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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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Dynamic analysis  of wt Ad2 trafficking in HeLa  cells overexpressing dynamitin and comparison to control, nocodazole-treated HeLa  cells, and virus at the cell surface. Virus motilities in control HeLa cells (panels Aa  and Ba, 40 min p.i.), HeLa  cells overexpressing dynamitin (panels Ab and Bb, 40  min p.i.), cells treated with  nocodazole (panels Ac and  Bc, 40 min p.i.), and on the  cell surface (panel Ad and  Bd, 5 min p.i.) were measured as described in Fig. 5  A. Traces of a representative  particle are shown for each  condition in panel A merged  onto a DIC image taken at  the beginning of the TR recordings (shown in a1, b1, c1,  and d1). The particle traces  are derived from (a) 43  frames at 2.6-s intervals, (b)  37 frames at 2.1-s intervals,  (c) 246 frames at 1.5-s intervals, and (d) 97 frames at 2.6-s  intervals. Note that in the  early stages of infection (panels d1 and d2), highly motile  particles were occasionally  seen moving towards the nucleus with micrometer per  second speeds (see movie  available at http://www.unizh.  ch/∼cellbio/jcb1999-1.html).  These fast-moving particles  may be contained in an endocytic vesicle or had just  reached the cytosol. B shows  cumulative distributions of  ES for each of the conditions  as described in Fig. 5. In  panel Ba, 100% ES = 1,677,  100% distance = 721.4 μm;  panel Bb 100% ES = 810,  100% distance = 244.9 μm;  panel Bc, 100% ES = 608,  100% distance = 40.6 μm;  panel Bd, 100% ES = 448,  100% distance = 11.5 μm.  Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-  1.html Bar, 5 μm.
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Figure 9: Dynamic analysis of wt Ad2 trafficking in HeLa cells overexpressing dynamitin and comparison to control, nocodazole-treated HeLa cells, and virus at the cell surface. Virus motilities in control HeLa cells (panels Aa and Ba, 40 min p.i.), HeLa cells overexpressing dynamitin (panels Ab and Bb, 40 min p.i.), cells treated with nocodazole (panels Ac and Bc, 40 min p.i.), and on the cell surface (panel Ad and Bd, 5 min p.i.) were measured as described in Fig. 5 A. Traces of a representative particle are shown for each condition in panel A merged onto a DIC image taken at the beginning of the TR recordings (shown in a1, b1, c1, and d1). The particle traces are derived from (a) 43 frames at 2.6-s intervals, (b) 37 frames at 2.1-s intervals, (c) 246 frames at 1.5-s intervals, and (d) 97 frames at 2.6-s intervals. Note that in the early stages of infection (panels d1 and d2), highly motile particles were occasionally seen moving towards the nucleus with micrometer per second speeds (see movie available at http://www.unizh. ch/∼cellbio/jcb1999-1.html). These fast-moving particles may be contained in an endocytic vesicle or had just reached the cytosol. B shows cumulative distributions of ES for each of the conditions as described in Fig. 5. In panel Ba, 100% ES = 1,677, 100% distance = 721.4 μm; panel Bb 100% ES = 810, 100% distance = 244.9 μm; panel Bc, 100% ES = 608, 100% distance = 40.6 μm; panel Bd, 100% ES = 448, 100% distance = 11.5 μm. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999- 1.html Bar, 5 μm.

Mentions: To quantitate the effects of dynamitin overexpression on Ad2 trafficking, we compared the extents and frequencies of the minus and plus end–directed ES in dynamitin overexpressing HeLa cells with control and nocodazole-treated cells. Virus motility at the cell surface was, in addition, determined to account for cytosol-independent particle motion. As shown by an ES population analysis (Fig. 9 B) and exemplified by representative particle traces (Fig. 9 A), dynamitin overexpression significantly reduced the extents and also the frequencies of the minus end–directed ES. The mean minus end–directed speed of 0.22 μm/s was significantly smaller than the plus end–directed mean speed of 0.30 μm/s (Tables I and II), resulting in a plus end–directed population speed of 0.06 μm/s compared with a minus end–directed speed of 0.03 μm/s in control cells (Fig. 9 B, panels a and b) (Table I). As pointed out earlier, not all minus end–directed transport of Ad2 was blocked by dynamitin overexpression. This was illustrated also by the maximal minus end–directed ES (∼2 μm/s) and a mean minus end speed, which was significantly higher than in nocodazole-treated cells (Table II). This result may either reflect the heterogenous expression levels of dynamitin or, alternatively, indicate some form of dynein/dynactin-independent minus end transport of Ad2. In contrast to the effect of dynamitin on the minus end migration of Ad2, dynamitin overexpression did not significantly affect the mean plus end–directed velocity, as illustrated by the predominantly peripheral virus localization in these cells (Fig. 8 A and Fig. 9 A, panel b2) (Tables I and II). However, dynamitin overexpression significantly increased the frequency of plus end motions to 31.5 from 25.5% in control cells or 25.8% in GFP-expressing cells (legend of Table I, chi square test at 95% confidence). Interestingly, dynamitin overexpression also significantly increased the fraction of immobile particles to 50.9 from 41.4% in control cells. The validity of our chi square analysis and of the sample volumes were confirmed by the fact that there were no significant differencies between the plus, the minus, and the no movement frequencies in control HeLa cells compared with GFP-expressing HeLa cells. That the expression of GFP had no significant effects on Ad2 trafficking was further supported by the observation that the mean minus- and plus end–directed speeds were identical in GFP-expressing cells and control cells (Tables I and II, one-sided t test, 95% confidence).


