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.