Membrane skeleton regulation of cell shape and interactions in lens development

细胞形状的膜骨架调节和晶状体发育中的相互作用

• 批准号: 7898748
• 负责人: Velia M Fowler
• 金额: $ 46.9万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7898748
• 关键词: Actins; Adult; Age; Alleles; Anterior; Architecture; Binding; Biochemical; Biological Assay; Cataract; Cell Differentiation process; Cell Maturation; Cell Shape; Cell membrane; Cell-Cell Adhesion; Cells; Cellular Structures; Chickens; Collaborations; Complex; Crystalline Lens; Crystallins; Data; Defect; Development; Disease; Dissociation; Embryo; Employee Strikes; Erythrocytes; Family; Filament; Goals; Growth; Growth and Development function; Heart; Human; Individual; Inherited; Knock-out; Knockout Mice; Laboratories; Lasers; Lead; Length; Lens Fiber; Life; Maintenance; Measures; Mechanics; Membrane; Microfilaments; Minus End of the Actin Filament; Molecular; Morphogenesis; Mus; Mutation; Myosin Heavy Chains; Optics; Pattern; Phenotype; Precipitation; Progress Reports; Property; Protein Isoforms; Proteins; Proteolysis; Proteolytic Processing; Protocols documentation; Regulation; Regulation of Cell Shape; Research; Role; Scanning; Shapes; Spectrin; Structure; Testing; Tissues; Transgenes; Transgenic Mice; Tropomyosin; Work; adhesion receptor; age related; base; depolymerization; design; fiber cell; genetic regulatory protein; in vivo; insight; lens; lens cortex; lens morphogenesis; lens protein; lens transparency; light scattering; membrane skeleton; migration; monomer; mouse model; mutant; postnatal; programs; promoter; protein function; public health relevance; response

DESCRIPTION (provided by applicant): The ocular lens is comprised of successive layers of extremely long thin fiber cells whose shapes, ordered hexagonal packing and regular membrane structure are critical for lens transparency and focusing. The broad, long-term objectives of this research are to elucidate the role of the spectrin-based membrane skeleton in fiber cell morphogenesis, lens optical quality and mechanical properties, and how defects in the membrane skeleton may contribute to progressive, age-dependent declines in lens functions. This proposal will study tropomodulins (Tmods), a family of actin regulatory proteins that function cooperatively with tropomyosins (TMs) to cap actin filament pointed ends, regulating actin filament turnover and stability in the membrane skeleton, contributing to cell shapes, interactions and mechanical properties. The mouse lens contains two Tmods, Tmod1 and Tmod3, which are both associated with fiber cell membranes. To investigate Tmod functions in vivo in the lens, we have generated knockout mice with targeted deletions in Tmod1 and Tmod3. Recent work from our laboratory has shown that lenses lacking Tmod1 develop normally but demonstrate striking defects in their cortical fiber cells, including abnormal fiber cell shapes with disordered cellular packing in the anterior cortex. Levels of a membrane-associated, short 3-TM isoform are reduced selectively in the absence of Tmod1, concomitant with depolymerization of actin filaments and reduction of 12-spectrin on membranes. It is hypothesized that Tmod1 and the short 3-TM function to stabilize the actin filament linkers in the membrane skeleton, thus controlling its integrity and long-range organization. This is expected to be critical for anchoring of adhesion receptors to control fiber cell shapes, interactions and ordered packing. Increased levels of Tmod3 in embryonic and postnatal but not adult lenses indicates that Tmod3 may compensate for the absence of Tmod1 in lens development and initial growth, leading to age-dependent progression of fiber cell disorder. Further, Tmod3 (but not Tmod1) is proteolysed in adult lens fiber cells, suggesting a unique role for Tmod3 degradation in membrane skeleton remodeling in fiber cell elongation and maturation. The specific aims are: (1) To investigate the role of Tmod1 in controlling membrane skeleton assembly, stability and associations with adhesion receptors by biochemical and morphological analyses of lenses from Tmod1 knockout mice, (2) To investigate the expression and function of Tmod3 in lens fiber cells and its role in compensating for absence of Tmod1 by molecular, biochemical and morphological analyses of lens development and growth in Tmod1 and Tmod3 knockout mice, (3) To investigate the consequences of loss of Tmod1 or Tmod3 for lens optical quality and mechanical stiffness by functional assays on living lenses ex vivo. Completion of these studies will elucidate the molecular and cellular basis for membrane skeleton regulation of fiber cell shapes and interactions during lens development, growth, and ageing, and how this influences lens optical quality and mechanical properties. PUBLIC HEALTH RELEVANCE: This project seeks to elucidate the molecular basis for membrane skeleton regulation of lens fiber cell shape, interactions and ordered hexagonal packing, and how this contributes to maintenance of lens transparency and focusing ability. Our studies will utilize transgenic mouse models in which deficiencies in specific membrane skeleton components lead to developmental or age-dependent abnormalities in lens fiber cell structures and interactions. These studies are expected to provide mechanistic insights into the molecular and cellular basis for normal lens optical quality and mechanical functions. Our studies on mouse lenses may also aid in understanding the causes of inherited human cataracts and loss of accommodative ability with age.

