Roles of motor proteins in cerebellar Purkinje neuron biology

肌球蛋白18 A是一种肌球蛋白2样蛋白，含有独特的N-和C-末端蛋白质相互作用结构域，与肌球蛋白2共组装。已知与肌球蛋白18 A结合的一种蛋白质是-Pix，它是Rac 1和Cdc 42的鸟嘌呤核苷酸交换因子（GEF），已显示通过激活肌动蛋白和肌球蛋白丝在树突棘中的组装来促进树突棘成熟。在这里，我们表明，肌球蛋白18 A集中在小脑浦肯野神经元的棘通过与肌球蛋白2的共组装，并通过在其N-末端延伸的肌动蛋白结合位点。miRNA介导的肌球蛋白18 A的敲低导致棘成熟中的显著缺陷，其由肌球蛋白18 A的RNAi免疫版本挽救。重要的是，-Pix与肌球蛋白18 A共同定位在棘中，并且其棘定位在肌球蛋白18 A敲低或其肌球蛋白18 A结合位点缺失时丢失。最后，我们发现，肌球蛋白18 A敲低浦肯野神经元的棘含有显着减少F-肌动蛋白和肌球蛋白2。总之，这些数据表明，肌球蛋白2和肌球蛋白18 A的混合丝形成一个复杂的-Pix在浦肯野神经元棘，促进脊柱成熟的肌动蛋白和肌球蛋白丝下游的-Pixs GEF活动的组装。 肌球蛋白X（Myosin X，MX）是一种高度保守的脊椎动物特异性非常规肌球蛋白，其尾部结构域包含PIP 3特异性PH结构域、微管结合MyTH 4结构域和整合素结合FERM结构域。MX主要与丝状伪足（通常称为丝状伪足肌球蛋白）的形成和维持、整合素在质膜中的转运以及有丝分裂和减数分裂纺锤体的正确定位有关。有趣的是，神经元表达全长MX（FL-MX）和无头版本（Hdl-MX），并且两者似乎在放射状胶质细胞迁移的不同方面发挥作用（这导致新皮层中的大多数神经元和胶质细胞）。MX还参与netrin-1受体DCC向神经突尖端的转运，从而调节轴突寻路。在我们过去的努力，以确定浦肯野神经元（PN），小脑的主神经元的另一种非传统的肌球蛋白（肌球蛋白Va）的功能，我们开发了新的工具来研究这个复杂的神经元。有趣的是，PN表达的MX水平比其他CNS神经元高得多。此外，PNs是独特的，在没有神经支配的情况下进行丝状伪足到树突棘的转换，也许是因为它们表达高水平的丝状伪足肌球蛋白。为了开始研究MX在PN中的功能，我们在发育PN中表达了GFP标记的FL-MX。时间推移成像显示，MX定位于树突丝状伪足的尖端，然后沿着这些高度运动的树突丝状伪足移动，直到它定位于树突棘。为了扩展这些观察结果，我们创建了一种针对FL-MX和Hdl-MX的MX条件性敲除（cKO）小鼠。全身MX KO显示出部分胚胎致死性，存活的小鼠表现出各种缺陷，包括小尺寸、融合的趾和白色腹部斑点。胚胎表型包括露脑畸形和严重发育缺陷。这些数据表明，MX是小鼠胚胎发生的关键，它可能在神经管闭合中起着关键作用。为了获得MX在PN中的特异性作用，我们将MX cKO小鼠与L7-PCP cre小鼠杂交，L7-PCP cre小鼠在PN中特异性表达cre重组酶。获得的小鼠将接受各种测试，从测量动物协调性，到在原位、切片和培养中评估PN结构和功能。 总之，这些方法应该揭示MX功能在这个复杂神经元中的关键方面。

Myosin 18A is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18A is -Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here we show that myosin 18A concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A. Importantly, -Pix co-localizes with myosin 18A in spines, and its spine localization is lost upon myosin 18A knockdown or when its myosin 18A binding site is deleted. Finally, we show that the spines of myosin 18A knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A form a complex with -Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of -Pixs GEF activity. Myosin X (MX) is a highly conserved, vertebrate-specific unconventional myosin whose tail domain contains a PIP3-specfic PH domain, a microtubule-binding MyTH4 domain, and an integrin-binding FERM domain. MX has been linked primarily to the formation and maintenance of filopodia (it is commonly referred to as the filopodial myosin), the transport of integrins in the plasma membrane, and the proper positioning of mitotic and meiotic spindles. Interestingly, neurons express both full length MX (FL-MX) and a headless version (Hdl-MX), and both appear to function in different aspects of radial glia migration (which give rise to most neurons and glia in the neocortex). MX has also been implicated in the transport of the netrin-1 receptor DCC to the tips of neurites, thereby regulating axonal path-finding. In our past efforts to define the function of another unconventional myosin (myosin Va) in Purkinje neurons (PN), the master neuron of the cerebellum, we developed novel tools to study this complex neuron. Interestingly, PNs express much higher levels of MX than other CNS neurons. Moreover, PNs are unique in undergoing filopodia-to-dendritic spine conversion without innervation, perhaps because they express high levels of this filopodial myosin. To begin to address the function of MX in PNs, we expressed GFP-tagged FL-MX in developing PNs. Time lapse imaging showed that MX localizes to the tips of dendritic filopodia, and then moves along these highly motile dendritic filopodia until it localizes to dendritic spines. To extend these observations, we have created a MX conditional knockout (cKO) mouse that targets both FL-MX and Hdl-MX. The whole-body MX KO shows partial embryonic lethality, and mice that survive exhibit a variety of defects including small size, fused digits and white belly spotting. Embryonic phenotypes include exencephaly and gross developmental defects. These data demonstrate that MX is critical for mouse embryogenesis, and that it probably plays a pivotal role in neural tube closure. To access the role of MX specifically within PNs, we are crossing our MX cKO mouse with L7-PCP cre mice, which express cre recombinase specifically in PNs. The mice obtained will be subjected to a variety of tests, from measuring animal coordination, to accessing PN structure and function in situ, in slices, and in culture. Together, these approaches should reveal the critical aspects of MX function in this complex neuron.