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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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Overexpression of full-length, wild-type eGFP-myosin-Vb causes coalescence of peripheral endocytic compartments and inhibits perinuclear accumulation of transferrin. HeLa cells were transiently transfected with full-length, wild-type myosin-Vb tagged with eGFP and imaged 24 h after transfection. (A) Diagram depicting predicted results. (B, C) Colocalization of actin on enlarged compartments (arrows) with eGFP-myosin-Vb. (D, E, F) In cells expressing high levels of eGFP-myosin-Vb coincident with exposure to transferrin (arrows), large, peripheral organelles decorated with myosin-Vb also contain transferrin. (G, H, I, J, K, L) In cells exposed to a 1-min pulse of transferrin 24 h after transfection and 10 min before imaging, transfected cells (arrows) contain large, peripheral organelles decorated with myosin-Vb that lack transferrin. Cells expressing lower levels of myosin-Vb (arrowheads, panel K; too low to be seen in panel J) accumulate less transferrin than the surrounding untransfected cells (arrowheads). Bar, 15 μm.
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Figure 2: Overexpression of full-length, wild-type eGFP-myosin-Vb causes coalescence of peripheral endocytic compartments and inhibits perinuclear accumulation of transferrin. HeLa cells were transiently transfected with full-length, wild-type myosin-Vb tagged with eGFP and imaged 24 h after transfection. (A) Diagram depicting predicted results. (B, C) Colocalization of actin on enlarged compartments (arrows) with eGFP-myosin-Vb. (D, E, F) In cells expressing high levels of eGFP-myosin-Vb coincident with exposure to transferrin (arrows), large, peripheral organelles decorated with myosin-Vb also contain transferrin. (G, H, I, J, K, L) In cells exposed to a 1-min pulse of transferrin 24 h after transfection and 10 min before imaging, transfected cells (arrows) contain large, peripheral organelles decorated with myosin-Vb that lack transferrin. Cells expressing lower levels of myosin-Vb (arrowheads, panel K; too low to be seen in panel J) accumulate less transferrin than the surrounding untransfected cells (arrowheads). Bar, 15 μm.

Mentions: Figure 2 and Additional files 2, 3, 4, 5, 6, 7, 8 show transferrin accumulation in peripheral compartments as a function of the overexpression level of eGFP-myosin-Vb, which the dynamic tethering hypothesis predicts will cause the coalescence and caging of peripheral endosomes by actin (Fig. 2A). A coalescence of actin around the enlarged peripheral endosomes is shown by the colocalization of myosin-Vb and actin (Fig. 2B,C).


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)

Overexpression of full-length, wild-type eGFP-myosin-Vb causes coalescence of peripheral endocytic compartments and inhibits perinuclear accumulation of transferrin. HeLa cells were transiently transfected with full-length, wild-type myosin-Vb tagged with eGFP and imaged 24 h after transfection. (A) Diagram depicting predicted results. (B, C) Colocalization of actin on enlarged compartments (arrows) with eGFP-myosin-Vb. (D, E, F) In cells expressing high levels of eGFP-myosin-Vb coincident with exposure to transferrin (arrows), large, peripheral organelles decorated with myosin-Vb also contain transferrin. (G, H, I, J, K, L) In cells exposed to a 1-min pulse of transferrin 24 h after transfection and 10 min before imaging, transfected cells (arrows) contain large, peripheral organelles decorated with myosin-Vb that lack transferrin. Cells expressing lower levels of myosin-Vb (arrowheads, panel K; too low to be seen in panel J) accumulate less transferrin than the surrounding untransfected cells (arrowheads). Bar, 15 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2533098&req=5

Figure 2: Overexpression of full-length, wild-type eGFP-myosin-Vb causes coalescence of peripheral endocytic compartments and inhibits perinuclear accumulation of transferrin. HeLa cells were transiently transfected with full-length, wild-type myosin-Vb tagged with eGFP and imaged 24 h after transfection. (A) Diagram depicting predicted results. (B, C) Colocalization of actin on enlarged compartments (arrows) with eGFP-myosin-Vb. (D, E, F) In cells expressing high levels of eGFP-myosin-Vb coincident with exposure to transferrin (arrows), large, peripheral organelles decorated with myosin-Vb also contain transferrin. (G, H, I, J, K, L) In cells exposed to a 1-min pulse of transferrin 24 h after transfection and 10 min before imaging, transfected cells (arrows) contain large, peripheral organelles decorated with myosin-Vb that lack transferrin. Cells expressing lower levels of myosin-Vb (arrowheads, panel K; too low to be seen in panel J) accumulate less transferrin than the surrounding untransfected cells (arrowheads). Bar, 15 μm.
Mentions: Figure 2 and Additional files 2, 3, 4, 5, 6, 7, 8 show transferrin accumulation in peripheral compartments as a function of the overexpression level of eGFP-myosin-Vb, which the dynamic tethering hypothesis predicts will cause the coalescence and caging of peripheral endosomes by actin (Fig. 2A). A coalescence of actin around the enlarged peripheral endosomes is shown by the colocalization of myosin-Vb and actin (Fig. 2B,C).

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
Related in: MedlinePlus