描述（由申请人提供）：眼用透镜由连续层的极长的薄纤维单元组成，其形状、有序的六边形堆积和规则的膜结构对于透镜的透明度和聚焦是关键的。这项研究的广泛的，长期的目标是阐明纤维细胞形态发生，透镜的光学质量和机械性能的血影蛋白为基础的膜骨架的作用，以及如何在膜骨架的缺陷可能会导致渐进的，年龄依赖性下降的透镜功能。该提案将研究原肌调节蛋白（Tmods），一个肌动蛋白调节蛋白家族，与原肌球蛋白（TM）合作，以帽肌动蛋白丝尖端，调节肌动蛋白丝在膜骨架中的周转和稳定性，有助于细胞形状，相互作用和机械性能。小鼠透镜含有两个Tmod，Tmod 1和Tmod 3，它们都与纤维细胞膜相关。为了研究Tmod在体内透镜中的功能，我们产生了Tmod 1和Tmod 3靶向缺失的敲除小鼠。我们实验室最近的工作表明，缺乏Tmod 1的晶状体发育正常，但在其皮质纤维细胞中表现出明显的缺陷，包括异常的纤维细胞形状和前皮质中的无序细胞包装。一个膜相关的，短的3-TM亚型的水平降低选择性在Tmod 1的情况下，伴随着解聚的肌动蛋白丝和减少12-血影蛋白的膜。据推测，Tmod 1和短3-TM的功能，以稳定肌动蛋白丝连接器在膜骨架，从而控制其完整性和远程组织。预计这对于粘附受体的锚定以控制纤维细胞形状、相互作用和有序包装是至关重要的。Tmod 3在胚胎和出生后晶状体中的水平增加，但在成人晶状体中没有，表明Tmod 3可能补偿透镜发育和初始生长中Tmod 1的缺失，导致纤维细胞疾病的年龄依赖性进展。此外，Tmod 3（而不是Tmod 1）在成人透镜纤维细胞中被蛋白水解，这表明Tmod 3降解在纤维细胞伸长和成熟的膜骨架重塑中具有独特的作用。具体目标是：（1）通过对Tmod 1基因敲除小鼠晶状体的生物化学和形态学分析，研究Tmod 1在控制膜骨架组装、稳定性以及与粘附受体的结合中的作用。（2）通过分子生物学方法，研究Tmod 3在透镜纤维细胞中的表达和功能，Tmod 1和Tmod 3基因敲除小鼠透镜发育和生长的生物化学和形态学分析。（3）通过离体活体晶状体功能测定研究Tmod 1或Tmod 3基因缺失对透镜光学质量和机械刚度的影响。这些研究的完成将阐明纤维细胞形状和相互作用在透镜发育，生长和老化过程中的膜骨架调节的分子和细胞基础，以及这如何影响透镜的光学质量和机械性能。公共卫生关系：本研究旨在阐明透镜纤维细胞形态、相互作用和有序六方堆积的膜骨架调节的分子基础，以及这如何有助于保持透镜的透明度和聚焦能力。我们的研究将利用转基因小鼠模型，其中特定膜骨架成分的缺陷导致透镜纤维细胞结构和相互作用的发育或年龄依赖性异常。这些研究有望为正常透镜光学质量和机械功能的分子和细胞基础提供机制见解。我们对小鼠晶状体的研究也可能有助于了解遗传性人类白内障和随着年龄增长而丧失晶状体能力的原因。