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Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

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Abstract

Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

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Abbreviations

AIS:

Axon initial segment

EGFP:

Enhanced green fluorescent protein

LDCV:

Large dense core vesicle

mGFP/mRFP:

Monomeric green/red fluorescent protein

NPY:

Neuropeptide Y

SEM:

Standard error of the mean

SG:

Secretory granule

DIV:

Days in vitro

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Acknowledgments

The imaging was performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen. The authors are grateful to W. Almers for providing NPY-EGFP and NPY-mRFP and to the University of Bergen for financial support including research fellowships. H.-H. G. acknowledges grants from the Meltzer Foundation.

Author information

Correspondence to Hans-Hermann Gerdes.

Additional information

Claudia Margarethe Bittins and Tilo Wolf Eichler have contributed equally.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

NPY-EGFP labeled LDCVs in living neurons at 12 DIV. Cells were transfected with NPY-EGFP and a vector encoding mCherry (not shown). Images were taken at 1 Hz for a duration of 100 s. Scale bar 50 µm (AVI 3300 kb)

MyosinVa-tail-mCherry in living neurons at 12 DIV. Cells were double transfected with NPY-EGFP (not shown) and myosinVa-tail-mCherry. Images were taken at 1 Hz for a duration of 100 s. Scale bar 50 µm (AVI 6414 kb)

Dual-color time lapse recording of a neuron at 12 DIV, transfected with NPY-EGFP (green) and myosinVa-tail-mCherry (red) showing cotransport of the two proteins. Images were taken at 1 Hz for a duration of 100 s. Scale bar, 50 µm (AVI 2794 kb)

NPY-EGFP labeled LDCVs in living neurons at 12 DIV. Cells were transfected with NPY-EGFP and a vector encoding mCherry (not shown). Images were taken at 1 Hz for a duration of 100 s. Scale bar 50 µm (AVI 3300 kb)

MyosinVa-tail-mCherry in living neurons at 12 DIV. Cells were double transfected with NPY-EGFP (not shown) and myosinVa-tail-mCherry. Images were taken at 1 Hz for a duration of 100 s. Scale bar 50 µm (AVI 6414 kb)

Dual-color time lapse recording of a neuron at 12 DIV, transfected with NPY-EGFP (green) and myosinVa-tail-mCherry (red) showing cotransport of the two proteins. Images were taken at 1 Hz for a duration of 100 s. Scale bar, 50 µm (AVI 2794 kb)

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Bittins, C.M., Eichler, T.W., Hammer, J.A. et al. Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles. Cell Mol Neurobiol 30, 369–379 (2010) doi:10.1007/s10571-009-9459-2

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Keywords

  • Axonal transport
  • Hippocampal neurons
  • Large dense core vesicles
  • Myosin Va
  • Neuropeptide Y