Spatiotemporal Analysis of CD82-mediated Integrin Adhesion

 描述(申请人提供)：造血干细胞/祖细胞(HSPC)与细胞微环境或“小生境”的相互作用提供基于黏附的信号，这是维持HSPC增殖、分化和存活所必需的。尽管HSPC移植治疗血癌取得了临床上的成功，但HSPC与其微环境相互作用的调控机制在很大程度上仍不清楚。因此，识别调节HSPC与微环境黏附的机制和分子，为提高HSPC的移植治疗提供临床靶点是至关重要的。本研究的目的是确定CD82通过调节整合素的聚集和激活来调节整合素介导的HSPC黏附的机制。我们将验证CD82通过调节整合素聚集和激活来调节HSPC黏附和归巢的假设。在特定的目标1中，我将使用Western印迹和流式细胞术分析来量化β1对配体的反应的激活水平，以解决β表达和特定的CD82突变如何调节CD1的激活。此外，我将使用超分辨率成像(SRI)来解析总的和激活的β1整合素的膜蛋白组织。对于特定的目的2，我将在过继转移实验中使用突变的CD82和原代人类CD34+细胞来评估CD82如何调节HSPC的归巢和小生境定位。此外，我将使用SRI来量化在HSPC与利基细胞的接触部位B1和CD82的分子组织，以确定CD82如何调节整合素组织来响应微环境。这项建议意义重大，因为我们希望将CD82及其调控伙伴确定为加强HSPC移植治疗的分子靶点。这项建议的创新之处在于，它将尖端成像技术与活体分析相结合，以提供对Tetraspanins如何调控HSPC归巢的多尺度理解。此外，我的研究采取了一种独特的方法，针对整合素的上游调控因子CD82来控制HSPC的归巢。因此，这项建议将提供对新型分子调节剂的洞察，这些分子调节剂可以在移植前针对增强HSPC的粘附性。

 DESCRIPTION (provided by applicant): Hematopoietic stem/progenitor cell (HSPC) interactions with the cellular microenvironment or "niche" provide adhesion based signaling, which is necessary for the maintenance of HSPC proliferation, differentiation, and survival. Despite the clinical successes from using HSPC transplantation for the treatment of blood cancers, the mechanisms that regulate HSPC interactions with their microenvironment remain largely unknown. As such, it is critical that we identify the mechanisms and molecules that regulate HSPC adhesion to the microenvironment to provide clinical targets to enhance HSPC transplantation therapies. The objective of this proposal is to determine the mechanism by which CD82 regulates integrin-mediated HSPC adhesion by modulating integrin clustering and activation. We will test the hypothesis that CD82 regulates HSPC adhesion and homing by modulating integrin clustering and activation. In Specific Aim 1, I will use Western blot and flow cytometry analyses to quantify β1 activation levels in response to ligand to address how CD82 expression and specific CD82 mutations regulate β1 activation. Furthermore, I will use super-resolution imaging (SRI) to resolve the membrane protein organization of total and activated β1 integrins. For Specific Aim 2, I will use mutant CD82 and primary human CD34+ cells in adoptive transfer experiments to assess how CD82 regulates the homing and niche localization of HSPCs. Additionally, I will use SRI to quantify the molecular organization of B1 and CD82 at the HSPC contact site with niche cells to identify how CD82 regulates integrin organization in response to the microenvironment. This proposal is significant because we expect to identify CD82 and its regulatory partners as molecular targets to enhance HSPC transplantation therapies. This proposal is innovative in that it combines cutting-edge imaging techniques with in vivo analyses to provide a multi-scale understanding of how tetraspanins regulate HSPC homing. Moreover, my study takes a unique approach by targeting an upstream regulator of integrins, CD82, for the control HSPC homing. As such, this proposal will provide insight into novel molecular regulators that can be targeted to enhance the adhesive properties of HSPCs prior to transplantation.