Capping Actin growth in Erythroid and Nonerythroid Cells

限制红系和非红系细胞中肌动蛋白的生长

• 批准号: 8279205
• 负责人: Velia M Fowler
• 金额: $ 47.38万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279205
• 关键词: Abnormal Red Blood Cell; Actins; Anemia; Anemia, Hemolytic, Congenital; Binding; Biochemistry; Biogenesis; Biological Assay; Blood Circulation; Bone Marrow; CD34 gene; Cell Membrane Structures; Cell Shape; Cell membrane; Cell physiology; Cells; Cellular Morphology; Chemicals; Complex; Defect; Diagnosis; Dissociation; Electron Microscopy; Embryo; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Evaluation; Family; Fetal Liver; Filament; Flow Cytometry; Fluorescence Microscopy; Goals; Growth; Hematopoietic stem cells; Hemolytic Anemia; Human; In Vitro; Knock-out; Knockout Mice; Length; Link; Membrane; Membrane Proteins; Membrane Structure and Function; Microfilaments; Minus End of the Actin Filament; Modeling; Molecular; Mus; Mutagenesis; Mutation; Patients; Pharmaceutical Preparations; Phosphorylation; Physiology; Polymers; Population; Property; Protein Kinase C; Proteins; Regulation; Research; Rhodamine; Role; Screening procedure; Shapes; Site; Spectrin; Structure; Testing; Time; Total Internal Reflection Fluorescent; Tropomyosin; Work; actin 2; adducin; base; crosslink; erythroid differentiation; fetal; in vivo; in vivo Model; insight; light microscopy; liquid chromatography mass spectrometry; membrane biogenesis; membrane skeleton; monomer; mouse model; novel; peripheral blood; polymerization; progenitor; reconstitution; small hairpin RNA; tropomodulin

DESCRIPTION (provided by applicant): The long-range goal of this research is to determine how actin filament dynamics is regulated in the spectrin-actin lattice of the membrane skeleton to create the red blood cell (RBC) with its specialized membrane, unique biconcave shape and deformability properties. Mutations in membrane skeleton components produce human hereditary hemolytic anemias with fragile RBC membranes, due to defective membrane biogenesis and/or weakened interactions in the membrane skeleton, thereby leading to reduced stability in the circulation. The spectrin-actin lattice consists of spectrin tetramers attached to short actin filaments at junctional complexes (JCs), forming the strands and vertices of a quasi-hexagonal lattice. The short RBC actin filaments are capped by tropomodulin-1 (Tmod1) at their pointed ends and 1/2-adducin at their barbed ends, while tropomyosins (TMs) span along the filament length, binding to Tmod1 at the pointed filament end. Despite the importance of RBC actin filaments as structural nodes in the spectrin latice and JC anchorage sites for membrane proteins, the role of actin monomer-polymer dynamics in membrane skeleton structure and stability, or biogenesis during erythroid terminal differentiation is not understood. This proposal focuses on Tmods, a unique family of TM-regulated, actin filament pointed end capping proteins that control actin dynamics and stability, cell morphology and physiology. Absence of Tmod1 in mouse RBCs leads to a mild compensated sphero-elliptocytic anemia with osmotically fragile RBCs, and a disrupted membrane skeleton with mis- regulated actin filament lengths. In contrast, absence of Tmod3 leads to fetal anemia with embryonic lethality at E14.5-15.5 due to impaired definitive erythropoiesis. Unlike Tmod1, which is a strong actin filament pointed end capper, Tmod3 sequesters actin monomers but is a weak filament capper. We hypothesize that Tmod1 regulates actin dynamics to maintain filament lengths and membrane skeleton structure in mature RBCs, while Tmod3 regulates actin monomer pols to control membrane skeleton assembly during erythroid terminal differentiation. The Specific Aims are: 1) To dissect the molecular and structural basis for Tmod1 and Tmod3 differential regulation of actin dynamics in vitro; 2) To define actin dynamics in normal human RBCs, and in wild-type, Tmod1-null and 1/2-adducin-null mouse RBCs, and to screen as yet undiagnosed human hemolytic anemia patients for defects in TMODs; 3) To determine the cellular basis for the Tmod3-null mouse fetal liver anemia, and test a requirement for Tmod3 in erythroblasts using bone marrow reconstitution and a RBC- specific conditional knockout for Tmod3; 4) To investigate Tmod1 and Tmod3 functions in membrane skeleton assembly during erythroid terminal differentiation from cultured human CD34+ cells isolated from peripheral blood. Our studies of actin dynamics will define a novel control point for membrane skeleton assembly and stability in human and mouse RBCs and their precursors, providing insights into RBC membrane biogenesis and survival in normal contexts and in human hemolytic anemias.

