Fig. 4. Expression of Cdc42^V12 by human melanocytes induces dendricity and multiple melanosome-containing filopodia. (a) Cdc42^V12 expressed using adenovirus is functionally active. Melanocytes (10⁶ cells) were infected with adenovirus vector expressing Cdc42^V12 (lane 3) or virus alone (lane 2) for 5 days at 37°C. Infection efficiency was approximately 50%. Whole cell lysates were incubated with GST-PBD, and bound proteins were analyzed by western blotting with anti-Cdc42 antibodies. Positive controls consisted of cell lysates pre-loaded with GTPS (lane 1). Cdc42^V12-expressing cells show increased levels of PBD-bound Cdc42. The band migrating at 35 kDa represents the GST-fusion protein. Total cell lysates blotted for Cdc42 show increase Cdc42 in Cdc42^V12-expressing cells. (b) Cells expressing Cdc42^V12 (detected by green fluorescence) show a highly dendritic morphology reminiscent of neural cells (1-3). Cells infected with empty vector show a bipolar morphology typical of melanocytes grown in keratinocyte growth medium, which lack phorbol esters (4). Bar, 20 µm (1-3); bar, 5 µm (4). (c) An Cdc42^V12-infected cell is identified by expression of GFP tag (1). Examination of the same cell stained with antibodies to TRP-1 (2) shows numerous melanosomes within the filopodia (arrowheads) compared with cells expressing empty vector (3,4). Bar, 20 µm. (d) The images shown were captured under DIC optics and show tips of melanocyte dendrites with numerous filopodia (1,2). Co-culture of Cdc42^V12- expressing melanocytes and non-infected keratinocytes (3) shows multiple filopodia (arrowheads) arising from a Cdc42^V12-expressing melanocyte in contact with a keratinocyte (KC). Images (4,5) show empty-vector-expressing melanocyte dendrite tips with a normal complement of filopodia (arrowhead).