FUNCTIONS FOR MYOSINS-I OF THE INTESTINAL BRUSH BORDER

基于肌动蛋白的细胞骨架在广泛的细胞现象中起着重要作用，包括细胞形状、极性、运动性、细胞信号转导机制、细胞器和mRNA的运输和定位以及细胞膜的机械和生理特性的调节。位于肠上皮细胞顶端、微绒毛或刷状缘（BB）下的肌动蛋白基细胞骨架装置是自然界中最高级有序的细胞骨架阵列之一。几十年来，BB细胞骨架一直是研究肌动蛋白细胞骨架的组织、组成和功能特性的模型系统。拟议的研究将集中在几个肌动蛋白为基础的分子马达（肌球蛋白），在脊椎动物和果蝇的BB细胞骨架的主要组成部分。研究的肌球蛋白是I类肌球蛋白，是由BB肌球蛋白-I（BBMI）组成的15种已知结构微绒毛的肌球蛋白之一，其含有几个结合的钙调蛋白轻链，是最好的生化特征肌球蛋白之一，但关于其在体内的功能还不清楚。建议的功能包括这种肌球蛋白在维持顶端膜极性通过囊泡运输或促进营养吸收的微绒毛运动的产生或通过机械化学调节BB膜中的膜转运蛋白的作用。这些和其他假设将通过缺乏BBMI基因的小鼠的表型表征进行测试（小鼠由合作者制造）。研究将包括BBMI-/-小鼠在肠细胞形态、极性、细胞骨架组成和稳定性方面的表型表征。将检查对肠道功能的影响，包括营养转运和对肠道病原体的反应以及粘膜损伤。BBMI的体内动力学将通过在肠上皮细胞系Caco 2BBe中观察GFP标记的BMIO来确定。在果蝇中，肠道BB含有两种I类肌球蛋白，肌球蛋白-Ib（MIb）和肌球蛋白-Ia（MIa）。初步分析表明，这两种肌球蛋白都是必需的。缺乏MIb的动物，像BBMI一样将BB膜拴在下面的细胞骨架上，会随着一龄幼虫的生长停滞而死亡，这是一种饥饿的表型。这表明MIb在营养吸收中的关键作用。MIb和MIa的表征将包括多个突变等位基因的产生和对上皮细胞组织、极性、BB结构/组成的影响的表型评估以及对肠道生理学的影响的评估。

The actin-based cytoskeleton plays essential roles in a wide range of cellular phenomena including generation of cell shape, polarity , motility, cell signal transduction mechanisms, transport and localization of organelles and mRNAs, and modulation of the mechanical and physiological properties of cell membranes. The actin-based cytoskeletal apparatus that underlies the apical, microvillous or brush border (BB) of the intestinal epithelial cell is among the most high ordered cytoskeletal arrays in nature. The BB cytoskeleton has served for decades as a model system to investigate the organization, composition and functional properties of the actin-based cytoskeleton. The proposed studies will focus on several actin-based molecular motors (myosins) that are major components of the BB cytoskeleton in both vertebrates and Drosophila. The myosins to be investigated are class I myosins, one of fifteen known structurally microvillous by bridges composed of BB myosin-I (BBMI), which contains several bound calmodulin light chains, is among the best biochemically characterized myosins, yet nothing is known regarding its functions in vivo. Proposed functions include a role for this myosin in maintaining apical membrane polarity through vesicle transport or facilitation of nutrient absorption either by generation of microvillar movements or through mechanochemical regulation of membrane transporters in the BB membrane. These and other hypotheses will be tested through the phenotypic characterization of mice lacking the gene for BBMI (mice were made by collaborators). Studies will include phenotypic characterization BBMI-/- mice with respect to eneterocyte morphology, polarity, cytoskeletal composition and stability. Effects on intestinal functions including nutrient transport and response to intestinal pathogens and mucosal injury will be examined. In vivo dynamics of BBMI will be determined through visualization of GFP-tagged BMIO in the intestinal epithelial cell line, Caco2BBe. In Drosophila, the intestinal BB contains two class-I myosins, myosin-Ib (MIb) and myosin-Ia (MIa). Preliminary analysis suggests that both of these myosins are essential. Animals lacking MIb, which like BBMI tethers the BB membrane to underlying cytoskeleton, die as growth arrested first instar larvae, a phenocopy of starvation. This suggests a critical role for MIb in nutrient absorption. Characterization of MIb and MIa will include generation of multiple mutant alleles and phenotypic assessment of effects on epithelial cell organization, polarity, BB structure/composition and assessment of effects on gut physiology.