Role of cytoplasmic dynein in melanosome transport in human melanocytes

Cytoplasmic dynein is a microtubule-associated retrograde-directed motor mo lecule for transport of membrane-bound organelles. To determine whether cyt oplasmic dynein is expressed in melanocytes, we performed reverse transcrip tase polymerase chain reaction using melanocyte cDNA and primers complement ary to human brain cytoplasmic dynein heavy chain. A polymerase chain react ion product of the expected molecular size was generated and the identity w as confirmed by sequence analysis. Western blotting of total melanocyte pro teins reacted with an anti-intermediate chain cytoplasmic dynein antibody i dentified the appropriate 74 kDa band. To determine whether cytoplasmic dyn ein plays a role in melanosome transport, duplicate cultures were treated w ith cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides a nd the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport o f melanosomes from the cell body into the dendrites, whereas melanosome dis tribution was not affected in sense-treated melanocytes. To determine wheth er ultraviolet irradiation modifies cytoplasmic dynein expression, melanocy te cultures were exposed to increasing doses of solar-simulated irradiation , equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm(2) induced cytoplasmic dynein protein, w hereas doses of 30 mJ per cm(2) or more were associated with decreased leve ls of cytoplasmic dynein compared with sham-irradiated controls. Our data s how that cytoplasmic dynein participates in retrograde melanosomal transpor t in human melanocytes and suggest that the altered melanosomal distributio n in skin after sun exposure is due, at least in part, to decreased cytopla smic dynein levels resulting in augmented anterograde transport.