Spatiotemporal Analysis of CD82-mediated Integrin Adhesion

 描述（由申请人提供）：造血干/祖细胞（HSPC）与细胞微环境或“小生境”的相互作用提供了基于粘附的信号传导，这是维持HSPC增殖、分化和存活所必需的。尽管使用HSPC移植治疗血癌取得了临床成功，但调控HSPC与其微环境相互作用的机制在很大程度上仍然未知。因此，我们必须确定调控HSPC粘附于微环境的机制和分子，以提供临床靶点来增强HSPC移植治疗。本提案的目的是确定CD 82通过调节整合素聚集和活化来调节整合素介导的HSPC粘附的机制。我们将检验CD 82通过调节整合素聚集和活化来调节HSPC粘附和归巢的假设。在具体目标1中，我将使用蛋白质印迹和流式细胞术分析来定量β1激活水平对配体的反应，以解决CD 82表达和特异性CD 82突变如何调节β1激活。此外，我将使用超分辨率成像（SRI）来解析总整合素和活化的β1整合素的膜蛋白组织。对于特定目标2，我将在过继转移实验中使用突变型CD 82和原代人CD 34+细胞，以评估CD 82如何调节HSPC的归巢和生态位定位。此外，我将使用SRI来量化B1和CD 82在HSPC接触位点与小生境细胞的分子组织，以确定CD 82如何调节整合素组织以响应微环境。这一提议意义重大，因为我们期望将CD 82及其调控伙伴鉴定为增强HSPC移植治疗的分子靶点。该提议是创新的，因为它将尖端成像技术与体内分析相结合，以提供对四跨膜蛋白如何调节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，通过靶向整合素的上游调节因子CD 82，用于控制HSPC归巢。因此，该提案将提供对新型分子调节剂的深入了解，这些分子调节剂可以在移植前靶向增强HSPC的粘附特性。

 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.