Roles of motor proteins in cerebellar Purkinje neuron biology

运动蛋白在小脑浦肯野神经元生物学中的作用

• 批准号: 9157427
• 负责人: JOHN A HAMMER
• 金额: $ 52.5万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157427
• 关键词: Adolescent; Affinity Chromatography; Alleles; Ataxia; Binding; Biological Models; Biology; CMV promoter; Calcium; Cell physiology; Cells; Cerebellum; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Color; Cytoskeleton; DNA; Data; Dendritic Spines; Embryo; Endoplasmic Reticulum; Epitopes; Exhibits; Functional Imaging; Gelatin; Generations; Genes; Goals; ITPR1 gene; Immunoglobulin G; Immunoprecipitation; Initiator Codon; Knockout Mice; Length; Life; Link; Lysine; MYO5A gene; Mediating; Melanosomes; Messenger RNA; Methods; Molecular; Morphology; Motor; Mus; Myosin ATPase; Myosin Type V; Neurons; Null Lymphocytes; Organelles; Phosphotransferases; Play; Protein Binding Domain; Proteins; Recruitment Activity; Reporting; Role; Sampling; Site; Smooth Endoplasmic Reticulum; Source; Stem cells; Structure of purkinje fibers; Structure-Activity Relationship; Synapses; Synaptic plasticity; Techniques; Technology; Vertebral column; Work; Yeasts; base; calbindin-D28K; cellular imaging; density; embryonic stem cell; homologous recombination; human ITPR1 protein; in vivo; knockout animal; melanocyte; monolayer; mouse model; novel; phospholipase C beta; postnatal; promoter; receptor; tool; yeast two hybrid system

While the use of embryonic mixed primary cerebellar cultures has proven valuable for dissecting structure: function relationships in Purkinje Neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the available methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using specific extrinsic factors, we successfully differentiated mESCs into PNs without the use of a postnatal feeder-layer. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, PLCb4 and GRID2, all of which are PN-specific markers. Finally, using collagen, poly-l-lysine and gelatin as the extra cellular matrix allowed us to grow mESC-derived PNs in monolayers, which is crucial for live cell imaging. Current efforts are focused on expressing exogenous DNAs specifically in mESC-derived PNs using the PCP2/L7 PN-specific promoter. If this is successful, we will then attempt gene editing in stem cells using CRISPR, followed by complementation using exogenous DNA. Together, this technology would provide a scalable, high-throughput method for dissecting specific molecular mechanisms in PNs, especially when a KO mouse is not available. Myosin Va, a class V processive motor, transports tubules of smooth endoplasmic reticulum (SER) into the dendritic spines of cerebellar Purkinje neurons (PNs). These SER tubules provide the source of calcium downstream of mGluR1 activation that drives synaptic plasticity at parallel fiber: PN synapses. Calcium release from SER tubules is mediated by the IP3 receptor (type 1 inositol 1,4,5-trisphosphate receptor or IP3R1), a resident SER protein. The focus of this study was to identify the receptor on the surface of the SER for myosin Va. A yeast two-hybrid screen and immunoprecipitation analyses identified two potential receptor components that interact with both myosin Va and IP3R1: phospholipase C beta 4 (PLCβ4) and receptor-for-activated-kinase 1 (RACK1). Initial work has shown that both PLCβ4 and RACK1 are enriched in dendritic spines, and live cell imaging has shown that RACK1, like myosin Va, localizes at the tip of SER tubules that are moving into spines. Furthermore, the dendritic spines of PNs from IP3R1-null mice are devoid of SER, arguing that IP3R1 is required for the myosin Va-dependent translocation of SER into spines. Finally, the enrichment of PLCβ4 and RACK1 in dendritic spines is disrupted in both myosin Va knockdown cells and IP3R1-null cells. Together, these data suggest that myosin Va may be recruited to the SER via the IP3 receptor, with PLCβ4 and/or RACK1 providing a bridge linking the myosin to the receptor. Myosin Va, a processive class V motor, is involved in the transport of diverse cargos, including melanosomes, endoplasmic reticulum (ER), and mRNAs. The creation of tools to identify proteins that interact with myosin Va should increase our understanding of the cellular processes supported by this myosin. Towards that end, we report here the generation of a tandem affinity purification (TAP) tag knockin mouse at the MYO5A locus. A recombineering-based approach was used to insert via homologous recombination a TAP-tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and the FLAG epitope tag into MYO5A locus immediately after the initiation codon. Mice homozygous for the knockin allele, which express the TAP-tagged version of myosin Va (TAP-MyoVa) exclusively and under the control of the endogenous MYO5A promoter, exhibit normal coat color and no evidence of ataxia, arguing that TAP-MyoVa functions normally. Consistently, the dendritic spines of Purkinje neurons isolated from this mouse are fully loaded with ER, in contrast to the spines of Purkinje neurons from dilute (myosin Va null) mice, which are devoid of ER. Similarly, melanosomes are distributed normally in melanocytes from the TAP-tagged myosin Va mouse, in contrast to melanocytes from dilute mice, where the organelles are concentrated in the cell center. Moreover, introduction of a CMV promoter-driven TAP-Tag myosin Va construct into dilute melanocytes rescues melanosome distribution. Given this clear evidence that TAP-MyoVa is fully functional, we purified TAP-MyoVa and associated proteins directly from juvenile mouse cerebella excised from TAP-tagged mice and subjected the samples to mass spectroscopic analyses. Importantly, elutes contained several known myosin Va binding partners (list), further verifying that TAP-MyoVa is fully functional. Moreover, we found numerous novel interacting proteins. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this myosin in vivo.

