Dissecting the coupling of cell polarity and the stem cell cycle by chemical genetics

很明显，我们身体组织中干细胞的正确功能与这些组织发生恶性肿瘤的风险密切相关。在一些组织中，干细胞已被确定为癌症的起源细胞。因此，重要的是揭示保护干细胞不被驱使进入异常增殖的机制，以便能够预防、控制或最小化它们发生故障时的影响。干细胞的功能是维持组织，并通过不对称分裂来实现。这种分裂的结果是一个自我更新的干细胞和一个将分化的子细胞。并非所有干细胞都以这种方式分裂，但在许多干细胞中，一组进化上保守的分子（如PAR复合体）起作用，允许它们进行不对称分裂，并将命运信息传递给将分化的子细胞。一种旨在解释干细胞在癌症中的作用的假设认为，不对称干细胞分裂失败会导致错误的细胞，由于它们对正常的控制机制没有反应，因此无法控制。本研究旨在解决控制不对称干细胞分裂的分子机制，以防止这种情况的发生。PAR复合体的作用和细胞极性的建立是一个深入研究的主题，但主要集中在基本上不分裂的细胞，如上皮细胞、神经元或迁移细胞。在许多哺乳动物模型中，没有歧义地挑选出干细胞并对其进行实验操作的困难阻碍了理解极性在许多相关干细胞中的确切作用。此外，干细胞具有持续增殖的能力。在细胞分裂的背景下，通过标准的遗传分析（如使用突变体或敲低信使RNA）来研究极性，无法解决这一过程的动力学，因为它不提供时间控制。这一建议旨在解决核扩散与极性之间的联系。果蝇神经干细胞，被称为神经母细胞，来自一种遗传上易于处理的生物体，具有非常短的细胞周期，可以很容易地识别，易于活细胞成像，并且它们的不对称性受到诸如PAR复合物的保守蛋白质的控制。我们已经在果蝇中开发了化学遗传学，可以对激酶的活性进行特异性和急性抑制。我们将使用这种方法来具体解决细胞周期机制如何调节模型系统干细胞中的细胞极性。以细胞周期依赖的方式驱动干细胞极性的上游信号尚未得到充分研究。我们的方法首次允许暂时剖析关键激酶在干细胞极性，不对称性和细胞命运决定中的信号传导作用。一旦PAR复合体在有丝分裂的神经母细胞中实现极化定位，它就会驱动分子的不对称定位，如NOTCH信号调节因子NUMB，这些分子被完全分离到子细胞中，在那里它们指导分化。因此，命运决定因素的不对称定位也受到细胞周期的控制。这不仅适用于神经母细胞，也适用于其他哺乳动物干细胞，如小鼠乳腺干细胞、放射状胶质细胞和造血干细胞。干细胞允许像NUMB这样的分子不对称定位的分子机制是非常不清楚的。我们假设肌动球蛋白网络提供了这种模式信息。我们的初步结果进一步支持了这样一种观点，即命运决定因素的翻译后修饰是它们在肌动球蛋白皮层不对称保留所必需的。我们的目标是揭示在响应细胞周期依赖的线索中将磷酸化决定因素与干细胞皮层联系起来的分子。

It has become clear that the correct function of the stem cells in the tissues of our body are intimately linked to the risk of those tissues to develop malignancies. In some tissues stem cells have been identified as the cell-of-origin of cancer. It is therefore important to reveal the mechanisms that protect stem cells from being driven into aberrant proliferation to be able to prevent, control or minimize the impact if they malfunction. Stem cells function to maintain tissues and can do this through asymmetric division. The outcome of such a division is a self-renewed stem cell and a daughter cell that will differentiate. Not all stem cells divide in this way, but in many that do, a set of evolutionarily conserved molecules operates, such as the PAR complex, allowing them to divide asymmetrically and to transmit fate information to the daughter cell that will differentiate. One hypothesis aiming at explaining the role of stem cells in the context of cancer states that failed asymmetric stem cell divisions results in mis-specified cells, that can't be kept in check as they do not respond to the control mechanisms normally in place. This proposal aims at addressing the molecular mechanisms that control asymmetric stem cell division to prevent this from happening. The role of the PAR complex and the establishment of cell polarity is the subject of intense research, which is focussed however on cells that are largely none dividing such as epithelial cells, neurons or migratory cells. The difficulty to single out stem cells without ambiguity and to experimentally manipulate them in many mammalian models has hindered understanding the precise role of polarity in many relevant stem cells. Moreover, stem cells have the ability to continuously proliferate. Studying polarity in the context of cell division by standard genetic analysis such as the use of mutants or knockdown of the messenger RNA is unable to resolve the dynamics of the process as it does not provide temporal control. This proposal aims at addressing the link between proliferation and polarity.Drosophila neural stem cells, called neuroblasts, come from a genetically tractable organism, have a very short cell cycle, can be readily identified, are amenable to live cell imaging and their asymmetry is under the control of conserved proteins such as those of the PAR complex. We have developed chemical genetics in the fly that allow specific and acute inhibition of the activity of kinases. We will use this approach to specifically address how the cell cycle machinery regulates cell polarity in a model system stem cell. The upstream signals that drive stem cell polarity in a cell cycle dependent manner are understudied. Our approaches allow for the first time to temporally dissect the role of signalling by critical kinases on stem cell polarity, asymmetry and cell fate determination. Once the PAR complex achieves a polarized localization in neuroblasts in mitosis, it drives the asymmetric localization of molecules such as the NOTCH signalling regulator NUMB, that are exclusively segregated to daughter cells, where they instruct differentiation. Therefore, the asymmetric localization of fate determinants is also under cell cycle control. This is true for neuroblast, but also for other mammalian stem cells such those of the murine mammary gland, radial glia and haematopoietic stem cells. The molecular mechanism that patterns stem cells allowing the asymmetric localization of molecules such as NUMB are highly unclear. We hypothesise that the actomyosin network provides this patterning information. Our preliminary results further support the idea that posttranslational modification of fate determinants is required for their asymmetric retention at the actomyosin cortex. We aim at revealing the molecules that link phosphorylated determinants to the stem cell cortex in response to cell cycle dependent cues.