Results Design and Generation of Fluorescent Melanosomal Markers We sought to design a live-cell melanosomal marker by attaching a fluorescent protein tag to a melanosomal-specific protein. Cumulative displacement was calculated using the formula: Viral particles were concentrated from decanted supernatant using a Lenti-X Concentrator kit Clontech, Mountain View, CA according to manufacturer instructions. Significance testing was conducted asdescribed in Materials and Methods and is present in Supporting Information. B Images from a time-lapse series numbers indicate elapsed time in seconds; Movie S3 showing the movement of pigmented melanosomes and EGFP-Rab32 red and green, respectively, in colour combine. Boissy RE Melanosome transfer to and translocation in the keratinocyte. Images werethen obtained using a confocal microscope see Materials andMethods.
Expression of GFP-Rab27a in BF1 cells resulted in diffuse cytosolic fluorescence, with visible vesicular structures localized primarily at the cell periphery, likely a result of Rab27a binding to cortical actin  , . Two types of melanosomal movement were readily apparent: Visual inspection revealed that Rab27a-positive melansomes are more peripheral than Rabpositive and Rab38 melanosomes, consistent with previous observations 20 Figure 5A and Movie S6. Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes. We conclude that automated correlative analysis of organelle movement and peripheral membrane protein distribution is likely to be a powerful approach for use in future studies to address the role of these proteins in organelle transport. Pfeiffer , Jonathan M.
Insets show enlarged area depicted by the white dashed square. Greka for providing lentiviral mflanosome and protocols, and Dr. Melanocytes, neural crest-derived cells that produce melanin, reside primarily in the human epidermis and retinal pigmented epithelium but are also found within the cochlea brain and heart .
D Fluorescent protein intensities f 1f 2etc. Primary antibodies were detected using alexa andalexalabelled secondary antibodies, and Mocie distributionwas revealed using Texas red-conjugated phalloidin.
National Center for Biotechnology InformationU. Abstract Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes.
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. Schiaffino MV, Tacchetti C The ocular albinism type 1 OA1 protein and the evidence for an intracellular signal transduction system involved in melanosome biogenesis.
Melanosomal Dynamics Assessed with a Live-Cell Fluorescent Melanosomal Marker
Nat Cell Biol 4: They perform this function by melwnosome as compartment-specific molecular switches that cycle between active GTP- and inactive GDP-bound conformations. Insertion of the correct sequences of moevment into the respective plasmids was confirmed by DNA sequencing Macrogen Inc. M analysis requires that the number of objects in both channels to be approximately equal; it ranges between 0 and 1, with 1 being high colocalization.
The slope of the graphs reflects melanosomal speed, which is consistently slow for the green trace and consistently fast for the black trace, in agreement with the V avg values for these melanosomes. Analysis of the intensity of individual fluorophores underlying the changes in EGFP: Role of cytoplasmic dynein in melanosome transport in human melanocytes.
Previous studies suggested that Rab27a sequentially recruits the modular adaptor Mlph and alternative myosin motor MyoVa, and that this complex allows actin-dependent capture of melanosomes in peripheral dendrites Lavado A, Montoliu L New animal models to study the role of tyrosinase in normal retinal development.
Second, comparison of the speed of Rab27a-positive and Rab27a-negative melanosomes indicates that recruitment of Rab27a results in Mlph-dependent and MyoVa-dependent switching of melanosomes from MT- to actin-based transport.
The movie showsthe movement of EGFP-Rab27a containing structures green andmelanosomes pseudo-coloured red. This melnaosome corresponded to the melanosome speed distribution observed in non-rescued Mlph-null cells expressing EGFP-Rab27a Figures 1D and 6C,Dand is consistent with the release of a percentage of melanosomes from docking Notably, in the study of MyoVa function, MyoVa-null melanocytes manifest perinuclear melanosome clustering, rendering the vast majority of melanosomes in these cells untrackable.
While this movle makes melanin a very effective UV filter for the skin, it poses a challenge to the development of useful fluorescent melanosomal markers, as fluorophores in the luminal compartment are effectively shielded from receiving or emitting light. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. This suggested that MT- and actin-dependent melanosome transport steps are regulated by Rab7 and Rab27a, respectively.
TIRF microscopy minimizes the amount of light reaching fluorophores, thus minimizing the effects of incident light during quantification of melanosome motility.
Melanosomal Dynamics Assessed with a Live-Cell Fluorescent Melanosomal Marker
Support Center Support Center. Melansomes are visible as dark particles in phase contrast and as fluorescent particles in fluorescence imaging. PfeifferJonathan M. See online Supporting Information text for full details of statistical analysis of data. Horizontal line indicates median speed, outer bars indicate 25th—75th percentile range and circles indicate the 5th—95th percentile range for each population.
Toriello HV Pigmentary anomalies and hearing loss. The graphs in the right column plot pixel intensity of channel 1 versus pixel intensity of channel 2 as an additional means of relating the brightness data in both channels. Exp Dermatol 12 Suppl 25— Melanosome biogenesis is proposed to proceed via four morphologically defined maturation stages.
However, key aspects of their movement, like velocity, trajectory, and diffusion coefficient, have not been determined, primarily due to lack of technical tools. The data in these graphs are not normalized.
We evaluated the specificity of the melaonsome fusion proteins by colocalization with established immunocytochemical melanosomal markers including TRP-1 and Pmel Due to slightly higher correlation coefficients calculated for OA1-mCherry Table 1 and melanossome avoid potential artifacts arising from the anchoring of Rab27a-positive melanosomes to cellular cortical regions, we used fluorescently-tagged OA1 for subsequent studies.
Supporting Information Additional Supporting Information may be found in the online version of this article: Consistent with this, analysis of the chocolate coat colour mutant mouse revealed a role for Rab38, a non-ubiquitously expressed Rab, in the specialized trafficking pathways that underlie pigmentation 18 Several such studies revealed fundamental roles for Rab family small GTPases and their effectors in transport and biogenesis of melanosomes and other LROs Analysis of diffusion and flow in two-dimensional systems.
Boissy RE Melanosome transfer to and translocation in the keratinocyte. Scatter plots showing colocalization of fluorescent pixels from red and green channels in HEMs from corresponding sub-figures in Fig. Curr Opin Cell Biol.
Thus, improved understanding of melanosome biology has broader implication for our understanding of the biology of a range of related organelles. Two types of melanosomal movement were readily apparent: In response to UV exposure the fraction of melanosomes moving by active transport increases black vs.
Dynamics of EGFP-Rab32 containing structures green and melanosomes pseudo-coloured red in transfected melan-ink4a cells.