Motor proteins and cytoskeletal dynamics in lymphocytes, melanocytes and neurons

淋巴细胞、黑素细胞和神经元中的运动蛋白和细胞骨架动力学

• 批准号: 9572304
• 负责人: JOHN A HAMMER
• 金额: $ 81.81万
• 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/9572304
• 关键词: ANGPTL2 gene; Actins; Actomyosin; Adherent Culture; Adolescent; Affinity; Affinity Chromatography; Antigens; B-Lymphocytes; BAI1 gene; Binding; CD8B1 gene; Cell Adhesion; Cell physiology; Cells; Charge; Chemicals; Cholesterol; Coculture Techniques; Complex; Contracts; Cyclic Nucleotides; Cytoplasmic Granules; Data; Defect; Dendritic Spines; Discrimination; Dissection; Distal; Dynein ATPase; Endoplasmic Reticulum; Exhibits; Extracellular Matrix; Feedback; Filament; Formulation; Frequencies; Generations; ITPR1 gene; Immunoglobulin G; Initiator Codon; Integrins; Knockout Mice; Length; Ligands; Light; Lighting; Lymphocyte; Lysosomes; MYO5A gene; Medial; Melanosomes; Membrane; Membrane Proteins; Methods; MicroRNAs; Microfilaments; Microscopy; Minus End of the Microtubule; Molecular; Morphology; Motor; Mus; Myelin Basic Proteins; Myosin ATPase; Myosin S-2; Myosin Type II; Neurons; Nonmuscle Myosin Type IIB; Nuclear Magnetic Resonance; Organelles; Ovalbumin; Peptides; Peripheral; Phosphorylation; Play; Point Mutation; Positioning Attribute; Protein Binding Domain; Proteins; Radial; Receptor Signaling; Recruitment Activity; Resolution; Role; Sampling; Side; Signal Transduction; Signaling Molecule; Site; Stem cells; Structure; Structure-Activity Relationship; Synapses; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Tyrosine; Vertebral column; base; calbindin-D28K; cell motility; density; dynein light chain; embryo tissue; embryonic stem cell; high resolution imaging; homologous recombination; immunological synapse; immunological synapse formation; improved; in vivo; keratinocyte; late endosome; mechanotransduction; melanocyte; monolayer; mouse model; nerve stem cell; neurofilament; novel; phosphoric diester hydrolase; promoter; relating to nervous system

Upon antigen recognition, actin assembly and inward flow in the plane of the radially symmetric immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin LFA-1. Using two forms of structured-illumination microscopy (3D-SIM and TIRF-SIM), we show that actin arcs populating the medial, lamella-like region of the IS (the pSMAC) arise from linear actin filaments generated by the formin mDia present at the distal edge of the IS. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS (the dSMAC), these linear filaments are organized by myosin II into concentric arcs that possess the anti-parallel organization required for contraction. Quantitative, fixed-cell 3D-SIM shows that open, active LFA-1 often aligns with arcs while TCR MCs commonly reside between arcs. Consistently, live-cell TIRF-SIM shows TCR MCs being swept inward by arcs. Disrupting actin arc function by blocking their formation via formin inhibition or their concentric organization and contraction via myosin II inhibition results in less centralized TCR MCs, miss-segregated integrin clusters, decreased T: B cell adhesion frequency, and diminished proximal TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport. Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS. Melanoregulin (Mreg), the product of the dilute suppressor locus, is a small, highly-charged, multiply-palmitoylated protein present on the limiting membrane of melanosomes. Previous studies have implicated Mreg in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these and related organelles by binding to RILP, a Rab7 effector that recruits the dynein motor complex. Here we shed new light on the possible molecular function of Mreg by solving its structure using nuclear magnetic resonance (NMR). The structure reveals bands of positive and negative charge that occupy opposite sides of the proteins surface, and that sandwich a putative, tyrosine-based (Y166) cholesterol recognition sequence (CRAC motif). We confirmed that cholesterol interacts with Mreg, as residues S163 and L168 within the CRAC motif show the largest NMR chemical shift upon cholesterol addition. Importantly, Mreg containing a function blocking point mutation within its CRAC motif (Y166I) still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Reversing the charge of three closely-spaced acidic residues (D177, E180, and D181) also inhibits Mregs ability to drive these organelles to microtubule minus ends, but only partially. We propose that cholesterol recognition alters Mregs orientation on the membrane in such a way as to allow it to interact with a component(s) involved in dynein recruitment (e.g. RILP), and that this interaction is further promoted by the acidic patch. Finally, we draw comparisons between Mreg and the protein ORP1L, which controls the microtubule minus end-directed transport, positioning and fate of late endosomes in part by recognizing both cholesterol and components that target dynein. The actin-based motor myosin Va transports numerous cargos, including the endoplasmic reticulum (ER) in cerebellar Purkinje neurons and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Towards that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of ER transport in Purkinje neurons and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it and associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va binding partners like dynein light chain and neurofilament light peptide were identified. Importantly, numerous novel interacting proteins were also identified, including 2',3'-cyclic-nucleotide 3'-phosphodiesterase, brain-specific angiogenesis inhibitor 1-associated protein 2, and myelin basic protein. 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. While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure: function relationships in cerebellar Purkinje Neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (ESCs) 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 mouse ESCs into PNs. Using a recently-described neural differentiation media, we generate monolayers of neural progenitor cells (NPs) from ESCs, which we differentiate into PN precursors using specific extrinsic factors. These precursors are then differentiated into mature PNs by co-culture with granule neuron (GN) precursors also derived from