Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle.
The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va 'receptor', and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing.
Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
X
X
Wu
K
K
Rao
M B
MB
Bowers
N G
NG
Copeland
N A
NA
Jenkins
J A
JA
Hammer
D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton; D003712:Dendrites / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018995:Myosin Heavy Chains; D024701:Myosin Type V; D015388:Organelles; D010641:Phenotype; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D007381:Intermediate Filament Proteins; C513082:Myo5a protein, mouse; D000075745:rab27 GTP-Binding Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D024701:Myosin Type V; C512962:Rab27a protein, mouse; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins