Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection
of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/17725 |
| Date | 23 September 2009 |
| Creators | Amsen, Eva |
| Contributors | Rotin, Daniela |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0023 seconds