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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 end– directed transport dominates over plus end–directed  transport of wt Ad2: analysis  in TC7/MAP4/MTB–GFP  cells (A–E) and TC7 parental cells (F). (A and B) Images of TR-Ad2 were recorded with intervals of 1.3 s  starting 30 min p.i. (time  stamp displaying minutes: seconds.centiseconds). One  particular virus particle (arrow) was followed in 104  subsequent frames. Frames  39–46 and 93–99 (with two  additional active particles)  are shown in A and B, respectively. (C) The distance  of the traced particle (A and  B, arrows) to the presumptive MTOC was plotted at  each frame, thus highlighting periods of activity and  relative immobility. (D) ES  of the traced particle were  calculated between subsequent frames and plotted as  micrometers per second indicating minus end–directed  movement as negative and  plus end–directed movement as positive speeds. (E)  Population analysis of motile particles (see Materials  and Methods). 958 ES from  19 different virus particles  and six different cells (see  also Table I) were measured, grouped into 0.1-μm/s  intervals, and then plotted as  either minus- (dark bars)  or plus end–directed (grey  bars) events. ES < 0.1 μm/s  were within the experimental error and plotted as 0 (inset, open bar). Inset, cumulative representation of the  minus- (dark bar) and plus  end–directed ES (grey bar,  100% = 958) and distances  (100% = 532.9 μm). For the  distance calculations, only  ES > 0.1 μm/s were included. (F) Population analysis of ES in parental TC7 cells not expressing MAP4–GFP with inset as in E (100% ES = 729, 100% distance = 495.6 μm). The  corresponding movie is available at http://www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
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Figure 5: Fast minus end– directed transport dominates over plus end–directed transport of wt Ad2: analysis in TC7/MAP4/MTB–GFP cells (A–E) and TC7 parental cells (F). (A and B) Images of TR-Ad2 were recorded with intervals of 1.3 s starting 30 min p.i. (time stamp displaying minutes: seconds.centiseconds). One particular virus particle (arrow) was followed in 104 subsequent frames. Frames 39–46 and 93–99 (with two additional active particles) are shown in A and B, respectively. (C) The distance of the traced particle (A and B, arrows) to the presumptive MTOC was plotted at each frame, thus highlighting periods of activity and relative immobility. (D) ES of the traced particle were calculated between subsequent frames and plotted as micrometers per second indicating minus end–directed movement as negative and plus end–directed movement as positive speeds. (E) Population analysis of motile particles (see Materials and Methods). 958 ES from 19 different virus particles and six different cells (see also Table I) were measured, grouped into 0.1-μm/s intervals, and then plotted as either minus- (dark bars) or plus end–directed (grey bars) events. ES < 0.1 μm/s were within the experimental error and plotted as 0 (inset, open bar). Inset, cumulative representation of the minus- (dark bar) and plus end–directed ES (grey bar, 100% = 958) and distances (100% = 532.9 μm). For the distance calculations, only ES > 0.1 μm/s were included. (F) Population analysis of ES in parental TC7 cells not expressing MAP4–GFP with inset as in E (100% ES = 729, 100% distance = 495.6 μm). The corresponding movie is available at http://www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.

Mentions: We conducted extensive particle tracking analysis in TC7 cells (an African green monkey kidney cell line) and in HeLa cells. The TC7 cells have a particularly large and flat cytoplasm and are well suited for studies of cytoplasmic movement of Ad since they efficiently internalize and target virus to the nucleus (data not shown). To determine the approximate position of the MTOC we used in some experiments a TC7 clone, which was stably transfected with a cDNA encoding a GFP hybrid of the MAP4 MT-binding domain (MAP4/MTB–GFP; Bulinski et al., 1997; Nguyen et al., 1997). This cell line was viable in our hands for more than 20 passages (Olson et al., 1995). TR-labeled Ad2 was internalized at 37°C for 30 min and the coverslip was mounted to a temperature-controlled heating chamber placed on an inverted fluorescence microscope. Fluorescence images were recorded in the TR channel at 1.2– 2.6-s frame intervals over several min as described in Materials and Methods. We use the term ES to define directional movement of particles between two subsequent frames, negative ES indicating movement to the MTOC (minus end) and positive ES to the periphery (plus end). At the end of the recordings the MAP4/MTB–GFP fluorescence was captured. A representative experiment is shown in Fig. 5 where we traced three different particles. One peripheral particle (arrow) was followed throughout the experiment and two MTOC-neighboring particles were traced at the end of the experiment in frames 92–98 (Fig. 5 B, arrowheads). The peripheral particle moved ∼30 μm in a quasi-straight direction towards the MTOC. In Fig. 5, C and D we plotted the distances of the particle from the MTOC and the ES for each frame. The ES defined two distinct types of activity periods. The first type of activity occurred between frames 39–44 and 68–79 and contained exclusively MTOC-directed motions. This activity resulted in efficient particle advancement towards the MTOC and typically lasted for several seconds reaching elementary speeds of 0.17–2.63 μm/s. The other type of activity was characterized by rapidly alternating minus- and plus end– directed ES up to 0.5 μm/s at nearly equal extents and frequencies (Fig. 5, frames 1–38, 45–67, and 80–104). Such activities resulted either in a net minus end–directed speed of 2–4 μm/min (frames 44–68), or yielded no net movement (frames 1–38 and 80–104). Note that from frames 80–104, two additional MTOC-near particles still showed net minus- (Fig. 5 B, solid arrowhead) and plus end–directed (Fig. 5 B, open arrowhead) movements, indicating that cells and viruses were still motion competent.


