FUNCTIONS FOR MYOSINS-I OF THE INTESTINAL BRUSH BORDER

肌动蛋白为基础的细胞骨架在广泛的细胞现象中发挥重要作用，包括细胞形状、极性、运动、细胞信号转导机制的产生、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、微绒毛或刷状边缘（BB）的肌动蛋白基细胞骨架装置是自然界中最有序的细胞骨架阵列之一。几十年来，BB细胞骨架一直被用作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。拟议的研究将集中于几种基于肌动蛋白的分子马达（肌球蛋白），它们是脊椎动物和果蝇BB细胞骨架的主要组成部分。待研究的肌球蛋白为I类肌球蛋白，是已知的15种由BB肌球蛋白-I （BBMI）组成的微绒毛状结构中的一种，它含有几个结合的钙调蛋白轻链，是生物化学特征最好的肌球蛋白之一，但其在体内的功能尚不清楚。提出的功能包括这种肌球蛋白通过囊泡运输或通过产生微绒毛运动或通过对BB膜中膜转运蛋白的机械化学调节来促进营养吸收，从而维持根尖膜极性。这些和其他假设将通过缺乏BBMI基因的小鼠的表型特征来验证（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。对肠道功能的影响包括营养转运和对肠道病原体和粘膜损伤的反应。通过在肠上皮细胞系Caco2BBe中可视化gfp标记的BMIO来确定BBMI的体内动态。在果蝇中，肠道BB含有两种i类肌球蛋白，myosin-Ib （MIb）和myosin-Ia （MIa）。初步分析表明，这两种肌凝蛋白都是必不可少的。缺乏MIb的动物，像BBMI一样将BB膜系在底层的细胞骨架上，作为生长受阻的一龄幼虫死亡，这是饥饿的一种表现。这表明MIb在营养吸收中起着关键作用。MIb和MIa的表征将包括多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，以及对肠道生理的影响评估。

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.