Melanocyte - Keratinocyte Interactions and Intercellular Communication in the Skin

Rachel L. Belote

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Abstract

Melanocytes are the, neural crest derived, pigment producing cells found in human skin. Through interactions with keratinocytes and the basement membrane, melanocytes reside is a defined ratio and spatial distribution with in the basal layer of the epidermis. In coordination with keratinocytes, they distribute pigment containing organelles called melanosomes throughout the layers of the epidermis where they serve as the main photo-protective mechanism against UV induced DNA damage across the layers of the skin. General melanocyte-keratinocyte interactions involved in melanocyte homeostasis such as cell proliferation, pigment production and pigment transfer and have been well studied. However, little is known about the interactions between keratinocytes and melanocytes at the single cell level. I first describe the optimization of a human primary melanocyte - keratinocyte co-culture system that mimics physiological conditions of human skin. In addition, I have developed a novel tissue culture system that can be used to study individual melanocytes with an intact human epidermis. I have shown that the stratified structure of the epidermis including proper localization of K14 positive basal layer keratinocytes and melanocytes is maintain for several days in culture. Using the primary human melanocyte-keratinocyte co-culture, I have provided novel insight into how melanocytes transfer melanosomes to keratinocytes. I show that keratinocytes phagocytose melanocyte derived packages that contain both the melanocyte plasma membrane and melanosome membrane. My findings are consistent with both the shedding - phagocytosis and cytophagocytosis models of melanosome transfer. In addition, I have provided evidence that melanocyte dendrite degradation is a mechanism by which melanocyte derived packages are produced. Using high resolution imaging of primary melanocyte and keratinocyte co-cultures, I have discovered that melanocytes form spine-like protrusions from their dendrites that are similar to the shape and size of neuronal dendritic spines. These structures were found at sights of contact between melanocytes and keratinocyte. Likewise, I have shown that keratinocyte membrane extensions, termed filopodia or microvilli in previous studies, contacted and/or wrapped around melanocyte dendrites. In an effort to characterize the melanocyte protrusions and keratinocyte extensions, I have investigated the presence of known synaptic proteins, SNAP25, synaptophysin, syntaxin 1A , PSD-95 and AMPA-type glutamate receptor GluR2. I provide evidence that all of these genes are expressed in the epidermis. Through RT-PCR I showed that melanocytes express SNAP25, synaptophysin, syntaxin 1A , PSD-95 and keratinocytes express syntaxin 1A and PSD-95. Using immunofluorescence staining of fixed frozen neonatal foreskin, I found that synaptophysin co-localizes with melanocyte markers TRP1/CKIT as well as the basal keratinocyte marker K14. Using the genetically encoded Ca2+ sensor, GCaMP6f, I showed that melanocytes in co-culture with keratinocytes respond to 100μM α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a AMPA-type glutamate receptor agonist, with an increase in Ca2+ concentration. In addition, I observed spontaneous fluctuations in Ca2+ in the absence of external stimulation. These fluctuations were observed in 3 distinct types: whole cell Ca2+ increase, confined regions of Ca2+ increase within dendrite shafts, or in localized burst in the melanocyte spine-like structures. In conclusion, I have provided novel insight into how melanocytes and melanocytes interact and prosed that melanocyte -keratinocyte communication can occur via specialized structures on the cell surface.