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因子以及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，我们能够表征极性调节剂和Scribble极性复合体的成员（致命巨型幼虫同源物1:Llgl1），并为其在HSC生物学调节中的作用提供证据。Llgl1的缺失导致造血干细胞（HSC）的竞争优势，Llgl1 KO HSC显示出更高程度的抗逆性。尽管细胞周期增加，但Llgl1 KO HSC并未耗尽。此外，原发性患者原细胞中Llgl1的低表达与急性髓系白血病患者的预后差和总生存期降低有关。本研究旨在探讨Scribble极性复合物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。我们将研究其基因失活对稳态造血的影响，以及通过骨髓连续移植到受体宿主中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表征。在第二步，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，分析纤维连接蛋白包被表面上的触发和自发细胞极化，以及骨髓的体内二维显微镜。Scribble复合体成员的细胞内相互作用将通过全局基因表达谱分析（在转录水平上使用Scribble敲除与野生型HSC）和质谱分析（用于蛋白质-蛋白质相互作用）进行分析。提出的实验将允许在造血干细胞Scribble极性复合物的功能和结构表征。这将加深我们对通过极性机制调控干细胞生物学和命运的认识。我们和其他人已经提供了极性复合物成员在造血干细胞白血病前期转化中的作用及其预后相关性的第一个证据。这可能导致通过干扰极性网络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.