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: 7326845
• 负责人: Velia M Fowler
• 金额: $ 68.84万
• 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 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7326845
• 关键词: Abnormal Red Blood Cell; Accounting; Actins; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Bleeding time procedure; Blood Platelets; Breeding; Cell Survival; Cell membrane; Cells; Chemicals; Chimera organism; Complex; Cytoskeleton; Data; Electron Microscopy; End Point; Endothelial Cells; Erythrocytes; Erythroid; Erythroid Progenitor Cells; Exhibits; Facility Construction Funding Category; Family; Family member; Filament; Filopodia; Fluorescence Microscopy; Gel; Gene Targeting; Genes; Genetic; Globin; Goals; Grant; Growth; Hematology; Hemolytic Anemia; Human; Immunofluorescence Immunologic; In Vitro; Knock-out; Knockout Mice; Label; Laboratories; Lead; Length; Life; Measures; Mediating; Megakaryocytes; Membrane; Microfilaments; Minus End of the Actin Filament; Modeling; Molecular; Mouse Strains; Mus; Mutagenesis; Mutation; N-terminal; Numbers; Osmotic Fragility test; Phase; Phenotype; Physiology; Platelet Activation; Platelet Count measurement; Progress Reports; Property; Protein Isoforms; Proteins; Pyrenes; Range; Rate; Regulation; Research; Research Personnel; Rest; Role; Shapes; Skeleton; Striated Muscles; Structure; System; Testing; Thin Filament; Thrombus; Time; Tissues; Tropomyosin; base; cell motility; cellular imaging; crosslink; in vivo; member; monomer; mouse model; mutant; novel; polymerization; programs; promoter; pyrene; receptor; receptor coupling; size

Actin filament lengths are precisely regulated and very stable in the red blood cell (RBC) membrane skeleton, while in platelets, actin filament lengths are dynamically regulated during receptor- mediated actin assembly as platelets extend lamellipodia during thrombogenesis. The broad, long term objective of this research is to elucidate how actin filament length and turnover is controlled in RBCs and platelets. An additional objective is to determine the role of actin filament length regulation in RBC membrane skeleton biogenesis and stability, and how aberrant filament length regulation may result in abnormal RBCs and hemolytic anemias. This proposal focuses on molecular mechanisms and in vivo functions of tropomodulins (Tmods), tropomyosin (TM)-regulated actin filament pointed end-capping proteins in RBCs (Tmodl) and platelets (TmodS). The specific aims are: (1) To investigate the molecular basis for Tmodl or TmodS binding to RBC or platelet TMs, and for TM-regulated actin capping. Biochemical and biophysical assays will be used to identify TM isoform-specific binding and TM-actin capping domains and a novel TmodS monomer-binding site. (2) To establish the in vivo function of Tmodl in regulation of RBC actin filament length, membrane skeleton assembly and stability using mouse models. Tmodl and TmodS conditional knockout mice will be generated followed by breeding with a v,LCR-ppr- Cre mouse to produce single or double knockouts for Tmodl and/or TmodS in RBCs and platelets. RBC phenotypes will be examined by hematology, ektacytometry,membrane skeleton assembly, and electron microscopy of actin filament lengths in membrane skeletons. (3) To investigate an in vivo function for TmodS in receptor-mediated actin assembly during activation and spreading of platelets. Platelet function in TmodS-deficient mouse platelets will be evaluated by hematological analyses, bleeding times, and thrombus formation under flow ex vivo. A role for TmodS in actin assembly, shape change and spreading will be evaluated by biochemical assays, immunofluorescence and electron microscopy, and by time-lapse interference reflection and fluorescence microscopy of lamellipodia and filopodia protrusior in living platelets. Molecular requirements for TmodS function will be tested using a permeabilized platelet model for receptor-coupled platelet activation, by introduction of TmodS or Tmodl domains and mutants.

肌动蛋白丝长度受到精确调节，并且在红细胞（RBC）中非常稳定 膜骨架，而在血小板中，肌动蛋白丝长度在受体期间动态调节 介导的肌动蛋白组件作为血小板在血栓形成过程中延伸薄片。长期的长期 这项研究的目的是阐明肌动蛋白丝的长度和周转方式如何在RBC和 血小板。另一个目标是确定肌动蛋白丝长度调节在RBC中的作用 膜骨架生物发生和稳定性，以及异常长度调节可能导致 异常RBC和溶血性贫血。该提议着重于分子机制和体内 肌动蛋白（TMODS），肌动蛋白（TM）调节的肌动蛋白丝指向终端封顶的功能 RBC（TMODL）和血小板（TMOD）中的蛋白质。具体目的是：（1）研究分子 与RBC或血小板TMS结合的TMODL或TMOD的基础，以及TM调节的肌动蛋白上限。 生化和生物物理测定将用于识别TM同工型特异性结合和TM-肌动蛋白 上限域和新型TMODS单体结合位点。 （2）建立TMODL的体内功能 在调节RBC肌动蛋白长度，使用小鼠模型的膜骨架组件和稳定性。 TMODL和TMODS将产生有条件的敲除小鼠，然后用V，LCR-PPR-繁殖 CRE鼠标以RBC和血小板中的TMODL和/或Tmods产生单个或双重敲除。 RBC 表型将通过血液学，ektacytermetry，膜骨架组件和电子检查 膜骨骼中肌动蛋白丝长度的显微镜。 （3）研究用于体内功能 血小板激活和扩散期间受体介导的肌动蛋白组件中的Tmods。血小板功能 在TMODS缺陷型小鼠中，小鼠血小板将通过血液学分析，出血时间和 流动流动下的血栓形成。 TMOD在肌动蛋白组装，形状变化和 扩散将通过生化测定，免疫荧光和电子显微镜以及 通过延时干扰反射和lamellipodia的荧光显微镜和丝状肌肌肌肌 在活血小板中。 TMODS功能的分子要求将使用透化血小板测试 通过引入Tmods或Tmodl结构域和突变体的受体偶联血小板激活模型。