CELL BIOLOGY OF MELANOCYTE MIGRATION AND LOCALIZATION

我的长期目标是确定 是表皮中黑素细胞迁移和定位的基础。 黑素细胞在表皮中的迁移和定位对于 在皮肤发育过程中建立正常的色素沉着， 创伤后皮肤的重新色素沉着。 本文中概述的实验 该提案将测试黑素细胞迁移是 由特异性受体-配体相互作用决定， 黑素细胞在表皮中的定位部分地由 整合素受体 我们已经证明胎儿和新生儿的黑素细胞 表达整合素和整合素受体介导黑素细胞 连接到纤连蛋白。 我们还表明，胎儿和新生儿 黑素细胞与纤连蛋白的相互作用不同， 整合素的数量，这表明，发育调节 存在黑素细胞-基质相互作用。 最后，我们已经表明，TGF- β，bFGF和干细胞因子（SCF）调节整合素的表达， 黑素细胞，并且已经显示用TGF-β治疗黑素细胞 和SCF导致黑素细胞对细胞外 基质（ECM）蛋白。 因为肝细胞生长因子，一种黑素细胞有丝分裂原， 直接影响黑素细胞迁移的实验，以确定 将执行此现象的机制。 具体目标1将 重点定义基质蛋白、受体和调节因子 其控制胎儿和新生儿黑素细胞迁移。 黑素 将由Boyden评价细胞外基质蛋白的迁移 在平面模型中的小室分析和延时视频显微镜;作用 黑素细胞整合素和糖胺聚糖以及TGF-β、bFGF、SCF 然后测定黑素细胞迁移中的HGF。 一旦我们 定义了控制该模型中迁移的参数，我们将 研究黑色素细胞在胶原细胞中的迁移， 黑素细胞在胎儿期的迁移环境。 在每个实验中， 将比较胎儿和新生儿的黑素细胞， 这些差异可能对管理移徙很重要。 我们 建立了一个皮肤等效物（SE）模型，以研究黑素细胞-角质形成细胞 胎儿和新生儿表皮的相互作用，并已表明， 黑素细胞的数量和位置由角质形成细胞决定， 这些因素可能是发育调节的。 整合素α 2 和alpha3最近被证明在发育过程中 调节角质形成细胞的细胞-细胞受体。 具体目标2重点 在确定这些角质形成细胞整合素的作用， 胎儿和新生儿表皮中黑素细胞的定位和数量。 最初的实验将涉及阻断研究与抗整合素 抗体对角质形成细胞-黑素细胞共培养物的影响。 确认并延长 根据这些观察结果，我们将用整合素α 2 和α 3，并确定对黑素细胞附着到这些细胞的影响。 角质形成细胞 最后探讨整合素在黑素细胞中的作用 在复层表皮中的数量和位置，我们将构建SE 与转染的角质形成细胞，并确定增加的效果 角质形成细胞整合素表达对黑素细胞-角质形成细胞相互作用的影响。

My long term objectives are to determine the molecular mechanisms which underlie melanocyte migration and localization in the epidermis. Melanocyte migration and localization in the epidermis are critical for establishing normal pigmentation during cutaneous development, and for repigmentation of skin after trauma. The experiments outlined in this proposal will test the hypotheses that migration of melanocytes is determined by specific receptor-ligand interactions and that the localization of melanocytes in the epidermis is determined, in part, by integrin receptors. We have shown that fetal and neonatal melanocytes express integrins and that integrin receptors mediate melanocyte attachment to fibronectin. We have also shown that fetal and neonatal melanocytes interact differently with fibronectin and express different amounts of integrins, suggesting that developmental regulation of melanocyte-matrix interactions exist. Finally, we have shown that TGF- beta, bFGF and stem cell factor (SCF) regulate integrin expression in melanocytes, and have shown that treatment of melanocytes with TGF-beta and SCF result in alterations in melanocyte affinity for extracellular matrix (ECM) proteins. Because HGF, a melanocyte mitogen, has been shown to directly affect melanocyte migration, experiments to determine the mechanisms of this phenomenon will be performed. Specific aim 1 will focus on defining the matrix proteins, receptors, and regulatory factors which control fetal and neonatal melanocyte migration. Melanocyte migration on extracellular matrix proteins will be evaluated by Boyden chamber assays and time-lapse videomicroscopy in a planar model; the role of melanocyte integrins and glycosaminoglycans and of TGF-beta, bFGF, SCF and HGF in melanocyte migration will then be determined. Once we have defined the parameters that control migration in this model, we will study melanocyte migration in collagen cells, which recapitulates the environment of migrating melanocytes in fetal life. In each experiment, fetal and neonatal melanocytes will be compared to identify developmental differences which may be important for regulating migration. We developed a skin equivalent (SE) model to study melanocyte-keratinocyte interactions in fetal and neonatal epidermis, and have shown that melanocyte number and position is determined by keratinocytes, and that these factor(s) are probably developmentally regulated. Integrins alpha2 and alpha3 have recently been shown to function as developmentally regulated cell-cell receptors for keratinocytes. Specific aim 2 focuses on determining the role of these keratinocyte integrins in the localization and number of melanocytes in fetal and neonatal epidermis. Initial experiments will involve blocking studies with anti-integrin antibodies on keratinocyte-melanocyte co-cultures. To confirm and extend these observations, we will transfect keratinocytes with integrins alpha2 and alpha3 and determine the effect on melanocyte attachment to these keratinocytes. Finally, to evaluate the role of integrins on melanocyte number and position in the stratified epidermis, we will construct SEs with the transfected keratinocytes and determine the effect of increased keratinocyte integrin expression on melanocyte-keratinocyte interactions.