Roles of motor proteins in cerebellar Purkinje neuron biology

肌凝蛋白18A是一种肌凝蛋白2样蛋白，含有独特的N端和c端蛋白相互作用域，与肌凝蛋白2共同组装。已知与肌球蛋白18A结合的一种蛋白质是-Pix，一种针对Rac1和Cdc42的鸟嘌呤核苷酸交换因子（GEF），已被证明通过激活脊柱中肌动蛋白和肌球蛋白丝的组装来促进树突棘成熟。在这里，我们发现肌凝蛋白18A通过与肌凝蛋白2的共组装以及通过其n端延伸的肌动蛋白结合位点集中在小脑浦肯野神经元的脊柱中。mirna介导的myosin 18A的敲低导致脊柱成熟的显著缺陷，这是由rnai免疫版本的myosin 18A拯救的。重要的是，-Pix在脊柱中与肌凝蛋白18A共定位，当肌凝蛋白18A敲低或其肌凝蛋白18A结合位点被删除时，其脊柱定位丢失。最后，我们发现肌凝蛋白18A敲除的浦肯野神经元的棘含有显著减少的F-actin和肌凝蛋白2。总之，这些数据表明，肌凝蛋白2和肌凝蛋白18A的混合细丝在浦肯野神经元棘中与-Pix形成复合物，通过增强-Pix GEF活性下游的肌动蛋白和肌凝蛋白细丝的组装来促进脊柱成熟。

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.