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Filopodia are conduits for melanosome transfer to keratinocytes

Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

View larger version (132K): [in a new window]   Fig. 1. Melanocytes extend long filopodia from dendrite tips that transport melanosomes to keratinocytes. (a) Two melanocyte dendrites with prominent filopodia (fp; arrowhead) are shown in images taken at 8 seconds. The arrowheads point to thin structures consistent with filopodia. (b) A melanosome (circle) is present in a filopodia (fp) and moves towards the keratinocyte membrane (KM; outlined in hatched line). Over the course of 16 seconds the melanosome has moved along a filopodia towards the keratinocyte membrane. (c) The tip of a melanocyte dendrite is shown with multiple connections with a keratinocyte membrane (KM; outlined in hatched line). A melanosome (circle) moves towards the KM over the course of 40 seconds. (d) Two sequential images captured 5 minutes after treatment of cells with nocodazole are shown. Melanosomes have redistributed towards the melanocyte cell body, leaving a dendrite that appears empty. Filopodia (arrowhead) were not affected by nocodazole treatment.

View larger version (135K): [in a new window]   Fig. 2. Melanosomes are transported to the keratinocyte along filopodia. (a) A scanning view of a melanocyte dendrite (MD) contacting a keratinocyte (KC) is shown. The boxed area is shown in detail in sequential images taken every 8 seconds from movies made from this area (jcs.biologists.org/supplemental or www.urmc.rochester.edu/derm/scottmovies.html ). A filopodia arising from the lateral aspect of the dendrite is either attached or inserted into the keratinocyte membrane. A string of melanosomes (approximately six of them) moves in single file toward the keratinocyte (2-5). The arrowhead indicates the leading melanosome. The last image (6) shows the same melanocyte in which the melanocyte dendrite (arrowhead) is markedly attenuated. A long filopodia (arrow) is shown in which three melanosomes are present. (b) A scanning view of a melanocyte dendrite (MD) adjacent to a keratinocyte (KC) is shown. The hatched line delineates the KC membrane in the upper right hand corner. The boxed area is shown in detail in images that span 152 seconds. A string of melanosomes (approximately four of them; arrowhead) is present within a projection arising from the side of the body of the melanocyte dendrite. These projections were frequently observed in melanocytes and were shorter and thicker than filopodia. Other similar projections are present (asterisks). (c) Melanocytes and keratinocytes separately labeled with DiI (red fluorescence) and DiO (green fluorescence), respectively, were co-cultured and irradiated. Representative images taken with filters to detect both green and red fluorescence in the range of the two dyes are shown. Sham-irradiated cells showed approximately 1% of keratinocytes with yellow fluorescence (arrows; 1). 24 hours after irradiation approximately 10% of keratinocytes show yellow fluorescence (arrow) when viewed with filters to detect DiI and DiO, indicating membrane fusion (2). Bar, 50 µm.

View larger version (94K): [in a new window]   Fig. 3. Electron micrographs of melanocyte-keratinocyte co-cultures and human skin in vivo demonstrate melanosomes within filopodia. Electron micrographs of co-cultures of melanocyte and keratinocytes revealed numerous long thin projections arising from melanocyte (MC) dendrites (a,b), most of which were cut in cross section (arrowhead; c,d). A longitudinal section of a filopodia is shown in (b). Occasionally we detected osmophilic structures consistent with melanosomes within cross sections of filopodia (c,d). Melanosomes were aligned along the base of filopodia (e) and were present along the length of areas of contact between melanocytes and keratinocyte (KC; f). In human skin in vivo thin structures arising from melanocyte dendrites (MD), consistent with filopodia (fp), were easily identified (g,h) (arrows), many of which contained melanosomes (*). Figures (i,j) show enlarged images from the boxed area of parts (g,h), respectively. The arrows show the presence of melanosomes within filopodia. Magnification x5000 (a); x40,000 (b-d); x17,000 (e); x15,000 (f), x4000 (g,h).

View larger version (55K): [in a new window]   Fig. 4. Expression of Cdc42V12 by human melanocytes induces dendricity and multiple melanosome-containing filopodia. (a) Cdc42V12 expressed using adenovirus is functionally active. Melanocytes (106 cells) were infected with adenovirus vector expressing Cdc42V12 (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). Cdc42V12-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 Cdc42V12-expressing cells. (b) Cells expressing Cdc42V12 (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 Cdc42V12-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 Cdc42V12- expressing melanocytes and non-infected keratinocytes (3) shows multiple filopodia (arrowheads) arising from a Cdc42V12-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).

View larger version (51K): [in a new window]   Fig. 5. PAK1, Cdc42 and myosin Va localization in human melanocytes. (a) Immunofluorescence microscopy for Cdc42 (1) and PAK1 (2) was performed on cultured human melanocytes. Cdc42 displayed a vesicular pattern with localization along the length of the melanocyte dendrite as well as in the Golgi area. PAK1, in contrast, was distributed diffusely in the cytosol, in the Golgi area as well as in the dendrites. Bar, 30 µm. (b) The results of double labeling of melanocytes with antibodies to TRP-1 (mel-5) and PAK1 are shown. Images (1,4) show mel-5 localization; images (2,5) show PAK1 localization, images (3,6) are cells viewed with filters to detect both fluorescein-isothiocyanate and Texas Red. PAK1 and TRP-1 colocalize predominantly in the peri-nuclear area (3); however colocalization is also observed along the melanocyte dendrite (6; arrowhead). Bar, 30 µm (1-3); bar, 10 µm (4-6). (c) Melanocytes stained with antibodies to myosin Va show the expected localization of myosin Va at the tips of melanocyte dendrites, consistent with the presence of melanosomes at this site. Myosin Va staining was also detected along the length and at the tips of melanocyte filopodia. Bar, 2 µm.

View larger version (63K): [in a new window]   Fig. 6. PAK1 and N-WASP are present on enriched melanosome fractions. (a,b) Electron microscopy on human melanosomes (HMS) showed that enriched melanosome factions contained primarily stage III (arrows) and stage IV (arrowhead) melanosomes (a; Magnification, 20,000). Western blotting on HMS for transferrin receptor (b) was performed to assess purity of the preparation. HMS extracts (20 µg) run on a 10% gel for transferrin receptor are negative, suggesting that few contaminating endosomes are present in the melanosome preparation. Positive controls consisted of melanocyte whole cell lysate. (c) Western blots of lysates run on 15% gels (20 µg/lane; PAK1, 70 µg/lane; N-WASP) of human melanosome fraction (HMS) and whole cell lysates of human melanocytes (HMC) for PAK1 and N-WASP are shown. Positive controls consisted of Jurkat cell lysates (PAK1) and rat brain lysates (N-WASP). A strong immunoreactive band for PAK1 and N-WASP at the expected molecular weights are present.