The fundamental roles of axonal actin during neuronal growth and longevity

轴突轴内的肌动蛋白细胞骨架长期以来一直被忽视，但超分辨率显微镜现在使其易于研究。在这里，我们将使用果蝇遗传学来剖析轴突肌动蛋白的作用，建立在有希望的初步数据的基础上，这些数据表明轴突生长和维持轴突MTs的重要功能。轴突是神经元的电缆状突起，连接神经系统，对其功能不可或缺。尽管轴突很重要，但人们对其形成和维持的基本机制仍知之甚少。重要的理解将来自于对肌动蛋白和微管（MT）细胞骨架的研究，这是轴突生长和维持所绝对需要的。肌动蛋白和MTs是丝状蛋白聚合物，它们排列成细胞内支架，维持细胞结构和调节细胞动力学。到目前为止，研究主要集中在神经元突出的细胞骨架网络上：首先，运动生长锥（GCs）中丰富的肌动蛋白网络在发育过程中指导轴突伸长；其次，形成轴突结构骨干的mt束，需要在生物体的一生中（如人类的几十年）建立和维持轴突。进一步的重要作用可能来自轴突轴中的肌动蛋白细胞骨架，但这种肌动蛋白很难可视化并且被广泛忽视。最近，小鼠神经元的超分辨率显微镜为轴突肌动蛋白的研究提供了精确的模板。这些研究揭示了肌动蛋白短丝束排列成周期性图案的环，围绕轴突MT束，理想地调节其动力学。值得注意的是，我们在果蝇的神经元上使用超分辨率显微镜时发现了类似的重复模式，这表明这些结构是进化保守的和功能相关的。我们对两类基因和药物操作的实验进一步支持了这种相关性：一类预计会影响轴突轴中的肌动蛋白，另一类维持肌动蛋白环，但两者都清楚地从gc中去除肌动蛋白。这些处理对轴突延伸有不同的影响，这有力地支持了轴突肌动蛋白具有促进生长作用的模型。轴突肌动蛋白的这种作用将为轴突生长模型引入新的机制概念，从而为解开神经生物学中尚未解决的基本问题提供了新的机会。此外，我们对两类肌动蛋白操作的实验表明，轴突肌动蛋白还有第二个作用，即维持轴突mt。因此，当mt通过特定的遗传操作不稳定时，额外去除轴突肌动蛋白会消除它们的增殖，轴突收缩并最终消失。这一令人惊讶的新发现同样开辟了新的机会，我们相信它不仅对轴突的生长，而且对轴突的退化和分支都有潜在的影响。为了将轴突肌动蛋白的试验数据转化为对轴突生物学的实质性理解，我们将利用果蝇神经元提供的独特遗传和实验机会，我们在这方面有10年的经验。因此，我们已经单独或联合研究了果蝇轴突生长和GC调节过程中的约40种肌动蛋白和MT调节因子。这为我们对本项目的研究提供了坚实而独特的知识基础。在这里，我们将1)使用超分辨率显微镜结合我们的肌动蛋白操作来功能性验证轴突肌动蛋白环模型，2)使用微流体室，精细的实时成像和牵引前显微镜来证明生长促进作用，3)通过关注肌动蛋白依赖的MT聚合和MT向前滑动来揭示潜在的机制，以及4)证明轴突肌动蛋白在体内的相关性。

The actin cytoskeleton within axon shafts has long been neglected, but supra-resolution microscopy has now made it amenable to investigation. Here we will use Drosophila genetics to dissect the roles of axonal actin, building on promising pilot data which suggest important functions in axon growth and in maintaining axonal MTs. Axons are the cable-like protrusions of neurons which electrically wire the nervous system and are indispensable for its function. In spite of their importance, the fundamental mechanisms which underpin the formation and maintenance of axons remain poorly understood. Important understanding will come from work on the actin and microtubule (MT) cytoskeleton which is absolutely required for the growth and maintenance of axons. Actin and MTs are filamentous protein polymers which arrange into intracellular scaffolds maintaining cell architecture and mediating cell dynamics. So far, research has primarily been focussed on the prominent cytoskeletal networks of neurons: firstly, abundant actin networks in motile growth cones (GCs) which guide axon elongation during development; secondly, bundles of MTs which form the structural backbones of axons and are required to establish and then maintain axons for an organism's lifetime (i.e. decades in humans). Further important roles are likely to come from the actin cytoskeleton in axon shafts, but this actin has been difficult to visualise and has been widely neglected. Recently, supra-resolution microscopy of mouse neurons delivered a precise template for studies of axonal actin. These studies revealed bundles of short actin filaments arranged into periodically patterned rings which surround the axonal MT bundles, ideal to regulate their dynamics. Notably, we find similar repetitive patterns when using supra-resolution microscopy on neurons of the fruitfly Drosophila, suggesting that these structures are evolutionary conserved and functionally relevant. Such relevance is further supported by our experiments with two classes of genetic and drug manipulations: one class is expected to affect actin in axon shafts, the other to maintain actin rings, but both clearly remove actin from GCs. These treatments have differential effects on axon extension which strongly support a model in which axonal actin has growth-promoting roles. Such a role of axonal actin would introduce novel mechanistic concepts into models of axon growth, thus providing new opportunities to unravel this still unresolved, fundamental problem in neurobiology. Furthermore, our experiments with the two classes of actin manipulations suggest that axonal actin has a second role, which is to maintain axonal MTs. Thus, when MTs are destabilised through specific genetic manipulation, additional removal of axonal actin eliminates their proliferation and axons retract and eventually vanish. This surprising and novel finding likewise opens up new opportunities, and we believe that it will have potential implications not only for axon growth but also for axon degeneration and branching. To turn our pilot data on axonal actin into substantial understanding of axon biology, we will capitalise on the unique genetic and experimental opportunities provided by fly neurons, for which we have 10 years of experience. Thus, we have already investigated ~40 actin and MT regulators of Drosophila, alone or in combinations, during axon growth and GC regulation. This provides us with a solid and unique knowledge base for the research on this project. Here, we will 1) use supra-resolution microscopy in combination with our actin manipulations to functionally validate the model of axonal actin rings, 2) proof the growth-promoting roles using micro-fluid chambers, refined live imaging and traction fore microscopy, 3) unravel the underlying mechanisms by focussing on actin-dependent MT polymerisation and forward sliding of MTs, and 4) demonstrate the relevance of axonal actin in vivo.