Limits...
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.

Show MeSH

Related in: MedlinePlus

Microtubule-dependent transport of Ad2 to the  MTOC/nuclear envelope. (A) TR-labeled Ad2 was bound to  HeLa cells in the cold and internalized for 15 (panel a) or 60 min  (panel b). Cells were labeled for γ-tubulin and analyzed by confocal laser scanning microscopy (shown are the sections that contained the perinuclear punctate γ-tubulin signal only). Virus  particles (red) and γ-tubulin (green) are pseudocolored. (B) TR-labeled Ad2 was bound to HeLa cells in the cold in the absence  (panels a and b) or presence of either 20 μM nocodazole (panels  c–f) or 25 nM taxol (panels g and h). Drug treatment included a  30-min preincubation with drugs before virus binding. Cells were  warmed to 37°C in the presence or absence of drugs for 75 min  (panels a–d, and h) or treated with nocodazole for the same time  followed by an incubation without drug for 75 min (panels e and  f). Cells were fixed in pFA and stained for lamins A, -B, and -C  using anti-rabbit FITC and analyzed by confocal microscopy for  TR and FITC fluorescence. Complete stacks of optical sections  are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132937&req=5

Figure 2: Microtubule-dependent transport of Ad2 to the MTOC/nuclear envelope. (A) TR-labeled Ad2 was bound to HeLa cells in the cold and internalized for 15 (panel a) or 60 min (panel b). Cells were labeled for γ-tubulin and analyzed by confocal laser scanning microscopy (shown are the sections that contained the perinuclear punctate γ-tubulin signal only). Virus particles (red) and γ-tubulin (green) are pseudocolored. (B) TR-labeled Ad2 was bound to HeLa cells in the cold in the absence (panels a and b) or presence of either 20 μM nocodazole (panels c–f) or 25 nM taxol (panels g and h). Drug treatment included a 30-min preincubation with drugs before virus binding. Cells were warmed to 37°C in the presence or absence of drugs for 75 min (panels a–d, and h) or treated with nocodazole for the same time followed by an incubation without drug for 75 min (panels e and f). Cells were fixed in pFA and stained for lamins A, -B, and -C using anti-rabbit FITC and analyzed by confocal microscopy for TR and FITC fluorescence. Complete stacks of optical sections are shown.

Mentions: Previous EM studies have detected Ads in close association with MTs both in vivo and in vitro (data not shown) (Dales and Chardonnet, 1973; Luftig and Weihing, 1975; Weatherbee et al., 1977; Miles et al., 1980). To ask whether MTs are actually needed for directional transport of virus to the nucleus, we first tested if virus could be detected at the MTOC in cultured epitheloid HeLa cells. In these cells the MTOC predominantly contains the minus ends of MTs and is located in the perinuclear area as confirmed with an anti–γ-tubulin immunostaining and confocal microscopy (Fig. 2 A). As expected (i.e., Moudjou et al., 1996), γ-tubulin was found all over the cytoplasm but was concentrated at a perinuclear location (Fig. 2 A, green dot). Of a total of 101 examined cells, 75% had a perinuclear enrichment of incoming virus at 60 min postinfection (p.i.). At 15 min postinternalization no enrichment was observed.


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)

Microtubule-dependent transport of Ad2 to the  MTOC/nuclear envelope. (A) TR-labeled Ad2 was bound to  HeLa cells in the cold and internalized for 15 (panel a) or 60 min  (panel b). Cells were labeled for γ-tubulin and analyzed by confocal laser scanning microscopy (shown are the sections that contained the perinuclear punctate γ-tubulin signal only). Virus  particles (red) and γ-tubulin (green) are pseudocolored. (B) TR-labeled Ad2 was bound to HeLa cells in the cold in the absence  (panels a and b) or presence of either 20 μM nocodazole (panels  c–f) or 25 nM taxol (panels g and h). Drug treatment included a  30-min preincubation with drugs before virus binding. Cells were  warmed to 37°C in the presence or absence of drugs for 75 min  (panels a–d, and h) or treated with nocodazole for the same time  followed by an incubation without drug for 75 min (panels e and  f). Cells were fixed in pFA and stained for lamins A, -B, and -C  using anti-rabbit FITC and analyzed by confocal microscopy for  TR and FITC fluorescence. Complete stacks of optical sections  are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Microtubule-dependent transport of Ad2 to the MTOC/nuclear envelope. (A) TR-labeled Ad2 was bound to HeLa cells in the cold and internalized for 15 (panel a) or 60 min (panel b). Cells were labeled for γ-tubulin and analyzed by confocal laser scanning microscopy (shown are the sections that contained the perinuclear punctate γ-tubulin signal only). Virus particles (red) and γ-tubulin (green) are pseudocolored. (B) TR-labeled Ad2 was bound to HeLa cells in the cold in the absence (panels a and b) or presence of either 20 μM nocodazole (panels c–f) or 25 nM taxol (panels g and h). Drug treatment included a 30-min preincubation with drugs before virus binding. Cells were warmed to 37°C in the presence or absence of drugs for 75 min (panels a–d, and h) or treated with nocodazole for the same time followed by an incubation without drug for 75 min (panels e and f). Cells were fixed in pFA and stained for lamins A, -B, and -C using anti-rabbit FITC and analyzed by confocal microscopy for TR and FITC fluorescence. Complete stacks of optical sections are shown.
Mentions: Previous EM studies have detected Ads in close association with MTs both in vivo and in vitro (data not shown) (Dales and Chardonnet, 1973; Luftig and Weihing, 1975; Weatherbee et al., 1977; Miles et al., 1980). To ask whether MTs are actually needed for directional transport of virus to the nucleus, we first tested if virus could be detected at the MTOC in cultured epitheloid HeLa cells. In these cells the MTOC predominantly contains the minus ends of MTs and is located in the perinuclear area as confirmed with an anti–γ-tubulin immunostaining and confocal microscopy (Fig. 2 A). As expected (i.e., Moudjou et al., 1996), γ-tubulin was found all over the cytoplasm but was concentrated at a perinuclear location (Fig. 2 A, green dot). Of a total of 101 examined cells, 75% had a perinuclear enrichment of incoming virus at 60 min postinfection (p.i.). At 15 min postinternalization no enrichment was observed.

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