Mechanisms of axon guidance during development

我们在实验室中使用的实验系统是通过受体Notch调节Abl酪氨酸激酶信号网络来调节轴突生长和引导。在过去的一年里，我们发表了两篇论文，极大地促进了我们对这种分子机器的理解。 首先，作为分析Notch在细胞形态发生中作用的推论，有必要了解细胞形态发生和细胞骨架如何调节Notch。在Hunter等人中，我们发现肌动球蛋白通过两种方式调节Notch信号转导机制。首先，它直接参与产生拉力，在与其配体Delta结合时拉伸Notch胞外域，从而暴露细胞外蛋白酶裂解部位，并启动Notch受体激活的蛋白水解级联反应。其次，我们发现(出乎意料的)Notch的激活对相关质膜的刚性非常敏感：如果没有一个坚硬的表面来拉动，Delta内吞显然不能产生足够的力量来暴露Notch蛋白酶裂解部位，从而启动信号传递。这很可能是最近结果背后的分子机制之一，该结果表明，在选择干细胞自我更新或分化时，干细胞生态位的局部膜刚性。 第二，我们更直接地攻击了Notch-Abl信号通路的分子性质，特别是它的启动事件与典型的核Notch信号的异同。在Kannan等人中，我们发现激活Notch-Abl信号需要与经典的Notch信号完全相同的蛋白水解级联：正是相同的Notch切割事件将Notch胞内域从膜释放到细胞核以构建转录反式激活复合体，也诱导Notch与Abl网络组件的膜拴复合体的分解，从而通过激活Notch-CSL核信号的相同机制局部失活Abl信号。这两台机器的不同之处在于，Notch-Abl复合体使用了一小部分专门的Notch蛋白，这种蛋白在膜旁的酪氨酸残基上被磷酸化。这为我们提供了Notch蛋白分子子集上的特定生化处理，这些分子通过Abl局部向细胞骨架发出信号，此外还揭示了Notch局部抑制Abl网络信号的分子机制。 在对Notch本身进行分子研究的同时，我们还对依赖Notch的轴突--果蝇翅膀的TSM1神经元--的生长和引导机制进行了实时成像分析。在我们在BioRxiv上发表的一篇论文(目前正在其他地方进行审查)中，我们对影响轴突生长的肌动蛋白动力学进行了详细的、高分辨率的分析，分析了它们与生长锥运动的关系，以及Abl网络信号对它们的调节。我们已经证明，肌动蛋白的组织是ABL的关键靶点，而肌动蛋白又通过局部控制细丝动力学来控制轴突的生长。特别是，在体内，TSM1的远端轴突维持着肌动蛋白的局部积聚。肌动蛋白的质量经历了内在的、随机的大小波动，但Abl(可能是对外部引导信号的反应)将空间偏差引入这些波动。这会导致肌动蛋白质量沿着确定的轨迹前进，在这样做的同时，获得肌动蛋白密度的区域也获得了促进丝足延伸的能力，而失去肌动蛋白的区域则失去了维持丝足的能力，从而转化为平滑、明确的轴突干。在这个过程中，轴突会自动延长，并沿着定义的轨迹进行。我们目前正在扩展这些研究，调查其他信号分子的贡献，包括ABL网络的其他组件，对肌动蛋白动力学进行详细的计算模拟以剖析支撑这一细胞生物学的分子事件，并直接调查Notch激活对我们在发育中的生长锥中成像的细胞骨架事件的影响。

The experimental system we use in our lab is regulation of axon growth and guidance by the receptor Notch, via its regulation of the Abl tyrosine kinase signaling network. In the past year, we published two papers that significantly advance our understanding of this molecular machine. First, as a corollary to dissecting the role of Notch in cell morphogenesis, it became necessary to understand how cell morphogenesis and the cytoskeleton modulate Notch. In Hunter, et al., we found that actomyosin regulates the mechanism of Notch signaling in two ways. First, it is directly involved in generating the pulling force that stretches the Notch extracellular domain upon binding its ligand Delta, thereby exposing an extracellular protease cleavage site and initiating the proteolytic cascade of Notch receptor activation. Second, we found (unexpectedly) that activation of Notch is profoundly sensitive to the rigidity of the associated plasma membrane: without a rigid surface against which to pull, Delta endocytosis evidently cannot produce sufficient force to expose the Notch protease cleavage site and thereby initiate signaling. This is likely to be one of the molecular mechanisms underlying recent results implicating local membrane rigidity of the stem cell niche in the choice of stem cells to self-renew or differentiate. Second, we attacked more directly the molecular nature of the Notch-Abl signaling pathway, and in particular the similarities and differences of its initiating events with those of canonical, nuclear Notch signaling. In Kannan, et al., we found that activation of Notch-Abl signaling requires precisely the same proteolytic cascade as does canonical Notch signaling: the very same Notch cleavage event that releases the Notch intracellular domain from the membrane to transit to the nucleus to build a transcriptional transactivating complex, also induces disassembly of membrane-tethered complexes of Notch with components of the Abl network, thereby locally inactivating Abl signaling by the same mechanism that activates Notch-CSL nuclear signaling. What distinguishes these two machines is that Notch-Abl complexes employ a small, specialized population of Notch protein that is phosphorylated on juxtamembrane tyrosine residues. This provides us with a specific biochemical handle on that subset of Notch protein molecules that signal locally to the cytoskeleton via Abl, in addition to revealing the molecular mechanism by which Notch locally suppresses Abl network signaling. In parallel to these molecular studies of Notch itself, we have also advanced our live-imaging analysis of the mechanism of growth and guidance of a Notch-dependent axon, the TSM1 neuron of the Drosophila wing. In a paper that we have released on bioRxiv (and that is currently under review elsewhere), we have performed a detailed, high resolution analysis of the actin dynamics that underly axon growth, their relationship to growth cone motility, and their regulation by Abl network signaling. What we have shown is that actin organization is the key target of Abl, and that actin, in turn, controls axon growth by locally controlling filopodial dynamics. In particular, in vivo, the distal axon of TSM1 maintains a local accumulation of actin. This mass of actin undergoes intrinsic, stochastic fluctuations in size, but Abl (presumably in response to external guidance signals) introduces a spatial bias into those fluctuations. This causes the actin mass to advance along a defined trajectory, and as it does, regions that gain actin density also gain the ability to promote filopodial extension, while regions that lose their actin lose the ability to maintain filopodia and thus convert to smooth, definitive, axon shaft. In the process, the axon automatically lengthens, and does so along a defined trajectory. We are currently extending these studies by investigating the contributions of other signaling molecules, including other components of the Abl network, performing detailed computational simulations of actin dynamics to dissect the molecular events that underly this cell biology, and investigating directly the effect of Notch activation on the cytoskeletal events we have been imaging in the developing growth cone.