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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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(A-H) Overexpression of eGFP-myosin-Vb tail causes a slight shift in Rab4 distribution, but has little effect on Rab5 distribution. HeLa cells were transiently transfected with eGFP-myosin-Vb tail fragment (B,C,D,F,G,H) and mRFP-Rab4 (A,B,C,D) or mRFP-Rab5 (E,F,G,H) and imaged after overnight incubation. Bar, 15 μm.
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Figure 6: (A-H) Overexpression of eGFP-myosin-Vb tail causes a slight shift in Rab4 distribution, but has little effect on Rab5 distribution. HeLa cells were transiently transfected with eGFP-myosin-Vb tail fragment (B,C,D,F,G,H) and mRFP-Rab4 (A,B,C,D) or mRFP-Rab5 (E,F,G,H) and imaged after overnight incubation. Bar, 15 μm.

Mentions: Since mosaic endosomes have been observed with every possible combination of Rab4, Rab5, and Rab11a [35], we examined Rab4 and Rab5 distribution. Overexpression of the myosin-Vb tail produced a slight alteration in the distribution of Rab4 (Fig. 6A,B,C,D), but no significant effect on Rab5 distribution (Fig. 6F,G,H), which is puzzling given the association between EEA1 and Rab5 [36].


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)

(A-H) Overexpression of eGFP-myosin-Vb tail causes a slight shift in Rab4 distribution, but has little effect on Rab5 distribution. HeLa cells were transiently transfected with eGFP-myosin-Vb tail fragment (B,C,D,F,G,H) and mRFP-Rab4 (A,B,C,D) or mRFP-Rab5 (E,F,G,H) and imaged after overnight incubation. Bar, 15 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: (A-H) Overexpression of eGFP-myosin-Vb tail causes a slight shift in Rab4 distribution, but has little effect on Rab5 distribution. HeLa cells were transiently transfected with eGFP-myosin-Vb tail fragment (B,C,D,F,G,H) and mRFP-Rab4 (A,B,C,D) or mRFP-Rab5 (E,F,G,H) and imaged after overnight incubation. Bar, 15 μm.
Mentions: Since mosaic endosomes have been observed with every possible combination of Rab4, Rab5, and Rab11a [35], we examined Rab4 and Rab5 distribution. Overexpression of the myosin-Vb tail produced a slight alteration in the distribution of Rab4 (Fig. 6A,B,C,D), but no significant effect on Rab5 distribution (Fig. 6F,G,H), which is puzzling given the association between EEA1 and Rab5 [36].

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