Molecular characteristics of the apical recycling system

顶端回收系统的分子特征

• 批准号: 8539175
• 负责人: JAMES Richard GOLDENRING
• 金额: $ 8.71万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539175
• 关键词: Address; Apical; Binding; Brush Border; Caco-2 Cells; Celiac Disease; Cell Polarity; Cell model; Cells; Characteristics; Child; Clinical; Complex; Cytomegalovirus Infections; Diarrhea; Digestion; Disease; Enterocytes; Enzymes; Europe; Family; Family member; Food; In Vitro; Intestinal Absorption; Intestines; Investigation; Kidney; Knockout Mice; Knowledge; Lead; Liquid substance; Maintenance; Mediating; Membrane; Modeling; Molecular; Monomeric GTP-Binding Proteins; Motor; Mutation; Myosin ATPase; Navajo; Pathogenesis; Pathway interactions; Patients; Phenotype; Physiology; Point Mutation; Proteins; Pump; Recycling; Regulation; Role; Small Intestines; Structure; Study Subject; Syndrome; System; Tissues; Tribes; Vacuole; Vesicle; Work; absorption; apical membrane; cellular microvillus; disease phenotype; experience; in vitro Model; in vivo; insight; intestinal epithelium; loss of function; loss of function mutation; membrane assembly; protein transport; trafficking

DESCRIPTION (provided by applicant): Investigations over the past 5 years have led to the identification of regions of myosin Vb (Myo5B) responsible for its interactions with multiple Rab proteins and other effectors. Our studies have determined distinct regions in myosin Vb that are responsible for binding to Rab11 family members (Rab11a, Rab11b and Rab25), Rab8a and Rab10. Recently, work from patients in Europe and from the Navajo tribe has identified mutations in Myo5B as the cause of the rare congenital diarrhea syndrome Microvillous Inclusion Disease (MVID). Children with MVID have intractable diarrhea due to an apparent loss of absorptive capacity in the small intestine. Morphologically, the small intestinal cells show loss of the apical brush border and the presence of intracellular inclusions that appear to represent intracellular apical membrane vacuoles. All of the patients from the Navajo tribe have a single point mutation that inactivates proper motor function. These findings suggest that mutations in Myo5B lead to alterations in proper trafficking of proteins to the apical membrane and the assembly of the apical brush border. Given our experience with dissecting out the effects of Myo5B mutations on trafficking, we now propose to evaluate the underlying abnormalities that result from loss of function Myo5B mutations in MVID. We hypothesize that Myo5B, in concert with Rab11a and Rab8a, is responsible for the establishment and maintenance of polarized apical membrane structures including the intestinal microvilli. To address this hypothesis, we will first establish an in vitro model of MVID through the reduction of endogenous Myo5B expression and re-expression of Myo5B missense point mutations in polarized CaCo-2 cells in culture. These studies will allow putative recapitulation of the disease phenotype in vitro by re-expression of the Myo5B Navajo mutation in cells with endogenous knockdown. This in vitro cell model can be evaluated for changes in microvillar assembly as well as alterations in cell polarity. In addition, we will be able to manipulate the phenotype in knockdown cells to evaluate particular Rab-associated pathways associated with Myo5B. Finally we will be able to compare observations in the in vitro system with alterations in tissues from patients with the Myo5B Navajo mutation. Second, we will examine the effects of loss of Myo5B in Myo5B knockout mice. By developing both constitutive and intestinal mucosal-targeted deletion models for Myo5B, we will be able to evaluate the effects of Myo5B inactivation on intestinal cell apical polarity and function. These effects will be compared with both in vitro models and the in vivo effects observed in patients with the Navajo mutation. Overall these studies will not only provide information on how mutations in Myo5B can lead to the sequelae of MVID, but will also determine how alterations in Myo5B- regulated trafficking can lead to alterations in intestinal cell polarity.

描述（由申请人提供）：过去5年中的调查导致了肌球蛋白VB（MyO5B）的识别，负责其与多种RAB蛋白和其他效应子的相互作用。我们的研究确定了肌球蛋白VB中的不同区域，这些区域负责与Rab11家族成员（RAB11A，RAB11B和RAB25），RAB8A和RAB10结合。最近，欧洲和纳瓦霍人部落的患者的工作已经确定了Myo5b中的突变是罕见的先天性腹泻综合征微文化包容性疾病（MVID）的原因。由于小肠的吸收能力明显丧失，患有MVID的儿童患有顽固性腹泻。从形态上讲，小肠细胞显示顶端刷边界的丧失以及似乎代表细胞内顶膜液泡的细胞内夹杂物的存在。来自纳瓦霍人部落的所有患者均具有单点突变，使运动功能失活。这些发现表明，Myo5b中的突变导致蛋白质适当地转移到顶膜和顶端刷子边界的组装中。鉴于我们剖析了Myo5b突变对贩运的影响的经验，我们现在建议评估MVID功能MYO5B突变丧失导致的潜在异常。我们假设Myo5b与Rab11a和Rab8a一致，负责建立和维护（包括肠道微绒毛）的极化顶膜结构。为了解决这一假设，我们将首先通过减少培养物中极化CACO-2细胞中的内源性myo5b表达和重新表达MyO5b错义点突变的重新表达来建立MVID的体外模型。这些研究将通过内源性敲低的细胞中的Myo5b Navajo突变在细胞中重新表达疾病表型，从而可以在体外提出疾病表型。可以评估这种体外细胞模型的微绒毛组件的变化以及细胞极性的改变。此外，我们将能够操纵敲低细胞中的表型，以评估与MyO5B相关的特定RAB相关途径。最后，我们将能够比较体外系统中的观察结果与MyO5B Navajo突变患者的组织改变。其次，我们将研究Myo5b敲除小鼠中MyO5b丢失的影响。通过为MyO5B开发构成和肠粘膜靶向的缺失模型，我们将能够评估Myo5b失活对肠细胞顶端极性和功能的影响。这些作用将与体外模型和在纳瓦霍突变患者中观察到的体内效应进行比较。总体而言，这些研究不仅将提供有关Myo5b中突变如何导致MVID后遗症的信息，而且还将确定MyO5B调节的运输的变化如何导致肠细胞极性的改变。