Motor proteins and cytoskeletal dynamics in T lymphocytes, B lymphocytes and melanocytes

T 淋巴细胞、B 淋巴细胞和黑素细胞中的运动蛋白和细胞骨架动力学

• 批准号: 10008805
• 负责人: JOHN A HAMMER
• 金额: $ 135.95万
• 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/10008805
• 关键词: 3-Dimensional; Actin-Binding Protein; Actins; Actomyosin; Adaptive Immune System; Adhesions; Antibodies; Antibody Response; Antigen-Presenting Cells; Antigens; Attenuated; Autoimmunity; B-Cell Activation; B-Lymphocytes; Binding; C-terminal; CD80 Antigens; Cell Line; Cells; Cellular Structures; Charge; Cholesterol; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Contracts; Distal; Dynein ATPase; Event; Filament; Functional disorder; Glass; Goals; Head; Hypersensitivity; Image; Immune; Immune system; Impairment; Infection; Integrins; Knock-in Mouse; Knock-out; Lead; Light; Lipid Bilayers; Lymphocyte antigen; Lymphoproliferative Disorders; Lysosomes; Major Histocompatibility Complex; Medial; Mediating; Melanosomes; Membrane; Membrane Proteins; Microfilaments; Minus End of the Microtubule; Molecular; Molecular Weight; Motor; Motor Activity; Movement; Myosin ATPase; Myosin S-2; N-terminal; NMR Spectroscopy; Nature; Nonmuscle Myosin Type IIA; Organelles; Pathology; Peptides; Peripheral; Physiological; Play; Point Mutation; Polymers; Predisposition; Process; Protein Binding Domain; Protein Isoforms; Proteins; Receptor Signaling; Receptors, Antigen, B-Cell; Resolution; Role; Side; Signal Transduction; Structure; Surface Antigens; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Tail; Testing; Tropomyosin; Tryptophan; Tyrosine; Viral; antigen binding; arm; base; cell motility; cell transformation; cellular imaging; cofilin; effector T cell; experimental study; imaging modality; immunological synapse; immunological synapse formation; in vivo; inhibitor/antagonist; keratinocyte; knock-down; late endosome; melanocyte; novel; prevent; recruit; response; uptake

B cells are a critical branch of the immune system and drive antibody-based protection. The strength of an antibody response is determined by the ability of B cells to extract and internalize membrane-bound antigens from antigen-presenting cells (APCs). Antigen uptake requires the formation of an immune synapse (IS) where B cell receptor-bound antigens are moved centripetally into a central cluster before being extracted from the APC and internalized. The force provided by the actin motor myosin 2A (M2A) powers antigen extraction. B cells that lack M2A activate aberrantly and mount weak antibody responses. However, the organization of the actomyosin network, its role in IS formation, and the mechanism by which M2A powers antigen extraction in B cells are unknown. Here we test the hypothesis that the actomyosin network drives the events of IS formation that promote B cell activation and antigen uptake. We first define the dynamic organization of actin and M2A at the IS using the super-resolution imaging modalities TIRF/SIM, 3D-SIM and Airyscan. On functionalized glass and planar lipid bilayers, the A20 B cell line forms concentric arcs in the medial portion of the IS. These arcs are rich in M2A based on immunostaining, and on imaging cells in which endogenous M2A was tagged with GFP using CRISPR. 3D-SIM imaging of APCs with primary splenic B cells isolated from M2A-GFP knock-in mice shows that M2A polarizes towards the IS. B cell IS studies are often performed using antigen stimulation only. in vivo, B cell integrins provide adhesion to APCs and, via an unknown mechanism, allow for IS formation and antigen uptake with weakly-stimulating antigens. Surprisingly, we found that actomyosin arc formation in primary B cells requires both antigen and integrin costimulation, conditions that reflect physiological B cell activation. These contractile actomyosin arcs are especially prominent in primary B cells such that the actomyosin arcs are the major actin structure at the IS. The contractile nature of the actomyosin arcs that dominate at the primary B cell IS may explain why integrin costimulation boosts B cell responses to weakly-stimulating antigens. Notably, integrin costimulation on bilayers produces actin arcs that sweep peripheral antigen clusters centripetally and is required for contracting low amounts of antigen to form the IS. Moreover, M2A