NPs using different extrinsic factors. The morphology of ESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, ESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLC4, PSD93 and myosin IIB-B2, all of which are PN-specific markers. Moreover, we show that ESC-derived PNs express proteins driven by the PN-specific promoter Pcp2/L7, form synapses with GNs as in primary cultures, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating ESCs into PNs should facilitate the dissection of molecular mechanisms in PNs.

抗原识别后，肌动蛋白组装和径向对称免疫突触（IS）的平面中的内部流动驱动T细胞受体微量群（TCR MCS）和整合素LFA-1的集中度。 使用两种形式的结构化 - 灌溉显微镜（3D-SIM和TIRF-SIM），我们表明肌动蛋白弧形填充了IS（PSMAC）的内侧，类似薄片的区域（PSMAC），是由由IS远端的formin MDIA产生的线性肌动蛋白细丝产生的。 在遍历IS（DSMAC）的外部ARP2/3产生的类似薄片的薄片样区域后，这些线性丝由肌球蛋白II组织成具有抗平行组织所需的抗平行组织的同心弧。 定量的固定细胞3D-SIM显示，开放的活性LFA-1通常与ARC对齐，而TCR MC通常位于弧之间。 一致地，活细胞TIRF-SIM显示TCR MC被ARC扫荡。 通过肌动蛋白抑制作用或同心组织阻止其形成，通过肌球蛋白II抑制作用来破坏肌动蛋白ARC的功能，导致较少集中的TCR MC，毫无原状的整合素簇，降低T：B细胞粘附频率，并降低近端TCR信号。 总之，我们的结果定义了直接推动TCR MC运输的主要肌动菌素收缩结构的起源，组织和功能意义。 机械转移是一种出现的，但仍然了解到T细胞激活的成分。 在这里，我们调查了散布于外周种超分子激活簇（PSMAC）免疫突触（IS）对T细胞受体（TCR）微量簇的微量簇运输和近端信号传导的收缩性肌动蛋白弧（PSMAC）区域所产生的配体依赖性贡献。 使用超级分辨率显微镜，OT1-CD8+小鼠T细胞和两个对TCR亲和力不同的椭圆蛋白（OVA）肽，我们表明，有组织的肌动蛋白肌球蛋白弧的产生取决于配体效应以及肌球蛋白2对肌动蛋白丝的能力。 尽管弱配体会诱导混乱的肌动蛋白弧，但强大的配体导致有组织的肌球蛋白弧与对张力敏感的CASL磷酸化良好相关，并且配体在IS IS中心的积累。 阻断肌球蛋白2的收缩力大大降低了强体和弱配体之间观察到的SRC和LAT磷酸化程度的差异，认为肌动蛋白弧中肌动蛋白2依赖性力产生有助于配体歧视。 总之，我们的数据与IS PSMAC区域中的肌动菌素弧促进了一种机械化学反馈机制的观点一致，该机制可以扩增IS临界信号分子的积累。 稀抑制剂基因座的甲那诺蛋白（MREG）是一种小的，高，高的，多层酰胺酰化的蛋白质，存在于黑色素体的极限膜上。 先前的研究已经暗示了MREG在黑素体从黑素细胞转移到角质形成细胞中的转移，并通过与RILP结合RILP（一种募集Dynein运动络合物的RAB7效应子）来促进这些和相关细胞器的微管减去这些和相关细胞器的终极转移。 在这里，我们通过使用核磁共振（NMR）求解MREG的分子功能的新灯。 该结构揭示了占据蛋白质表面相对边的正电荷带，并将其夹在推定的基于酪氨酸的基于酪氨酸（Y166）胆固醇识别序列（CRAC基序）。 我们确认胆固醇与MREG相互作用，因为CRAC基序中的残基S163和L168显示添加胆固醇时最大的NMR化学位移。 重要的是，在其CRAC基序中包含功能阻塞点突变（Y166I）内的MREG仍然靶向后期内体/溶酶体，但不再促进其微管减去终端导向的转运。 逆转三个紧密间隔的酸性残基（D177，E180和D181）的电荷也抑制了MREGS将这些细胞器驱动到微管负末端的能力，但仅部分部分。 我们建议胆固醇识别会改变膜上的MREG方向，以使其与参与动力蛋白募集的成分相互作用（例如RILP），并且通过酸性斑块进一步促进了这种相互作用。 最后，我们在MREG和蛋白质ORP1L之间进行比较，该蛋白ORP1L控制了微管减去最终导向的转运，后期内体的定位和命运，部分原因是识别靶向动力蛋白的胆固醇和成分。 基于肌动蛋白的运动肌球蛋白VA传输了许多碳，包括小脑Purkinje神经元中的内质网（ER）和黑素细胞中的黑素体。 识别与这种肌球蛋白相互作用的蛋白质是理解其细胞功能的关键。 为此，我们使用重新组合来通过同源重组插入串联亲和力纯化（TAP）标签（由蛋白A的IgG结合结构域组成，TEV裂解位点和FLAG标签在启动密码子后立即进入鼠标Myo5a基因座中。 重要的是，我们提供的证据表明，肌球蛋白VA（Tap-Myova）的Tag标签版本通常在Purkinje神经元中的ER传输和黑素细胞中的黑色素体定位方面起作用。 鉴于Tap-Myova具有完全功能性的和其他证据，我们将其纯化并直接从少年小鼠小脑中纯化，并将样品进行质谱分析。 正如预期的那样，鉴定出已知的肌球蛋白VA结合伙伴，例如Dynein轻链和神经丝光肽。重要的是，还鉴定了许多新型的相互作用蛋白，包括2'，3'-偶然核苷酸3'-磷酸二酯酶，脑特异性血管生成抑制剂1相关蛋白2和髓磷脂碱性蛋白。 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。 虽然由胚胎组织制备的混合原代小脑培养物已被证明对解剖结构很有价值：小脑Purkinje神经元（PNS）中的功能关系，但这种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（ESC）已成功地分化为PN。 这项研究的重点是简化小鼠ESC的分化为PN。 使用最近描述的神经分化培养基，我们从ESC产生了神经祖细胞（NP）的单层，我们使用特定的外部因素将其分化为PN前体。 然后，通过与颗粒神经元（GN）前体共培养也使用不同的外部因子衍生出来，将这些前体分化为成熟的PN。 ESC衍生的PNS的形态与原发性培养物中生长的PNs没有区别在于总形态，脊柱长度和脊柱密度。 此外，ESC衍生的PNS表达Calbindin D28K，IP3R1，IRBIT，PLC4，PSD93和肌球蛋白IIB-B2，所有这些都是PN特异性标记。 Moreover, we show that ESC-derived PNs express proteins driven by the PN-specific promoter Pcp2/L7, form synapses with GNs as in primary cultures, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel细胞外基质配方可重复产生有利于高分辨率成像的单层培养物。 我们改进的将ESC分为PNS的方法应促进PNS中分子机制的解剖。