Membrane skeleton regulation of cell shape and interactions in lens development

描述（由申请人提供）：晶状体由超长薄纤维细胞的连续层组成，其形状、有序的六边形填充和规则的膜结构对晶状体的透明度和聚焦至关重要。本研究的广泛、长期目标是阐明基于光谱的膜骨架在纤维细胞形态发生、晶状体光学质量和力学性能中的作用，以及膜骨架缺陷如何导致晶状体功能的进行性、年龄依赖性下降。该提案将研究原调节蛋白（Tmods），这是一个肌动蛋白调节蛋白家族，它与原肌球蛋白（TMs）协同作用，覆盖肌动蛋白丝的尖端，调节肌动蛋白丝在膜骨架中的转换和稳定性，影响细胞形状、相互作用和机械性能。小鼠晶状体包含两个Tmods， Tmod1和Tmod3，它们都与纤维细胞膜有关。为了在体内研究Tmod在晶状体中的功能，我们产生了Tmod1和Tmod3靶向缺失的敲除小鼠。我们实验室最近的研究表明，缺乏Tmod1的晶状体发育正常，但在其皮层纤维细胞中表现出明显的缺陷，包括纤维细胞形状异常，前皮层细胞堆积紊乱。在缺乏Tmod1的情况下，与膜相关的短3-TM异构体的水平选择性降低，同时肌动蛋白细丝解聚和膜上12-谱蛋白的减少。假设Tmod1和短的3-TM起稳定膜骨架中肌动蛋白丝连接物的作用，从而控制其完整性和远程组织。这对于粘附受体的锚定控制纤维细胞的形状、相互作用和有序包装是至关重要的。胚胎和出生后晶状体中Tmod3水平升高，而成人晶状体中没有Tmod3水平的升高，这表明Tmod3可能弥补了晶状体发育和初始生长中Tmod1的缺失，从而导致纤维细胞疾病的年龄依赖性进展。此外，Tmod3（而不是Tmod1）在成人晶状体纤维细胞中被蛋白水解，这表明Tmod3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Tmod1敲除小鼠晶状体的分子、生化和形态学分析，探讨Tmod1在控制膜骨架组装、稳定性和与粘附受体的关联中的作用；(2)通过Tmod1和Tmod3敲除小鼠晶状体发育和生长的分子、生化和形态学分析，探讨Tmod3在晶状体纤维细胞中的表达和功能，以及它在补偿Tmod1缺失中的作用。(3)通过活体晶状体功能分析，探讨Tmod1或Tmod3缺失对晶状体光学质量和机械刚度的影响。这些研究的完成将阐明晶状体发育、生长和老化过程中纤维细胞形状和相互作用的膜骨架调节的分子和细胞基础，以及这如何影响晶状体的光学质量和力学性能。公共卫生相关性：本项目旨在阐明晶状体纤维细胞形状、相互作用和有序六边形排列的膜骨架调节的分子基础，以及这如何有助于晶状体透明度和聚焦能力的维持。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晶状体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理解遗传性人类白内障的原因和随年龄增长而丧失的调节能力。

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.