inhibition abrogates the organization of actin arcs and prevents antigen centralization. Therefore, we have identified in primary B cells a novel actomyosin network, which comprises the major actin structure at the IS and promotes robust antigen centralization during IS formation. Current efforts are directed at defining the mechanism by which the actomyosin network drives antigen extraction from APCs using live-cell volumetric imaging. T cells are a critical arm of the adaptive immune system because they kill virally-infected or transformed cells and facilitate the function of other immune cells (Zhang & Bevan, 2011; Zhu et al., 2010). T cell dysfunction can lead to an array of severe pathologies including susceptibility to infection, lymphoproliferative disease, autoimmunity, and hypersensitivity (Walter & Santamaria, 2005; Zhu & Paul, 2008). T cell activation is a complex process involving recognition by the T cells unique T cell receptor (TCR) of specific peptide antigen bound to major histocompatibility complex (MHC) on the surface of an antigen-presenting cell (APC). This recognition can lead to long-term stable engagement with the APC and the formation of a highly-organized structure at the T cell: APC interface termed the immunological synapse (IS) (Dustin & Baldari, 2017). The IS itself is a multidomain structure divided into distal, peripheral, and central supramolecular activation complexes (dSMAC, pSMAC, cSMAC). TCR microclusters contact antigen-bearing MHC at the periphery of the IS and are then transported across the dSMAC and pSMAC to the cSMAC. This centripetal movement of microclusters is driven by the retrograde flow of an Arp2/3-generated, branched actin network in the dSMAC and the contraction of formin-generated, myosin 2-rich, concentric actin arcs in the pSMAC (Hammer et al., 2019; Murugesan et al., 2016; Yi et al., 2012; Ditlev et al., pre-print BioRxiv). Perturbation of either of these actin structures dampens TCR signaling and impairs subsequent T cell activation. The goal of this study is to characterize the contributions made by tropomyosin and myosin 18A to the organization, dynamics and function of the actomyosin arcs populating the pSMAC. Tropomyosins are actin-binding proteins that form head-to-tail polymers along the actin filament. Several tropomyosin isoforms have been shown to associate preferentially with linear, formin-generated filaments, where they serve to promote the recruitment and activation of myosin 2 and thwart cofilin-mediated filament disassembly (Gateva et al., 2017; Tojkander et al., 2011). In preliminary experiments, we find that the low-molecular weight tropomyosin isoform 4.1 associates with the pSMAC arcs, and that the tropomyosin inhibitor TR100 disrupts their organization. Myosin 18A is a myosin 2-like protein that lacks motor activity and contains unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. Importantly, myosin 18A co-assembles with myosin 2 to make mixed filaments (Billington et al., 2015), suggesting that myosin 18A serves to recruit proteins to these mixed filaments or attach them to cellular structures. Preliminary experiments show that the myosin 18A isoform myosin 18A is highly expressed in T cells and that it co-assembles with myosin 2 in the pSMAC arcs. Moreover, knockdown or CRISPR-mediated knockout of myosin 18A alters arc organization and attenuates proximal signaling. Current efforts are directed at further clarifying the roles played by tropomyosin and myosin 18A in arc organization and function, as well as in T cell effector functions. 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) spectroscopy. Mreg contains six -helices that form an elongated fishhook-like fold in which positive and negative charges occupy opposite sides of the proteins surface and sandwich a putative, tyrosine-based (Y166) cholesterol recognition sequence (CRAC motif). The absence or significant exchange broadening of 1H-15N crosspeaks for multiple residues within this putative CRAC motif and a proximal tryptophan sidechain resonance argue that this motif has functional importance. Consistently, 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. Moreover, wild type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, 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).

