Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va.
In mice a molecular motor of the myosin V class (designated myosin Va) is known to be the product of the dilute locus, where a mutation prevents melanosome transport in melanocytes. There is conflicting evidence about whether it has a role in dendrite outgrowth. We investigated its role by transiently transfecting antisense oligonucleotides to inhibit its expression in a melanocyte cell line. We demonstrated mRNA and protein expression of myosin Va in 3 mouse melanocyte lines and 1 human melanoma cell line, using RT-PCR and immunoblotting. Two splice variants were found in human cells whilst only the longer transcript, containing an additional exon, was present in mouse melanocyte lines. The shorter variant was detected in other mouse tissues. Myosin Va protein levels were similar in 3 melanocyte lines with differing amounts of pigmentation, indicating that expression of myosin Va is not tightly coupled to expression of melanin. Immunocytochemistry showed 2 types of myosin Va localisation. A punctate pattern of staining concentrated in the perinuclear region was indicative of organelle association, and the observation of occasional linear punctate staining aligned with F-actin bundles supported the idea that myosin Va has a role in transporting melanosomes along actin filaments. Staining was also intense at tips of dendrites and at sites of dendrite-cell contact, consistent with a possible role in dendrite growth. Transient transfection of antisense phosphorothioate oligodeoxynucleotides targeted against myosin Va mRNA reduced expression of myosin Va protein in cultured mouse melan-a melanocytes by over 70 % 20 h after transfection whereas a control (shuffled sequence) oligonucleotide did not. Upon trypsinisation and replating these cells the capacity of the transfected cells to extend new dendrites was reduced in the cells containing the specific antisense oligonucleotides but unaffected by the control oligonucleotide. Image analysis confirmed that the effect of transfection on morphology was statistically significant (P < 0.01). In contrast when cells were not trypsinised and replated following transfection so that previously existing dendrites could persist, the normal dendritic morphology continued to be observed. We conclude that in addition to its involvement in melanosome transport, myosin Va has a role in the extension of new dendrites by melanocytes but not in maintenance of pre-existing dendrites.
Edgar
A J
AJ
Division of Biomedical Sciences, Imperial College School of Medicine, London, UK.
Bennett
J P
JP
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D016373:DNA, Antisense; D003712:Dendrites / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000502:physiology*; D008544:Melanocytes / Q000502:physiology*; D008545:Melanoma / Q000378:metabolism*; D051379:Mice; D020409:Molecular Motor Proteins; D018995:Myosin Heavy Chains; D024701:Myosin Type V; D020033:Protein Isoforms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D016373:DNA, Antisense; D007381:Intermediate Filament Proteins; D020409:Molecular Motor Proteins; C513082:Myo5a protein, mouse; D020033:Protein Isoforms; C077511:MYO5A protein, human; D024701:Myosin Type V; D018995:Myosin Heavy Chains