OA1-mCherry and GFP-Rab27a localization in B16-F1 mouse melanoma cells.

<p>A. Fluorescent images of B16-F1 cells expressing OA1-mCherry (left panels), immunostained with anti-TRP-1 antibody (middle panels), and shown as overlay (right panels). B. Fluorescent images of B16-F1 cells expressing OA1-mCherry (left), immunostained with anti-Pmel17 antibody (middle), and shown as overlay (right). C. Fluorescent images of B16-F1 cells expressing GFP-Rab27a (left), immunostained with anti-TRP-1 antibody (middle), and shown as overlay (right). D. Fluorescent images of B16-F1 cells expressing GFP-Rab27a (left), immunostained with anti-Pmel17 antibody (middle), and shown as overlay (right). E. B16-F1 cells co-expressing OA1-mCherry (left) and GFP-Rab27a (middle), and shown as overlay (right). Bottom panels show enlarged area marked by white box in the respective top pannel. Calibration bars: 8 µm (top images), 2 µm (bottom images) for all panels.</p

Melanosomal dynamics measured by TIRF imaging of OA1-GFP labeled melanosomes.

<p>A. TIRF image of a distal cellular extension of a HEM cell expressing OA1-GFP. Calibration bar: 8 µm. White boxes are expanded and detailed in B and C. A time-lapse movie showing melanosomal movement in this region is available in Movie S1. B. TIRF image series tracking one melanosomes (red arrows) with high average velocity (0.34±0.05 µm/s). (see also Movie S2). Changes in the fluorescence intensity reflect changes in the z-position of the melanosome. Calibration bar: 4 µm. C. TIRF image series tracking one melanosome (red arrows) with low average velocity (0.09±0.01 µm/s). (see also Supplementary Materials, Movie 3). Calibration bar: 4 µm. D. Average velocity of individual melanosomes for particles traveling towards the cell periphery (outward, shown in black) or towards the nuclear region (inward, shown in gray). E. Trajectories of representative melanosomes exhibiting restricted mobility (green trace), fast movement along mostly linear x-y trajectories (black trace) or a combination of the modes of movement. The red trajectory corresponds to a melanosome that initially has restricted movement and then starts moving along a linear trajectory, while the blue trace corresponds to a melanosome that has initial fast and linear movement and then becomes stationary. F. Cumulative displacement of the four representative melanosomes shown in Fig. 4E as a function of time. The slope of the cumulative displacement reflects the speed of the movement. Melanosomes with restricted mobility have a small slope that reflects slow movement (green trace in 4E vs. 4F) and fast melanosomes have a high slope (black trace in 4E vs. 4F). The red trace has an initial small slope (t <20 s) that changes to a high slope due to increased mobility (red trace in 4E vs. 4F), while the blue trace illustrates a melanosome moving fast (t <8 s) and then slowing down (blue trace in 4E vs. 4F). G. The maximal displacement of individual melanosomes for outward- (black) and inward- (gray) moving particles. H. The maximal displacement of individual melanosomes is a linear function of average velocity.</p

Melanosomal Dynamics Assessed with a Live-Cell Fluorescent Melanosomal Marker

<div><p>Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin’s physiological response to UV radiation.</p> </div

Schematic representation of the fluorescent fusion protein OA1-mCherry and GFP-Rab27.

<p>Schematic representation of the fluorescent fusion protein OA1-mCherry and GFP-Rab27.</p

Melanosomal transfer imaged with OA1-GFP in live melanocyte/keratinocyte co-cultures.

