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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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Fast minus- and plus end–directed transport of endosomal ts1 virus. TR-labeled ts1 virus was added to TC7/MAP4/ MTB–GFP cells and TR images were recorded with intervals of  1.5 s starting 20 min p.i. The GFP signal at the beginning of the  experiment is indicated in A and TR virus of frame 39 in panel B  (time stamps indicating minutes:seconds.centiseconds). The first  TR-frame was recorded 31.22 s after the initial GFP frame. One  particular virus particle (white arrow in B) was followed in 72  subsequent frames as indicated by the black trace in A (white arrowhead indicating the particle position in frame 1 and black arrowhead indicating the position in frame 72). The particle distance to the presumptive MTOC is plotted in panel C and ES  (μm/s) are indicated in D. (E) Population analysis of motile ts1  particles as described in Fig. 5 E (100% ES = 1,410, 100% distance = 754.2 μm). The corresponding movie is available at http: //www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
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Figure 6: Fast minus- and plus end–directed transport of endosomal ts1 virus. TR-labeled ts1 virus was added to TC7/MAP4/ MTB–GFP cells and TR images were recorded with intervals of 1.5 s starting 20 min p.i. The GFP signal at the beginning of the experiment is indicated in A and TR virus of frame 39 in panel B (time stamps indicating minutes:seconds.centiseconds). The first TR-frame was recorded 31.22 s after the initial GFP frame. One particular virus particle (white arrow in B) was followed in 72 subsequent frames as indicated by the black trace in A (white arrowhead indicating the particle position in frame 1 and black arrowhead indicating the position in frame 72). The particle distance to the presumptive MTOC is plotted in panel C and ES (μm/s) are indicated in D. (E) Population analysis of motile ts1 particles as described in Fig. 5 E (100% ES = 1,410, 100% distance = 754.2 μm). The corresponding movie is available at http: //www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.

Mentions: To confirm that the above measured speeds were truly derived from cytosolic viruses, we analyzed the trafficking of an endosomal mutant Ad2, ts1, in TC7/MAP4/MTB– GFP cells and in parental TC7 cells. Unlike wt virus, ts1 had a population mean speed of −0.02 μm/s in TC7/ MAP4/MTB–GFP cells and +0.04 μm/s in TC7 cells indicating slow minus- and plus end–directed net movements (Table I). Accordingly, we have not observed ts1 virus accumulation near the MTOC up to 60 min p.i. Like wt virus, ts1 showed a tailed distribution of ES, with the majority in the range of 0.1–0.2 μm/s (Fig. 6 E). In both cell types, about 22.5% of the ES were less than 0.1 μm/s. A typical ts1 trace in a MAP4 overexpressing cell shows how a virus particle moved from a location proximal to the MTOC towards the periphery with ES of up to 1.7 μm/s and from there returned to an MTOC-near location with ES of up to 2.3 μm/s (Fig. 6). Taken together, the data illustrate a distinct dynamic behavior of wt and endosomal viruses further supporting the notion that wt Ad2 travels as a naked particle along MTs.


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)

Fast minus- and plus end–directed transport of endosomal ts1 virus. TR-labeled ts1 virus was added to TC7/MAP4/ MTB–GFP cells and TR images were recorded with intervals of  1.5 s starting 20 min p.i. The GFP signal at the beginning of the  experiment is indicated in A and TR virus of frame 39 in panel B  (time stamps indicating minutes:seconds.centiseconds). The first  TR-frame was recorded 31.22 s after the initial GFP frame. One  particular virus particle (white arrow in B) was followed in 72  subsequent frames as indicated by the black trace in A (white arrowhead indicating the particle position in frame 1 and black arrowhead indicating the position in frame 72). The particle distance to the presumptive MTOC is plotted in panel C and ES  (μm/s) are indicated in D. (E) Population analysis of motile ts1  particles as described in Fig. 5 E (100% ES = 1,410, 100% distance = 754.2 μm). The corresponding movie is available at http: //www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Fast minus- and plus end–directed transport of endosomal ts1 virus. TR-labeled ts1 virus was added to TC7/MAP4/ MTB–GFP cells and TR images were recorded with intervals of 1.5 s starting 20 min p.i. The GFP signal at the beginning of the experiment is indicated in A and TR virus of frame 39 in panel B (time stamps indicating minutes:seconds.centiseconds). The first TR-frame was recorded 31.22 s after the initial GFP frame. One particular virus particle (white arrow in B) was followed in 72 subsequent frames as indicated by the black trace in A (white arrowhead indicating the particle position in frame 1 and black arrowhead indicating the position in frame 72). The particle distance to the presumptive MTOC is plotted in panel C and ES (μm/s) are indicated in D. (E) Population analysis of motile ts1 particles as described in Fig. 5 E (100% ES = 1,410, 100% distance = 754.2 μm). The corresponding movie is available at http: //www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
Mentions: To confirm that the above measured speeds were truly derived from cytosolic viruses, we analyzed the trafficking of an endosomal mutant Ad2, ts1, in TC7/MAP4/MTB– GFP cells and in parental TC7 cells. Unlike wt virus, ts1 had a population mean speed of −0.02 μm/s in TC7/ MAP4/MTB–GFP cells and +0.04 μm/s in TC7 cells indicating slow minus- and plus end–directed net movements (Table I). Accordingly, we have not observed ts1 virus accumulation near the MTOC up to 60 min p.i. Like wt virus, ts1 showed a tailed distribution of ES, with the majority in the range of 0.1–0.2 μm/s (Fig. 6 E). In both cell types, about 22.5% of the ES were less than 0.1 μm/s. A typical ts1 trace in a MAP4 overexpressing cell shows how a virus particle moved from a location proximal to the MTOC towards the periphery with ES of up to 1.7 μm/s and from there returned to an MTOC-near location with ES of up to 2.3 μm/s (Fig. 6). Taken together, the data illustrate a distinct dynamic behavior of wt and endosomal viruses further supporting the notion that wt Ad2 travels as a naked particle along MTs.

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