描述（由申请人提供）：这项研究的远距离目标是确定如何在膜骨架的光谱 - 肌动蛋白晶格中调节肌动蛋白细丝动力学，以创建其具有专门的膜，独特的biconcove形状和可变形性能的红细胞（RBC）。膜骨骼成分的突变由于缺陷的膜生物发生和/或膜骨骼中的相互作用而导致循环稳定性降低，因此产生人类遗传性溶血性贫血和脆弱的RBC膜。光谱 - 肌动蛋白晶格由连接到肌动蛋白丝（JCS）的短肌动蛋白丝（JCS）的光谱四聚体组成，形成了准甲状腺饰晶格的链和顶点。短rbc肌动蛋白丝在其尖端末端被托托瘤蛋白-1（TMOD1）盖住，在其刺的末端，沿灯丝长度沿齿状末端（TMS）跨度为1/2- adducin，在尖丝末端与TMOD1结合。尽管RBC肌动蛋白丝作为膜蛋白的光谱纬度和JC锚定位点中的结构淋巴结很重要，但尚不理解肌动蛋白单体聚合物动力学在膜骨架结构和稳定性中的作用，或者在erythroid末端分化过程中的生物发生在膜骨架结构和稳定性中的作用。该提案的重点是TMODS，这是一个由TM调节的，肌动蛋白丝的独特家族，指向控制肌动蛋白动力学和稳定性，细胞形态学和生理学的末端封盖蛋白质。小鼠RBC中的TMOD1的不存在导致带有渗透脆弱的RBC的轻度补偿sphoro-胸细胞性贫血，并具有破坏的膜骨骼，具有错误调节的肌动蛋白丝长度。相比之下，由于确定性红细胞生成受损而导致E14.5-15.5的胎儿贫血的缺失导致胎儿贫血。与TMOD1是强大的肌动蛋白丝尖尾圆形器不同，TMOD3隔离器肌动蛋白单体，但是薄丝胶囊。我们假设TMOD1调节肌动蛋白动力学以维持成熟RBC中的细丝长度和膜骨架结构，而TMOD3调节肌动蛋白单体小体以控制红细胞末端分化过程中的膜骨骼组件。具体目的是：1）剖析体外TMOD1和TMOD3差异调节的分子和结构基础； 2）定义正常人RBC中的肌动蛋白动力学，以及野生型，TMOD1-NULL和1/2 adducin-null小鼠RBC，以及尚未诊断的人类溶血性贫血患者筛查TMODS缺陷； 3）确定TMOD3-NULL小鼠胎儿肝贫血的细胞基础，并使用骨髓重构和TMOD3的RBC特定条件敲除在红细胞中测试TMOD3的需求； 4）研究在从外周血分离的培养的人CD34+细胞的红细胞终末分化过程中，TMOD1和TMOD3在膜骨骼组装中的功能。我们对肌动蛋白动力学的研究将定义人和小鼠RBC及其前体中膜骨骼组装和稳定性的新型控制点，从而在正常情况下以及人类溶血性贫血中对RBC膜生物发生和生存提供了见解。