Roles of motor proteins in cerebellar Purkinje neuron biology

树突棘是神经元树突表面的小的、富含肌动蛋白的突起，作为兴奋性突触输入和记忆形成的位点。 以前的研究表明，肌球蛋白II，传统的非肌肉肌球蛋白，在脊柱的组织和功能。 在这里，我们探讨了小脑浦肯野神经元（PN）的肌球蛋白18 A（M18 A）-α和M18 A-β，两个剪接异构体的M18 A，最近发现的肌球蛋白超家族的成员，是类似的，但不同的，II类肌球蛋白。 这些区别包括在两种剪接亚型中的C-末端PDZ配体基序，以及在M18 A-α的情况下，具有明显的ATP非依赖性肌动蛋白结合位点的300个残基的N-末端延伸、富含Lys和Glu的区域（KE区域）和PDZ结构域。M18 A-β缺乏这种N-末端延伸的事实表明这两种亚型的不同功能。 使用GFP标记的M18 A-α和β的版本和一种新的基因转导系统，我们发现，M18 A-α，但不是M18 A-β，本地化显着的树突棘的PN在解离培养。 此外，M18 A-α的棘分布与F-肌动蛋白的棘分布广泛重叠，如使用F-肌动蛋白的新报告子（F-Tractin）所揭示的。 使用高度特异性的抗M18 A抗体，通过小脑组织切片的免疫电镜证实了M18 A-α的突触后棘定位。 重要的是，M18 A-a本身具有广泛靶向棘的N-末端延伸，认为它在很大程度上（如果不是完全的话）负责肌球蛋白棘定位。 功能阻断点突变显示M18 A-alpha N-末端延伸内的KE区域和PDZ结构域不决定脊靶向。 相反，ATP-非依赖性肌动蛋白结合位点与肌动蛋白丝的相互作用似乎驱动了M18 A-α的脊柱定位。 一致的是，在体外结合试验中使用M18 A-α的N-末端延伸表明，它结合F-肌动蛋白具有中等高的亲和力（305 - 105 nM）。 我们认为，M18 A-alpha可能在PN棘的发育、组织和/或功能中发挥重要作用。 未来的实验使用条件M18 A基因敲除小鼠，我们已经成功地创建，结合PN特异性表达的Cre重组酶，将使我们能够解决肌球蛋白的棘定位在PN的生理意义。

Dendritic spines are small, actin-rich protrusions on the surface of neuronal dendrites that serve as sites of excitatory synaptic input and memory formation. Previous studies have implicated myosin II, the conventional non-muscle myosin, in spine organization and function. Here we have explored the localization within cerebellar Purkinje neurons (PNs) of myosin 18A (M18A)-alpha and M18A-beta, two spliced isoforms of M18A, a recently discovered member of the myosin super family that is similar to, and yet distinct from, class II myosins. These distinctions include a C-terminal PDZ-ligand motif in both spliced isoforms, and in the case of M18A-alpha, a 300-residue N-terminal extension that harbors an apparent ATP-independent actin-binding site, a Lys- and Glu-rich region (KE region), and a PDZ domain. The fact that M18A-beta lacks this N-terminal extension suggests distinct functions for these two isoforms. Using GFP-tagged versions of M18A-alpha and beta and a novel system for gene transduction, we find that M18A-alpha, but not M18A-beta, localizes dramatically to the dendritic spines of PNs in dissociated culture. Moreover, M18A-alphas spine distribution overlaps extensively with that of F-actin, as revealed using a novel reporter for F-actin (F-Tractin). The postsynaptic, spine localization of M18A-alpha was confirmed by immuno-EM of cerebellar tissue sections using a highly specific anti-M18A antibody. Importantly, M18A-alphas N-terminal extension by itself targets extensively to spines, arguing that it is largely if not entirely responsible for the myosins spine localization. Function-blocking point mutations show that the KE region and the PDZ domain within M18A-alphas N-terminal extension do not determine spine targeting. Rather, the interaction of the ATP-independent actin-binding site with actin filaments appears to drive the spine localization of M18A-alpha. Consistently, in vitro binding assays using M18A-alphas N-terminal extension show that it binds F-actin with moderately high affinity (305 105 nM). We suggest that M18A-alpha may play important roles in the development, organization, and/or function of PN spines. Future experiments using a conditional M18A knockout mouse, which we have successfully created, in combination with the PN-specific expression of Cre recombinase, will alow us to address the physiological significance of the myosin's spine localization in PNs.