<p>1. Fluorescent images of melanosomal transfer in co-cultured OA1-GFP-expressing HEMs and HEKERs showing: (i) contact between a HEM cellular process and an HEKER cell body; (ii, iii) sequential detachment of two melanosomes from the HEM followed by transfer to the HEKER (ii, iii), and retraction of the cellular process (iv, v, vi). Insets show enlarged area depicted by the white dashed square. The arrows indicate positions of transferred melanosomes, Calibration bar: 20 µm (image), 8 µm (inset). 2. Corresponding phase contrast images of the co-cultured cells shown in (A). Insets show enlarged area depicted by the white dashed square. Arrows indicate positions of transferred melanosomes. 3. Schematic representation of the melanosomal transfer process shown in (A) and (B). Cartoon images show: (i) at t = 0 min, a HEM (M) cellular process containing melanosomes extends filopodia towards an HEKER (K); over the following 200 min, a filopodium extends over the HEKER plasma membrane, as more melanosomes are transported along all filopodia; (iii) the tip of the extended filopodium containing melanosomes (purple arrow) becomes detached from the HEM and continues to move within the HEKER; (iv) a second melanosome within the same filopodium (blue arrow) is then transferred to the HEKER as all filopodia begin to retract; (v, vi) filopodia retract over the next ∼500 min; (vii) finally, the cellular process retracts towards the HEM cell body. The two melanosomes remain associated with and continue to move within the HEKER. Time-lapse movies of phase and fluorescent images are available as Movie S4.</p

Melanosomal localization of OA1-GFP in primary human melanocytes.

<p>A. HEMs expressing OA1-GFP (left), immunostained with anti-Pmel17 antibody (middle), and shown as overlay (right). Calibration bar: 8 µm (image), 2 µm (inset). B. HEMs expressing OA1-GFP (left), immunostained with anti-TRP-1 antibody (middle), and shown as overlay (right). Calibration bar: 8 µm (image), 2 µm (inset). Inset shows the enlarged area marked by the dashed line. C. Colocalization coefficients for representative HEMs expressing OA1-GFP (green) and immunostained with anti-TRP-1 antibody (red). The Mander’s overlap coefficient (M, blue bars) and the split coefficients for the green (M1, green bars) and red (M2, red bars) images have values close to maximal. D. The Intensity Correlation Quotient (ICQ) for six representative HEMs expressing OA1-GFP (green) and immunostained with anti-TRP-1 antibody (red) has values close to maximal value of 0.5.</p

Changes in melanosomal dynamics in response to UV.

<p>A. TIRF images of the distal end of a HEM expressing OA1-GFP before (t = 0 s), during (t = 600 s) and after (t = 1200 s) stimulation with UV (200 mJ/cm²). Calibration bar: 4 µm. B. Representative Mean Square Displacement (MSD) calculated by tracking individual melanosomes before UV stimulation and represented as a function of time. C. Time dependence of MSD for representative melanosomes imaged after exposure to UV. D. Distribution of diffusion coefficients (D) of individual melanosomes tracked before (gray; n = 22) or after (black; n = 15) UV exposure. E. Mean velocities of melanosomes before (gray) and after (black) UV exposure. Velocities <0.03 µm/s are considered negligible and the corresponding melanosomes move primarily by restricted diffusion (hatched area). Velocities >0.03 µm/s correspond to melanosomes moving by both diffusion and directed movement (gray area). F. In response to UV exposure the fraction of melanosomes moving by active transport increases (black vs. gray bars with V >0.03 µm/s in Fig. 5E), while the fraction of melanosomes moving by restricted diffusion decreases (black vs. gray bars with V <0.03 µm/s in Fig. 5E). 67% of the melanosomes in Fig. 5E move by active transport after UV exposure compared to 41% in unstimulated HEMs (solid black bar vs. solid gray bar) and 33% of the melanosomes in Fig. 5E have restricted diffusion after UV exposure, compared to 59% of melanosomes before UV exposure (hashed black bar vs. hashed gray bar).</p

Colocalization correlation coefficients for OA1-mCherry, GFP-Rab27a, and TRP-1 or pMel17 in B16-F1 mouse melanoma cells.

<p>Mander’s Correlation Coefficient (R) and the Intensity Correlation Quotient (ICQ) for the cells shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043465#pone-0043465-g002" target="_blank">Fig. 2A–D</a>.</p