FUNCTIONS FOR MYOSINS-I OF THE INTESTINAL BRUSH BORDER

以肌动蛋白为基础的细胞骨架在细胞形态、极性、运动、细胞信号转导机制、细胞器和mRNAs的运输和定位以及细胞膜的机械和生理特性的调节等广泛的细胞现象中发挥着重要作用。基于肌动蛋白的细胞骨架位于肠上皮细胞的顶端、微绒毛或刷状边缘(BB)，是自然界中排列最有序的细胞骨架之一。几十年来，BB细胞骨架一直被用作研究基于肌动蛋白的细胞骨架的组织、组成和功能特性的模型系统。拟议的研究将集中在几个基于肌动蛋白的分子马达(肌球蛋白)，这些分子马达是脊椎动物和果蝇BB细胞骨架的主要组成部分。被研究的肌球蛋白是I类肌球蛋白，是由BB肌球蛋白-I(BBMI)组成的15种已知的结构微绒毛之一，它含有几个结合的钙调蛋白轻链，是生化特征最好的肌球蛋白之一，但对其在体内的功能尚不清楚。已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机械化学调节BB膜中的膜转运蛋白来促进营养吸收，从而维持顶膜的极性。这些和其他假设将通过缺乏BBMI基因的小鼠的表型特征来检验(小鼠是由合作者制造的)。研究将包括BBMI-/-小鼠关于内细胞形态、极性、细胞骨架组成和稳定性的表型特征。将检查对肠道功能的影响，包括营养物质的运输以及对肠道病原体和粘膜损伤的反应。BBMI的体内动力学将通过在肠道上皮细胞系Caco 2BBe中显示GFP标记的BMIO来确定。果蝇肠道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，就像BBMI一样，将BB膜系在下面的细胞骨架上，随着一龄幼虫的生长受阻而死亡，这是饥饿的一种表现。这表明MIB在养分吸收中起着关键作用。MIB和MIA的特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.