Mechanisms of axon guidance during development

Abl酪氨酸激酶是轴突生长的主要调节剂，并指导整个动物王国。它是肌动蛋白结构和动力学的关键调节因子，也是轴突生长锥中许多导向因子受体信号整合的关键点。正如我们在今年连续发表的两篇论文中所报道的那样，我们现在可以追踪Abl控制肌动蛋白结构的机制，从而追踪生长锥运动，一直追溯到它在肌动蛋白生物物理学中的根源。简而言之，我们之前的遗传学和生物化学表明，Abl控制线性肌动蛋白聚合与肌动蛋白分支的比率（通过其对肌动蛋白聚合酶Enabled和Rac GEF Trio的拮抗调节）。现在，通过对体内生长锥中的肌动蛋白进行高分辨率、定量、实时成像，我们揭示了这种调节的意义。由Abl维持的中间水平的肌动蛋白分支允许生长锥核心中的大量肌动蛋白在长度上振荡，但仅在与维持肌动蛋白分布的秩序一致的狭窄范围内。引导信号受体的作用是局部有利于肌动蛋白的延伸或分支（分别），我们发现这引入了一个空间偏差的肌动蛋白的振荡，从而导致肌动蛋白质量前进沿着一个可预测的轨迹。反过来，肌动蛋白的运动有利于在轴突注定要延伸的地方组装丝状伪足，并在生长锥注定要空出的地方拆卸丝状伪足。随着时间的推移，该机制产生生长锥的动态核心沿着由引导线索限定的轨迹的净运动。这种分子模型在轴突生长和引导的文献中是完全没有先例的，但是除了解释我们自己的观察之外，它还可以解释各种肌动蛋白调节蛋白的作用，并且还为轴突引导机制中许多长期存在的谜团提供了简单的解释。 正在进行的实验正在对我们的模型进行进一步的测试，既通过对其他Abl途径组分的遗传操作后观察到的表型进行成像，又通过对改变肌动蛋白动力学的各种参数的后果进行详细的计算模拟。此外，我们与合作者一起（1）开发先进显微镜的新方法，提高空间和时间分辨率，我们可以监测活体样品中的生长锥结构和动态，（2）开发新的图像分析方法，以自动跟踪轴突图像，以及（3）研究Abl活性的不受控制的偏移对于神经退行性疾病背景下的神经回路的产生和维持的意义。

Abl tyrosine kinase is a master regulator of axon growth and guidance throughout the animal kingdom. It is a key regulator of actin structure and dynamics, and a key point of integration of signals from many guidance cue receptors in axonal growth cones. As we reported in two back-to-back papers this year, we can now trace the mechanism by which Abl controls actin structure, and thereby growth cone motility, all the way back to its roots in actin biophysics. In brief, our previous genetics and biochemistry showed that Abl controls the ratio of linear actin polymerization to actin branching (through its antagonistic regulation of the actin polymerase, Enabled, and of the Rac GEF, Trio). Now, by performing high-resolution, quantitative, live imaging of actin in a growth cone advancing in vivo, we have revealed the significance of this regulation. The intermediate level of actin branching maintained by Abl allows a mass of actin in the core of the growth cone to oscillate in length, but only within narrow limits consistent with maintenance of order in the actin distribution. The role of guidance cue receptors is to locally favor actin extension or branching (respectively), which we show introduces a spatial bias to the oscillations of actin, and thus causes the actin mass to advance along a predictable trajectory. Motion of the actin, in turn, favors filopodial assembly in places where the axon is destined to extend, and disassembly of filopodia in places the growth cone is destined to vacate. Over time, this mechanism produces net motion of the dynamic core of the growth cone along the trajectory defined by guidance cues. This molecular model is entirely without precedent in the literature of axon growth and guidance, but in addition to explaining our own observations, it can account for the effects of a wide range of actin modulatory proteins, and also provides simple explanations for numerous longstanding mysteries in the mechanism of axon guidance. Ongoing experiments are performing further tests of our model, both by imaging the phenotypes observed upon genetic manipulation of other Abl pathway components, and by generating detailed computational simulations of the consequences of altering various parameters of actin dynamics. In addition, together with collaborators we are (1) developing novel methods of advanced microscopy that increase the spatial and temporal resolution with which we can monitor growth cone structure and dynamics in live samples, (2) developing novel methods of image analysis to automate tracing of axon images, and (3) investigating the implications that uncontrolled excursions of Abl activity have for the production and maintenance of neural circuits in the context of neurodegenerative disease.