface of HeLa cells in the cold and internalized for 29 min at 37°C. During this time virus is taken up into the cytoplasm but is not yet targeted to the nucleus (Greber et al., 1993, 1997). Cells were subsequently shifted to 4°C and incubated in the cold for up to 60 min postinternalization, whereas control cells were kept at 37°C. Since exposure of cells to reduced temperatures causes depolymerization of MTs, targeting was also analyzed in cells that had been pretreated with the MT-stabilizing drug taxol. In control cells the large majority of virus particles was directed to the nucleus during the 60-min internalization, in agreement with previous results (Fig. 1) (Greber et al., 1997). In contrast, at reduced temperatures, with or without taxol treatment, virus particles were found randomly distributed in the cytoplasm at 60 min. The same result was obtained if the cold incubation was extended up to 90 min (data not shown). Virus particles also remained cytoplasmic if cellular ATP levels were reduced after 30 min of internalization by shifting cells into a glucose-free medium containing 10 mM sodium azide and 20 mM 2-deoxy-d-glucose (data not shown). These results confirmed that incoming Ad2 does not move through the cytoplasm by diffusion, but uses some form of mediated 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