Investigating the role of the polarity regulator Scribble in maintenance and polarity formation of hematopoietic stem cells.

干细胞特性的进化保守调节以及RNA/DNA结合蛋白已被证明影响造血干细胞的极性和分裂。最近，我们能够鉴定一个极性调节因子和Scribble极性复合体(致死巨型幼虫同源1：Llgl1)的成员，并为其在HSC生物学调控中的作用提供了证据。Llgl1的缺失导致了造血干细胞的竞争优势，Llgl1 KO HSC显示出更高的抗应激能力。尽管细胞周期增加，但Llgl1 KO HSC并未耗尽。此外，Llgl1在原发患者原始细胞中的低表达与预后不良和急性髓系白血病患者总体生存率下降有关。在这项拟议的研究中，我们旨在研究Scribble极性复合体对造血干细胞的影响。我们将使用中央复合体成员Scribble的条件敲除小鼠模型来表征功能相关性。将研究其在稳态造血过程中基因失活的影响，以及系列骨髓移植到受者体内对干细胞生物学的影响。细胞周期分析将补充造血干细胞的功能特征。在第二步中，我们的目标是阐明Scribble复合体成员(Llgl1和Scribble)在调节HSC极性和骨髓-生态位相互作用中的作用。为了解决这些问题，我们将使用延时显微镜，分析纤维连接蛋白涂层表面触发和自发的细胞极化，以及体内2D骨髓显微镜。Scribble复合体成员的细胞内相互作用将通过全球基因表达谱(在转录水平上使用Scribble基因敲除与野生型HSC)和质谱学(针对蛋白质-蛋白质相互作用)进行分析。拟议的实验将允许对造血干细胞中Scribble极性复合体的功能和结构进行表征。这将加深我们对干细胞生物学和通过极性机制调节命运的了解。我们和其他人已经提供了第一个证据，证明了极性复合体成员在白血病前造血干细胞转化中的作用及其预后相关性。这可能导致通过干扰极性网络来靶向白血病干细胞的直接治疗意义。

Evolutionary conserved regulators of stem cell properties as well as RNA-/DNA-binding proteins have been shown to influence hematopoietic stem cell-polarity and -division. Recently, we were able to characterize a polarity regulator and member of the Scribble polarity complex (lethal giant larvae homolog 1: Llgl1) and provided evidence for its role in regulation of HSC biology. Loss of Llgl1 resulted in competitive advantage of hematopoietic stem cells (HSC) and Llgl1 KO HSC revealed a higher degree of stress-resistance. Despite an increase in cell cycle, Llgl1 KO HSC did not exhaust. Moreover, low expression of Llgl1 in primary patient blasts was associated with dismal prognosis and decreased overall survival of patients with acute myeloid leukemia. In the proposed study we aim to investigate the impact of Scribble polarity complex on hematopoietic stem cells. We will characterize the functional relevance using a conditional knockout mouse model for the central complex member Scribble. The influence of its genetic inactivation in steady state hematopoiesis will be investigated as well as the impact on stem cell biology by serial bone marrow transplantation into recipient hosts. Cell cycle analysis will complement the functional characterization of hematopoietic stem cells. In a second step, we aim to clarify the role of both Scribble complex members (Llgl1 and Scribble) in regulation of HSC polarity and bone marrow-niche interaction. To address these questions we will use time-lapse microscopy, analysis of triggered and spontaneous cell polarization on fibronectin coated surfaces as well as in-vivo 2D microscopy of the bone marrow. Intracellular interactions of Scribble complex members will be analyzed by global gene-expression profiling (on a transcriptional level using Scribble knockout versus wildtype HSC) and by mass-spectrometry (for protein-protein interactions). The proposed experiments will allow functional and structural characterization of Scribble polarity complex in hematopoietic stem cells. This will deepen our knowledge on regulation of stem cell biology and -fate through the polarity machinery. We and others already provided first evidence for a role for polarity complex members in pre-leukemic transformation of hematopoietic stem cells and their prognostic relevance. This may lead to direct therapeutic implications for targeting leukemic stem cells through interference with the polarity network.