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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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Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but  not release of a fluid-phase  endosomal marker into the  cytosol. (A) TR-labeled wt  virus was bound in the cold to  HeLa cells transiently transfected with a plasmid DNA  expressing eGFP (panels a–c,  arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing  plasmid (panels d–f). In the  latter case, 95% of the GFP  expressing cells also overexpressed dynamitin. Virus was  internalized for 60 min at  37°C and cells were processed for indirect immunofluorescence microscopy using  anti-dynamitin mAb 50.1 and  goat anti–mouse IgG-Cy5.  (B) wt Ad2 (panels a–c) or  ts1 (panels d–f) was bound at  50 μg/ml to the surface of  HeLa cells, which had been  transiently transfected with  dynamitin/p50 DNA. Virus  was internalized for 15 min in  the presence of FITC-labeled  10 K dextran (5 mg/ml), followed by a 15-min incubation  in the absence of dextran,  fixed with pFA, and then  stained for dynamitin using  mAb 50.1 and anti-mouse  IgG-Cy5 (panels b and e).  The distribution of dextran  was recorded in the FITC  channel (panels a and d,  transfected cells are indicated  by an arrow) and DIC images  of the same cells shown in  panels c and f.
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Figure 8: Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.

Mentions: The major minus end–directed motor complex of interphase cells is cytoplasmic dynein (Vallee and Sheetz, 1996; Hirokawa, 1998). The motor activity can be experimentally dislodged from cargo by overexpression of p50/dynamitin, a component of the dynein-associated dynactin complex (Echeverri et al., 1996; Burkhardt et al., 1997; Ahmad et al., 1998). We therefore tested if transient overexpression of human dynamitin in HeLa cells affected nuclear targeting of Ad2. Control cells transfected with eGFP-encoding plasmid DNA showed efficient nuclear targeting of TR-labeled Ad2 at 60 min p.i. (Fig. 8 A, panels a–c). HeLa cells cotransfected with GFP and dynamitin plasmids showed reduced nuclear targeting of Ad2, particularly at high levels of overexpression (Fig. 7 A, panels d–f). At moderate dynamitin overexpressions, variable levels of nuclear targeting occurred, perhaps due to incomplete inactivation of the dynactin complex.


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)

Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but  not release of a fluid-phase  endosomal marker into the  cytosol. (A) TR-labeled wt  virus was bound in the cold to  HeLa cells transiently transfected with a plasmid DNA  expressing eGFP (panels a–c,  arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing  plasmid (panels d–f). In the  latter case, 95% of the GFP  expressing cells also overexpressed dynamitin. Virus was  internalized for 60 min at  37°C and cells were processed for indirect immunofluorescence microscopy using  anti-dynamitin mAb 50.1 and  goat anti–mouse IgG-Cy5.  (B) wt Ad2 (panels a–c) or  ts1 (panels d–f) was bound at  50 μg/ml to the surface of  HeLa cells, which had been  transiently transfected with  dynamitin/p50 DNA. Virus  was internalized for 15 min in  the presence of FITC-labeled  10 K dextran (5 mg/ml), followed by a 15-min incubation  in the absence of dextran,  fixed with pFA, and then  stained for dynamitin using  mAb 50.1 and anti-mouse  IgG-Cy5 (panels b and e).  The distribution of dextran  was recorded in the FITC  channel (panels a and d,  transfected cells are indicated  by an arrow) and DIC images  of the same cells shown in  panels c and f.
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Figure 8: Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.
Mentions: The major minus end–directed motor complex of interphase cells is cytoplasmic dynein (Vallee and Sheetz, 1996; Hirokawa, 1998). The motor activity can be experimentally dislodged from cargo by overexpression of p50/dynamitin, a component of the dynein-associated dynactin complex (Echeverri et al., 1996; Burkhardt et al., 1997; Ahmad et al., 1998). We therefore tested if transient overexpression of human dynamitin in HeLa cells affected nuclear targeting of Ad2. Control cells transfected with eGFP-encoding plasmid DNA showed efficient nuclear targeting of TR-labeled Ad2 at 60 min p.i. (Fig. 8 A, panels a–c). HeLa cells cotransfected with GFP and dynamitin plasmids showed reduced nuclear targeting of Ad2, particularly at high levels of overexpression (Fig. 7 A, panels d–f). At moderate dynamitin overexpressions, variable levels of nuclear targeting occurred, perhaps due to incomplete inactivation of the dynactin complex.

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