Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes

Background Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporti ng the hypothesis that actin-based and microtubule-based motor proteins co- operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes , they move towards the cell periphery and back again, Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. Objectives We aimed to investigate the in vitro expression of the minus end -directed motor protein cytoplasmic dynein in normal human epidermal melano cytes, keratinocytes and dermal fibroblasts. Methods Reverse transcription-polymerase chain reaction and Northern blot a nalysis were used. In addition, an attempt to obtain insight into the subce llular localization of cytoplasmic dynein, immunofluorescence studies and i mmunogold electron microscopic studies were performed. Results The three different forms of cytoplasmic dynein heavy chain were ex pressed in all studied skin cells. Immunofluorescence staining showed simil ar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tip s, Double labelling with a melanosome marker showed apparent co-localizatio n of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear a rea and dendrite tips, For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A cl ose association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. Conclusions The colocalization of different subunits of the cytoplasmic dyn ein complex with melanosomes is consistent with the hypothesis that this mo tor protein supports minus end-directed melanosome movement along microtubu les.