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Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking.

Provance DW, Addison EJ, Wood PR, Chen DZ, Silan CM, Mercer JA - BMC Cell Biol. (2008)

Bottom Line: Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition.Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).All results favored the peripheral dynamic tethering hypothesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: McLaughlin Research Institute, Great Falls, MT, USA. billp@mri.montana.edu

ABSTRACT

Background: Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments.

Results: In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).

Conclusion: All results favored the peripheral dynamic tethering hypothesis.

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Inhibition of myosin-Vb after loading with transferrin does not prevent transit from perinuclear recycling endosomes. HeLa cells transiently expressing sensitized myosin-Vb were loaded with Alexa 546-transferrin, washed, incubated in growth medium for 30 min, and imaged for myosin-Vb and transferrin. (A) Diagram depicting predicted results; the sensitized mutant myosin-Vb is shown in red and PE-ADP is shown as a green circle. (B) Inhibition of accumulation of transferrin (red) added after myosin-Vb inhibition by microinjection of PE-ADP2. Injected cells have blue nuclei. (C, D, E, F, G, H) The cell expressing sensitized myosin-Vb (center, panel C) was immediately injected with PE-ADP and the same field was imaged 30 min later (F, G, H). Panels C and D are overlaid in panel E, and panels F and G are overlaid in panel H. Bar, 15 μm.
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Figure 3: Inhibition of myosin-Vb after loading with transferrin does not prevent transit from perinuclear recycling endosomes. HeLa cells transiently expressing sensitized myosin-Vb were loaded with Alexa 546-transferrin, washed, incubated in growth medium for 30 min, and imaged for myosin-Vb and transferrin. (A) Diagram depicting predicted results; the sensitized mutant myosin-Vb is shown in red and PE-ADP is shown as a green circle. (B) Inhibition of accumulation of transferrin (red) added after myosin-Vb inhibition by microinjection of PE-ADP2. Injected cells have blue nuclei. (C, D, E, F, G, H) The cell expressing sensitized myosin-Vb (center, panel C) was immediately injected with PE-ADP and the same field was imaged 30 min later (F, G, H). Panels C and D are overlaid in panel E, and panels F and G are overlaid in panel H. Bar, 15 μm.

Mentions: In a previous study, we used a chemical-genetic approach to show that induction of tight binding of sensitized myosin-Vb to actin, before addition of transferrin, prevented transferrin from accumulating in perinuclear compartments [20]. Our hypothesis is diagrammed in Fig. 3A, and the effect of inhibition before transferrin uptake, demonstrated previously, is shown in Fig. 3B. If myosin-Vb is required for transport from perinuclear compartments to the plasma membrane, then inducing tight binding of myosin-Vb to actin after transferrin loading should increase transferrin accumulation in perinuclear compartments, just as myosin-Vb tail overexpression does. We therefore transfected HeLa cells with Y119G sensitized mutant (Fig. 3) and wild-type control (not shown) myosin-Vb, loaded them with fluorescent transferrin, and microinjected the specific inhibitor of Y119G myosin-Vb, N6-(2-phenylethyl)-ADP (PE-ADP) [20]. Only cells with a punctate eGFP localization, representing lower expression levels, were chosen for microinjection. When PE-ADP was injected 10 min (data not shown) and 30 min (Fig. 3D,E,F) following the addition of transferrin, we still observed a decrease in fluorescence intensity in the perinuclear region of the transfected and injected cells (Fig. 3D,E,F) as well as rapid movement of transferrin when it did not colocalize with myosin-Vb (Additional file 9). These data, as well as the limited colocalization between transferrin and myosin-Vb, indicate that myosin-Vb activity is not required to transport transferrin from perinuclear compartments to the plasma membrane. These data are much more consistent with the peripheral tethering hypothesis, because the peripheral site of myosin-Vb function has been bypassed by loading with transferrin before induction of tight binding of myosin-Vb to actin.


Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking.

