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 supporting 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.


OBJECTIVE
We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts.


METHODS
Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed.


RESULTS
The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close 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 dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
Vancoillie
G
G
Department of Dermatology, University Hospital, De Pintelaan 185, 9000 Gent, Belgium.
Lambert
J
J
Mulder
A
A
Koerten
H K
HK
Mommaas
A M
AM
Van Oostveldt
P
P
Naeyaert
J M
JM
D000328:Adult; D018929:Cell Culture Techniques; D003593:Cytoplasm / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism; D012867:Skin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004398:Dyneins