CELL BIOLOGY OF MELANOCYTE MIGRATION AND LOCALIZATION

我的长期目标是确定 黑素细胞在表皮中的迁移和定位。 黑素细胞在表皮中的迁移和定位对 在皮肤发育过程中建立正常的色素沉积，并 创伤后皮肤再色素沉着。这篇文章中概述的实验 该提案将检验黑素细胞迁移是 由特定的受体-配体相互作用决定，并且 黑素细胞在表皮中的定位部分取决于 整合素受体。我们已经证明胎儿和新生儿的黑素细胞 表达整合素及其受体介导黑素细胞 依附于纤维粘连蛋白。我们还表明，胎儿和新生儿 黑素细胞与纤维连接蛋白的相互作用不同，并表达不同的 整合素的量，表明发育调节 黑素细胞与基质的相互作用是存在的。最后，我们已经证明了转化生长因子- β、碱性成纤维细胞生长因子和干细胞因子调节细胞整合素的表达 黑素细胞，并已表明转化生长因子-β处理黑素细胞 和SCF导致黑素细胞对细胞外的亲和力改变 基质(ECM)蛋白。因为HGF，一种黑素细胞有丝分裂原，已经被证明 为了直接影响黑素细胞的迁移，实验确定 将对这一现象的机制进行研究。具体目标1将 重点定义基质蛋白、受体和调节因子 控制胎儿和新生儿黑素细胞迁移的物质。黑素细胞 博伊登将评估细胞外基质蛋白的迁移 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转化生长因子-β、碱性成纤维细胞生长因子、干细胞因子的表达 而HGF在黑素细胞迁移中的作用将被确定。一旦我们有了 定义了在此模型中控制迁移的参数，我们将 研究黑素细胞在胶原细胞中的迁移，这概括了 胎儿生命中黑素细胞迁移的环境。在每个实验中， 胎儿和新生儿的黑素细胞将进行比较，以确定发育 差异，这可能对管理移民很重要。我们 开发了皮肤等效(SE)模型来研究黑素细胞-角质形成细胞 在胎儿和新生儿表皮中的相互作用，并表明 黑素细胞的数量和位置由角质形成细胞决定， 这些因素(S)可能是发育调节的。整合素α2 和alpha3最近被证明在发育过程中的作用 角质形成细胞的调节细胞-细胞受体。特定目标2重点 角质形成细胞整合素在角质形成过程中的作用 胎儿和新生儿表皮中黑素细胞的定位和数量。 初步实验将涉及用抗整合素阻断研究 角质形成细胞-黑素细胞共培养的抗体。确认和延期 在这些观察中，我们将整合素α2基因导入角质形成细胞 和Alpha3，并确定对黑素细胞与这些细胞黏附的影响 角质形成细胞。最后，评估整合素在黑素细胞中的作用 数量和位置，我们将构建SE 用转染法测定角质形成细胞的增敏效果 角质形成细胞整合素在黑素细胞-角质形成细胞相互作用中的表达。

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.