Provance DW, Addison EJ, Wood PR, Chen DZ, Silan CM, Mercer JA - BMC Cell Biol. (2008)

Inhibition of myosin-Vb after loading with transferrin does not prevent transit from perinuclear recycling endosomes. HeLa cells transiently expressing sensitized myosin-Vb were loaded with Alexa 546-transferrin, washed, incubated in growth medium for 30 min, and imaged for myosin-Vb and transferrin. (A) Diagram depicting predicted results; the sensitized mutant myosin-Vb is shown in red and PE-ADP is shown as a green circle. (B) Inhibition of accumulation of transferrin (red) added after myosin-Vb inhibition by microinjection of PE-ADP2. Injected cells have blue nuclei. (C, D, E, F, G, H) The cell expressing sensitized myosin-Vb (center, panel C) was immediately injected with PE-ADP and the same field was imaged 30 min later (F, G, H). Panels C and D are overlaid in panel E, and panels F and G are overlaid in panel H. Bar, 15 μm.
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Related In: Results  -  Collection

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Figure 3: Inhibition of myosin-Vb after loading with transferrin does not prevent transit from perinuclear recycling endosomes. HeLa cells transiently expressing sensitized myosin-Vb were loaded with Alexa 546-transferrin, washed, incubated in growth medium for 30 min, and imaged for myosin-Vb and transferrin. (A) Diagram depicting predicted results; the sensitized mutant myosin-Vb is shown in red and PE-ADP is shown as a green circle. (B) Inhibition of accumulation of transferrin (red) added after myosin-Vb inhibition by microinjection of PE-ADP2. Injected cells have blue nuclei. (C, D, E, F, G, H) The cell expressing sensitized myosin-Vb (center, panel C) was immediately injected with PE-ADP and the same field was imaged 30 min later (F, G, H). Panels C and D are overlaid in panel E, and panels F and G are overlaid in panel H. Bar, 15 μm.
Mentions: In a previous study, we used a chemical-genetic approach to show that induction of tight binding of sensitized myosin-Vb to actin, before addition of transferrin, prevented transferrin from accumulating in perinuclear compartments [20]. Our hypothesis is diagrammed in Fig. 3A, and the effect of inhibition before transferrin uptake, demonstrated previously, is shown in Fig. 3B. If myosin-Vb is required for transport from perinuclear compartments to the plasma membrane, then inducing tight binding of myosin-Vb to actin after transferrin loading should increase transferrin accumulation in perinuclear compartments, just as myosin-Vb tail overexpression does. We therefore transfected HeLa cells with Y119G sensitized mutant (Fig. 3) and wild-type control (not shown) myosin-Vb, loaded them with fluorescent transferrin, and microinjected the specific inhibitor of Y119G myosin-Vb, N6-(2-phenylethyl)-ADP (PE-ADP) [20]. Only cells with a punctate eGFP localization, representing lower expression levels, were chosen for microinjection. When PE-ADP was injected 10 min (data not shown) and 30 min (Fig. 3D,E,F) following the addition of transferrin, we still observed a decrease in fluorescence intensity in the perinuclear region of the transfected and injected cells (Fig. 3D,E,F) as well as rapid movement of transferrin when it did not colocalize with myosin-Vb (Additional file 9). These data, as well as the limited colocalization between transferrin and myosin-Vb, indicate that myosin-Vb activity is not required to transport transferrin from perinuclear compartments to the plasma membrane. These data are much more consistent with the peripheral tethering hypothesis, because the peripheral site of myosin-Vb function has been bypassed by loading with transferrin before induction of tight binding of myosin-Vb to actin.

Bottom Line: Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition.Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).All results favored the peripheral dynamic tethering hypothesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: McLaughlin Research Institute, Great Falls, MT, USA. billp@mri.montana.edu

ABSTRACT

Background: Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments.

Results: In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).

Conclusion: All results favored the peripheral dynamic tethering hypothesis.

Show MeSH