B 细胞是免疫系统的重要分支，负责驱动基于抗体的保护。抗体反应的强度取决于 B 细胞从抗原呈递细胞 (APC) 中提取和内化膜结合抗原的能力。抗原摄取需要形成免疫突触 (IS)，其中 B 细胞受体结合的抗原向心移动到中央簇，然后从 APC 中提取并内化。肌动蛋白运动肌球蛋白 2A (M2A) 提供的力为抗原提取提供动力。缺乏 M2A 的 B 细胞会异常激活并产生微弱的抗体反应。然而，肌动球蛋白网络的组织、其在 IS 形成中的作用以及 M2A 在 B 细胞中提取抗原的机制尚不清楚。在这里，我们测试了以下假设：肌动球蛋白网络驱动 IS 形成事件，从而促进 B 细胞激活和抗原摄取。我们首先使用超分辨率成像模式 TIRF/SIM、3D-SIM 和 Airyscan 定义肌动蛋白和 M2A 在 IS 的动态组织。在功能化玻璃和平面脂质双层上，A20 B 细胞系在 IS 的内侧部分形成同心弧。根据免疫染色和成像细胞，这些弧富含 M2A，其中内源性 M2A 使用 CRISPR 用 GFP 进行标记。对从 M2A-GFP 敲入小鼠分离的原代脾 B 细胞进行 APC 的 3D-SIM 成像显示，M2A 向 IS 极化。 B 细胞 IS 研究通常仅使用抗原刺激进行。在体内，B 细胞整合素提供对 APC 的粘附，并通过未知的机制，允许 IS 形成和弱刺激抗原的抗原摄取。令人惊讶的是，我们发现原代 B 细胞中肌动球蛋白弧的形成需要抗原和整合素共刺激，这是反映生理 B 细胞激活的条件。这些收缩性肌动球蛋白弧在原代 B 细胞中尤其突出，因此肌动球蛋白弧是 IS 处的主要肌动蛋白结构。在原代 B 细胞 IS 中占主导地位的肌动球蛋白弧的收缩性质可以解释为什么整合素共刺激会增强 B 细胞对弱刺激抗原的反应。值得注意的是，双层上的整合素共刺激产生向心扫过外周抗原簇的肌动蛋白弧，并且是收缩少量抗原形成IS所必需的。此外，M2A 抑制会破坏肌动蛋白弧的组织并阻止抗原集中。因此，我们在原代 B 细胞中发现了一种新型肌动球蛋白网络，该网络包含 IS 处的主要肌动蛋白结构，并在 IS 形成过程中促进抗原的稳健集中。目前的工作旨在确定肌动球蛋白网络利用活细胞体积成像驱动从 APC 中提取抗原的机制。 T 细胞是适应性免疫系统的重要组成部分，因为它们杀死病毒感染或转化的细胞并促进其他免疫细胞的功能（Zhang & Bevan，2011；Zhu et al.，2010）。 T 细胞功能障碍可导致一系列严重的病理，包括易受感染、淋巴组织增生性疾病、自身免疫和超敏反应（Walter & Santamaria，2005；Zhu & Paul，2008）。 T 细胞激活是一个复杂的过程，涉及 T 细胞独特的 T 细胞受体 (TCR) 识别与抗原呈递细胞 (APC) 表面的主要组织相容性复合物 (MHC) 结合的特定肽抗原。这种识别可以导致与 APC 的长期稳定结合，并在 T 细胞上形成高度组织化的结构：APC 界面，称为免疫突触 (IS) (Dustin & Baldari, 2017)。 IS 本身是一个多域结构，分为远端、外周和中央超分子激活复合物（dSMAC、pSMAC、cSMAC）。 TCR 微簇与 IS 外围的带有抗原的 MHC 接触，然后穿过 dSMAC 和 pSMAC 转运至 cSMAC。