Membrane skeleton regulation of cell shape and interactions in lens development

描述(申请人提供)：人工晶状体由连续的超长纤维细胞组成，纤维细胞的形状、有序的六角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。这项研究的长期目标是阐明基于血影蛋白的膜骨架在纤维细胞形态发生、晶状体光学质量和机械性能中的作用，以及膜骨架中的缺陷如何导致晶状体功能的进行性、年龄相关性下降。这项建议将研究原肌球蛋白(Tmods)，这是一个肌动蛋白调节蛋白家族，与原肌球蛋白(TMS)协同作用，覆盖肌动蛋白细丝的尖端，调节肌动蛋白细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。小鼠的晶状体含有两个Tmod，Tmod1和Tmod3，它们都与纤维细胞膜有关。为了研究Tmod在晶状体中的体内功能，我们建立了Tmod1和Tmod3靶向缺失的基因敲除小鼠。我们实验室最近的工作表明，缺乏Tmod1的晶状体发育正常，但其皮质纤维细胞存在显著缺陷，包括纤维细胞形状异常，前皮层细胞堆积紊乱。在没有Tmod1的情况下，膜相关的短3-TM亚型的水平选择性地降低，伴随着肌动蛋白细丝的解聚和膜上12-血影蛋白的减少。推测Tmod1和短3-TM的功能是稳定膜骨架中的肌动蛋白细丝连接物，从而控制其完整性和远程组织。这对于黏附受体的锚定以控制纤维细胞的形状、相互作用和有序堆积至关重要。Tmod3在胚胎和出生后晶状体中的水平升高，但在成人晶状体中不表达，这表明Tmod3可能弥补了晶状体发育和初始生长中Tmod1的缺失，导致了纤维细胞疾病的年龄相关性进展。此外，Tmod3(但不是Tmod1)在成人晶状体纤维细胞中被蛋白质降解，这表明Tmod3降解在膜骨架重塑中在纤维细胞的伸长和成熟中具有独特的作用。本研究的具体目的是：(1)通过对Tmod1基因敲除小鼠晶状体的生化和形态分析，研究Tmod1在调控膜骨架组装、稳定性以及与黏附受体的关系中所起的作用；(2)通过对Tmod1和Tmod3基因敲除小鼠晶状体发育和生长的分子、生化和形态分析，研究Tmod1在晶状体纤维细胞中的表达和功能及其在补偿晶状体生长发育中的作用；(3)通过对活体晶状体进行功能分析，研究Tmod1或Tmod3缺失对晶状体光学质量和机械硬度的影响。这些研究的完成将阐明晶状体发育、生长和老化过程中膜骨架调节纤维细胞形状和相互作用的分子和细胞基础，以及这如何影响晶状体的光学质量和机械性能。与公众健康相关：本项目旨在阐明膜骨架调节晶状体纤维细胞形状、相互作用和有序六角形堆积的分子基础，以及这如何有助于维持晶状体的透明度和聚焦能力。我们的研究将利用转基因小鼠模型，在该模型中，特定膜骨架成分的缺陷会导致晶状体纤维细胞结构和相互作用的发育或年龄相关性异常。这些研究有望提供对正常镜片光学质量和机械功能的分子和细胞基础的机械性见解。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和调节能力随年龄增长而丧失的原因。

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.