虽然使用胚胎混合原发小脑培养物已证明对解剖结构很有价值：Purkinje神经元（PNS）中的功能关系，但该技术在技术上具有挑战性，通常会产生很少的细胞。最近，尽管可用的方法也非常具有挑战性，但小鼠胚胎干细胞（MESC）已成功地分化为PN。这项研究的重点是简化MESC的分化为PN。使用特定的外部因素，我们在不使用产后喂食器层的情况下成功将MESC分化为PN。 MESC衍生的PNS的形态与在原发性培养中生长的PNS在总形态，脊柱长度和脊柱密度方面没有区别。此外，MESC衍生的PNS Express Calbindin D28K，IP3R1，PLCB4和Grid2，所有这些都是PN特异性标记。最后，使用胶原蛋白，聚-L-赖氨酸和明胶作为额外的细胞基质，使我们能够在单层中生长MESC衍生的PN，这对于活细胞成像至关重要。当前的努力专注于使用PCP2/L7 PN特异性启动子在MESC衍生的PN中专门表达外源DNA。如果这是成功的，我们将尝试使用CRISPR在干细胞中进行基因编辑，然后使用外源性DNA进行互补。总之，这项技术将提供一种可扩展的高通量方法，用于解剖PNS中的特定分子机制，尤其是在没有KO小鼠的情况下。 肌球蛋白VA是一种V级过程，将光滑的内质网（SER）的小管转运到小脑Purkinje神经元（PNS）的树突状棘中。这些Ser小管提供了MGLUR1激活下游钙的来源，该钙激活在平行纤维下驱动突触可塑性：PN突触。从SER小管中释放钙是由IP3受体（1型肌醇1,4,5-三磷酸受体或IP3R1）介导的，它是一种驻留的Ser蛋白。这项研究的重点是鉴定肌球蛋白VA的SER表面的受体。酵母的两杂化筛选和免疫沉淀分析鉴定了两个潜在的受体成分，它们与肌球蛋白VA和IP3R1和IP3R1：磷脂酶Cβ4（PLCβ4）（PLCβ4）（PLCβ4）和受体 - 型 - 激活-Activate-Activativated-Kinase 1（Rack1（Rack1））相互作用。最初的工作表明，PLCβ4和RACK1都富含树突状刺，活细胞成像表明，像肌球蛋白VA一样，RACK1位于移动到脊柱的Ser小管的尖端上。此外，IP3R1-NULL小鼠PNS的树突状刺没有SER，认为肌球蛋白VA依赖性SER依赖于SER需要IP3R1。最后，在肌球蛋白VA敲低细胞和IP3R1无效细胞中，PLCβ4和RACK1在树突状棘中的富集被破坏。总之，这些数据表明，肌球蛋白VA可以通过IP3受体募集到SER，PLCβ4和/或RACK1提供将肌球蛋白与受体联系起来的桥。 肌球蛋白VA是一种造成的V级电动机，参与了包括黑色素体，内质网（ER）和mRNA在内的各种cargos的运输。 创建识别与肌球蛋白VA相互作用的蛋白质的工具应增加我们对这种肌球蛋白支持的细胞过程的理解。 为此，我们在这里报告了在Myo5a基因座上的串联亲和力净化（TAP）标记型鼠标。 一种基于重组的方法用于通过同源重组插入由蛋白A的IgG结合结构域，TEV裂解位点和FLAG表位标签组成的TAP-TAG，并在发起密码子后立即进入MyO5A基因座。 纯合子的纯合子的小鼠专门表达了肌球蛋白VA（Tap-myova）的TAP标签版本，并且在内源性myo5a启动子的控制下，表现出正常的涂层颜色，并且没有共济失调的证据，认为Tap-Myova功能正常。 一致地，与没有ER的稀释（肌球蛋白va null）小鼠的Purkinje神经元的刺相比，从该小鼠中分离出的Purkinje神经元的树突状刺。 类似地，与来自稀释的小鼠的黑素细胞相比，黑色素体通常分布在带有Tag标记的肌球蛋白VA小鼠的黑素细胞中，而黑色素细胞在细胞中浓缩了细胞中心。 此外，将CMV启动子驱动的TAP-TAG肌球蛋白VA构建体引入稀黑素细胞中挽救了黑色素体分布。 鉴于这种明确的证据表明Tap-Myova具有完全功能性，我们直接从幼体小鼠中纯化了Tap-myova和相关的蛋白质，并从tap的小鼠中切除，并将样品对质谱分析进行。 重要的是，洗脱液中包含几个已知的肌球蛋白VA结合伙伴（列表），进一步验证了Tap-Myova功能完全正常。 此外，我们发现了许多新颖的相互作用蛋白。 这里创建的鼠标模型应促进识别新型肌球蛋白VA结合伙伴，这反过来应该促进我们对这种肌球蛋白在体内扮演的角色的理解。