微团簇的这种向心运动是由 dSMAC 中 Arp2/3 生成的分支肌动蛋白网络的逆行流动和 pSMAC 中福尔明生成的富含肌球蛋白 2 的同心肌动蛋白弧的收缩驱动的（Hammer 等，2019；Murugesan 等，2016；Yi 等，2012；Ditlev 等，预印本） BioRxiv）。这些肌动蛋白结构的扰动都会抑制 TCR 信号传导并损害随后的 T 细胞激活。本研究的目的是表征原肌球蛋白和肌球蛋白 18A 对 pSMAC 中肌动球蛋白弧的组织、动力学和功能的贡献。原肌球蛋白是肌动蛋白结合蛋白，沿着肌动蛋白丝形成头尾聚合物。几种原肌球蛋白亚型已被证明优先与线性、福尔马林生成的细丝结合，促进肌球蛋白 2 的募集和激活，并阻止肌动蛋白丝切蛋白介导的细丝分解（Gateva 等，2017；Tojkander 等，2011）。在初步实验中，我们发现低分子量原肌球蛋白亚型 4.1 与 pSMAC 弧相关，并且原肌球蛋白抑制剂 TR100 破坏了它们的组织。肌球蛋白 18A 是一种类似肌球蛋白 2 的蛋白质，缺乏运动活性，并含有独特的 N 和 C 末端延伸，其中包含可识别和未表征的蛋白质：蛋白质相互作用结构域。重要的是，肌球蛋白 18A 与肌球蛋白 2 共同组装形成混合丝 (Billington et al., 2015)，这表明肌球蛋白 18A 可以将蛋白质募集到这些混合丝中或将它们附着到细胞结构上。初步实验表明，肌球蛋白 18A 亚型肌球蛋白 18A 在 T 细胞中高表达，并与 pSMAC 弧中的肌球蛋白 2 共同组装。此外，肌球蛋白 18A 的敲低或 CRISPR 介导的敲除会改变弧组织并减弱近端信号传导。目前的努力旨在进一步阐明原肌球蛋白和肌球蛋白 18A 在弧组织和功能以及 T 细胞效应功能中所发挥的作用。 黑素调节蛋白 (Mreg) 是稀释抑制基因座的产物，是一种小的、高电荷的、多棕榈酰化的蛋白质，存在于黑素体的限制膜上。先前的研究表明，Mreg 参与了黑素体从黑素细胞到角质形成细胞的转移，并通过与 RILP（一种招募动力蛋白运动复合体的 Rab7 效应器）结合，促进这些及相关细胞器的微管负端定向运输。在这里，我们通过使用核磁共振 (NMR) 光谱解析 Mreg 的结构，为 Mreg 可能的分子功能提供了新的线索。 Mreg 包含六个 β 螺旋，形成细长的鱼钩状折叠，其中正电荷和负电荷占据蛋白质表面的相对两侧，并夹入假定的基于酪氨酸 (Y166) 的胆固醇识别序列（CRAC 基序）。该推定的 CRAC 基序内多个残基的 1H-15N 交叉峰的缺失或显着交换展宽以及近端色氨酸侧链共振表明该基序具有功能重要性。一致的是，在其 CRAC 基序 (Y166I) 内含有功能阻断点突变的 Mreg 仍然以晚期内体/溶酶体为目标，但不再促进其微管负末端定向运输。此外，野生型Mreg不会促进暂时耗尽胆固醇的细胞中晚期内体/溶酶体的微管减末端定向运输。最后，反转三个紧密排列的酸性残基（D177、E180 和 D181）的电荷也会抑制 Mregs 将这些细胞器驱动到微管负端的能力，但只是部分抑制。我们提出，胆固醇识别会改变膜上 Mregs 的方向，使其与参与动力蛋白募集的成分（例如 RILP）相互作用。