Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport

C.L. Robinson, R.D. Evans, D.A. Briggs, J.S. Ramalho, A.N. Hume

Research output: Contribution to journalArticle

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4 Downloads (Pure)

Abstract

Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here,we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes. © 2017. Published by The Company of Biologists Ltd.
Original languageEnglish
Pages (from-to)2056-2065
Number of pages10
JournalJournal Of Cell Science
Volume130
Issue number12
DOIs
Publication statusPublished - 15 Jun 2017

Fingerprint

Melanosomes
Kinesin
Myosins
Actins
Melanocytes
Microtubules
Dendrites
Confocal Microscopy
Organelles
Small Interfering RNA
Cluster Analysis
Mammals
Proteins

Keywords

  • Actin
  • Kinesin-1
  • Melanocyte
  • Microtubules
  • Myosin-Va
  • Organelle transport

Cite this

Robinson, C.L. ; Evans, R.D. ; Briggs, D.A. ; Ramalho, J.S. ; Hume, A.N. / Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport. In: Journal Of Cell Science. 2017 ; Vol. 130, No. 12. pp. 2056-2065.
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Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport. / Robinson, C.L.; Evans, R.D.; Briggs, D.A.; Ramalho, J.S.; Hume, A.N.

In: Journal Of Cell Science, Vol. 130, No. 12, 15.06.2017, p. 2056-2065.

Research output: Contribution to journalArticle

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T1 - Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport

AU - Robinson, C.L.

AU - Evans, R.D.

AU - Briggs, D.A.

AU - Ramalho, J.S.

AU - Hume, A.N.

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AB - Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here,we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes. © 2017. Published by The Company of Biologists Ltd.

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