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 end– directed transport dominates over plus end–directed  transport of wt Ad2: analysis  in TC7/MAP4/MTB–GFP  cells (A–E) and TC7 parental cells (F). (A and B) Images of TR-Ad2 were recorded with intervals of 1.3 s  starting 30 min p.i. (time  stamp displaying minutes: seconds.centiseconds). One  particular virus particle (arrow) was followed in 104  subsequent frames. Frames  39–46 and 93–99 (with two  additional active particles)  are shown in A and B, respectively. (C) The distance  of the traced particle (A and  B, arrows) to the presumptive MTOC was plotted at  each frame, thus highlighting periods of activity and  relative immobility. (D) ES  of the traced particle were  calculated between subsequent frames and plotted as  micrometers per second indicating minus end–directed  movement as negative and  plus end–directed movement as positive speeds. (E)  Population analysis of motile particles (see Materials  and Methods). 958 ES from  19 different virus particles  and six different cells (see  also Table I) were measured, grouped into 0.1-μm/s  intervals, and then plotted as  either minus- (dark bars)  or plus end–directed (grey  bars) events. ES < 0.1 μm/s  were within the experimental error and plotted as 0 (inset, open bar). Inset, cumulative representation of the  minus- (dark bar) and plus  end–directed ES (grey bar,  100% = 958) and distances  (100% = 532.9 μm). For the  distance calculations, only  ES > 0.1 μm/s were included. (F) Population analysis of ES in parental TC7 cells not expressing MAP4–GFP with inset as in E (100% ES = 729, 100% distance = 495.6 μm). The  corresponding movie is available at http://www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
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Figure 5: Fast minus end– directed transport dominates over plus end–directed transport of wt Ad2: analysis in TC7/MAP4/MTB–GFP cells (A–E) and TC7 parental cells (F). (A and B) Images of TR-Ad2 were recorded with intervals of 1.3 s starting 30 min p.i. (time stamp displaying minutes: seconds.centiseconds). One particular virus particle (arrow) was followed in 104 subsequent frames. Frames 39–46 and 93–99 (with two additional active particles) are shown in A and B, respectively. (C) The distance of the traced particle (A and B, arrows) to the presumptive MTOC was plotted at each frame, thus highlighting periods of activity and relative immobility. (D) ES of the traced particle were calculated between subsequent frames and plotted as micrometers per second indicating minus end–directed movement as negative and plus end–directed movement as positive speeds. (E) Population analysis of motile particles (see Materials and Methods). 958 ES from 19 different virus particles and six different cells (see also Table I) were measured, grouped into 0.1-μm/s intervals, and then plotted as either minus- (dark bars) or plus end–directed (grey bars) events. ES < 0.1 μm/s were within the experimental error and plotted as 0 (inset, open bar). Inset, cumulative representation of the minus- (dark bar) and plus end–directed ES (grey bar, 100% = 958) and distances (100% = 532.9 μm). For the distance calculations, only ES > 0.1 μm/s were included. (F) Population analysis of ES in parental TC7 cells not expressing MAP4–GFP with inset as in E (100% ES = 729, 100% distance = 495.6 μm). The corresponding movie is available at http://www.unizh.ch/∼cellbio/jcb1999-1.html Bar, 10 μm.
Mentions: We conducted extensive particle tracking analysis in TC7 cells (an African green monkey kidney cell line) and in HeLa cells. The TC7 cells have a particularly large and flat cytoplasm and are well suited for studies of cytoplasmic movement of Ad since they efficiently internalize and target virus to the nucleus (data not shown). To determine the approximate position of the MTOC we used in some experiments a TC7 clone, which was stably transfected with a cDNA encoding a GFP hybrid of the MAP4 MT-binding domain (MAP4/MTB–GFP; Bulinski et al., 1997; Nguyen et al., 1997). This cell line was viable in our hands for more than 20 passages (Olson et al., 1995). TR-labeled Ad2 was internalized at 37°C for 30 min and the coverslip was mounted to a temperature-controlled heating chamber placed on an inverted fluorescence microscope. Fluorescence images were recorded in the TR channel at 1.2– 2.6-s frame intervals over several min as described in Materials and Methods. We use the term ES to define directional movement of particles between two subsequent frames, negative ES indicating movement to the MTOC (minus end) and positive ES to the periphery (plus end). At the end of the recordings the MAP4/MTB–GFP fluorescence was captured. A representative experiment is shown in Fig. 5 where we traced three different particles. One peripheral particle (arrow) was followed throughout the experiment and two MTOC-neighboring particles were traced at the end of the experiment in frames 92–98 (Fig. 5 B, arrowheads). The peripheral particle moved ∼30 μm in a quasi-straight direction towards the MTOC. In Fig. 5, C and D we plotted the distances of the particle from the MTOC and the ES for each frame. The ES defined two distinct types of activity periods. The first type of activity occurred between frames 39–44 and 68–79 and contained exclusively MTOC-directed motions. This activity resulted in efficient particle advancement towards the MTOC and typically lasted for several seconds reaching elementary speeds of 0.17–2.63 μm/s. The other type of activity was characterized by rapidly alternating minus- and plus end– directed ES up to 0.5 μm/s at nearly equal extents and frequencies (Fig. 5, frames 1–38, 45–67, and 80–104). Such activities resulted either in a net minus end–directed speed of 2–4 μm/min (frames 44–68), or yielded no net movement (frames 1–38 and 80–104). Note that from frames 80–104, two additional MTOC-near particles still showed net minus- (Fig. 5 B, solid arrowhead) and plus end–directed (Fig. 5 B, open arrowhead) movements, indicating that cells and viruses were still motion competent.

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