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)

Dynamic analysis  of wt Ad2 trafficking in HeLa  cells overexpressing dynamitin and comparison to control, nocodazole-treated HeLa  cells, and virus at the cell surface. Virus motilities in control HeLa cells (panels Aa  and Ba, 40 min p.i.), HeLa  cells overexpressing dynamitin (panels Ab and Bb, 40  min p.i.), cells treated with  nocodazole (panels Ac and  Bc, 40 min p.i.), and on the  cell surface (panel Ad and  Bd, 5 min p.i.) were measured as described in Fig. 5  A. Traces of a representative  particle are shown for each  condition in panel A merged  onto a DIC image taken at  the beginning of the TR recordings (shown in a1, b1, c1,  and d1). The particle traces  are derived from (a) 43  frames at 2.6-s intervals, (b)  37 frames at 2.1-s intervals,  (c) 246 frames at 1.5-s intervals, and (d) 97 frames at 2.6-s  intervals. Note that in the  early stages of infection (panels d1 and d2), highly motile  particles were occasionally  seen moving towards the nucleus with micrometer per  second speeds (see movie  available at http://www.unizh.  ch/∼cellbio/jcb1999-1.html).  These fast-moving particles  may be contained in an endocytic vesicle or had just  reached the cytosol. B shows  cumulative distributions of  ES for each of the conditions  as described in Fig. 5. In  panel Ba, 100% ES = 1,677,  100% distance = 721.4 μm;  panel Bb 100% ES = 810,  100% distance = 244.9 μm;  panel Bc, 100% ES = 608,  100% distance = 40.6 μm;  panel Bd, 100% ES = 448,  100% distance = 11.5 μm.  Movies are available at http://www.unizh.ch/∼cellbio/jcb1999-  1.html Bar, 5 μm.
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Figure 9: Dynamic analysis of wt Ad2 trafficking in HeLa cells overexpressing dynamitin and comparison to control, nocodazole-treated HeLa cells, and virus at the cell surface. Virus motilities in control HeLa cells (panels Aa and Ba, 40 min p.i.), HeLa cells overexpressing dynamitin (panels Ab and Bb, 40 min p.i.), cells treated with nocodazole (panels Ac and Bc, 40 min p.i.), and on the cell surface (panel Ad and Bd, 5 min p.i.) were measured as described in Fig. 5 A. Traces of a representative particle are shown for each condition in panel A merged onto a DIC image taken at the beginning of the TR recordings (shown in a1, b1, c1, and d1). The particle traces are derived from (a) 43 frames at 2.6-s intervals, (b) 37 frames at 2.1-s intervals, (c) 246 frames at 1.5-s intervals, and (d) 97 frames at 2.6-s intervals. Note that in the early stages of infection (panels d1 and d2), highly motile particles were occasionally seen moving towards the nucleus with micrometer per second speeds (see movie available at http://www.unizh. ch/∼cellbio/jcb1999-1.html). These fast-moving particles may be contained in an endocytic vesicle or had just reached the cytosol. B shows cumulative distributions of ES for each of the conditions as described in Fig. 5. In panel Ba, 100% ES = 1,677, 100% distance = 721.4 μm; panel Bb 100% ES = 810, 100% distance = 244.9 μm; panel Bc, 100% ES = 608, 100% distance = 40.6 μm; panel Bd, 100% ES = 448, 100% distance = 11.5 μm. Movies are available at http://www.unizh.ch/∼cellbio/jcb1999- 1.html Bar, 5 μm.
Mentions: To quantitate the effects of dynamitin overexpression on Ad2 trafficking, we compared the extents and frequencies of the minus and plus end–directed ES in dynamitin overexpressing HeLa cells with control and nocodazole-treated cells. Virus motility at the cell surface was, in addition, determined to account for cytosol-independent particle motion. As shown by an ES population analysis (Fig. 9 B) and exemplified by representative particle traces (Fig. 9 A), dynamitin overexpression significantly reduced the extents and also the frequencies of the minus end–directed ES. The mean minus end–directed speed of 0.22 μm/s was significantly smaller than the plus end–directed mean speed of 0.30 μm/s (Tables I and II), resulting in a plus end–directed population speed of 0.06 μm/s compared with a minus end–directed speed of 0.03 μm/s in control cells (Fig. 9 B, panels a and b) (Table I). As pointed out earlier, not all minus end–directed transport of Ad2 was blocked by dynamitin overexpression. This was illustrated also by the maximal minus end–directed ES (∼2 μm/s) and a mean minus end speed, which was significantly higher than in nocodazole-treated cells (Table II). This result may either reflect the heterogenous expression levels of dynamitin or, alternatively, indicate some form of dynein/dynactin-independent minus end transport of Ad2. In contrast to the effect of dynamitin on the minus end migration of Ad2, dynamitin overexpression did not significantly affect the mean plus end–directed velocity, as illustrated by the predominantly peripheral virus localization in these cells (Fig. 8 A and Fig. 9 A, panel b2) (Tables I and II). However, dynamitin overexpression significantly increased the frequency of plus end motions to 31.5 from 25.5% in control cells or 25.8% in GFP-expressing cells (legend of Table I, chi square test at 95% confidence). Interestingly, dynamitin overexpression also significantly increased the fraction of immobile particles to 50.9 from 41.4% in control cells. The validity of our chi square analysis and of the sample volumes were confirmed by the fact that there were no significant differencies between the plus, the minus, and the no movement frequencies in control HeLa cells compared with GFP-expressing HeLa cells. That the expression of GFP had no significant effects on Ad2 trafficking was further supported by the observation that the mean minus- and plus end–directed speeds were identical in GFP-expressing cells and control cells (Tables I and II, one-sided t test, 95% confidence).

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