Role of actin bundlers during enterocyte differentiation

肌动蛋白成束剂在肠上皮细胞分化过程中的作用

• 批准号: 10661765
• 负责人: MATTHEW J TYSKA
• 金额: $ 44.88万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661765
• 关键词: Actins; Activities of Daily Living; Apical; Architecture; Area; Binding; Biological Models; Brush Border; Bundling; Cell Differentiation process; Cell Line; Cell membrane; Complex; Cultured Cells; Data; Defect; Electron Microscopy; Enterocytes; Epidermal Growth Factor Receptor Pathway Substrate 8; Epithelial Cells; Epithelium; Event; Exhibits; Filament; Fingers; Funding; Glycocalyx; Growth; Image; Imaging technology; In Vitro; Individual; Internet; Intestines; Knockout Mice; Label; Maps; Membrane; Microbe; Microfilaments; Mitotic spindle; Molecular; Morphogenesis; Nutrient; Nutrient availability; Pathology; Pathway interactions; Physiology; Polymers; Positioning Attribute; Property; Proteins; Qualifying; Role; Site; Small Intestines; Specific qualifier value; Structure; Surface; System; Technology; Testing; Time; Tissues; Villus; apical membrane; cellular microvillus; density; human disease; in vivo; insight; intestinal epithelium; light microscopy; malformation; mechanical force; mouse model; novel; nutrient absorption; pathogen; polymerization; reconstitution; recruit; solute; superresolution microscopy; uptake

SUMMARY During differentiation, enterocytes build an extensive apical array of microvilli known as the brush border, which serves to amplify the plasma membrane surface area available for nutrient absorption. An individual microvillus is simple in structure, consisting of a supporting core bundle of ~25 actin filaments that protrudes from the apical surface wrapped in membrane. In addition to serving as the sole site of nutrient uptake, brush border microvilli also provide an anchoring point for the glycocalyx and regulate interactions with luminal microbes. Although the brush border serves as the primary functional interface of the intestinal tract, mechanisms that drive the timely formation of microvilli during enterocyte differentiation remained unclear until recently. During our first funding period, we discovered several factors that control actin filament polymerization during microvilli formation, including the IRTKS/EPS8 complex. However, building stable microvilli also requires that actin filaments are organized into core bundles, which exhibit flexural rigidities high enough to deform the apical surface. How nascent enterocytes coordinate the fundamental activities of actin filament polymerization and bundling in space and time to build stable microvilli remains unknown. In recent preliminary studies, we used a proximity labeling approach to identify proteins within ~20 nm of IRTKS/EPS8 puncta during microvillus assembly; this screen led to our exciting discovery of Mitotic Spindle Positioning (MISP) as a new actin filament bundling protein in the brush border. MISP is expressed along the full crypt-villus axis, where it localizes to the apical surface. Closer inspection with super-resolution microscopy revealed that MISP exhibits strikingly specific enrichment on core bundle rootlets. In cultured cells, we found that MISP stabilizes and elongates rootlets, and recruits other canonical actin bundlers to these sites. Importantly, we found that purified MISP is sufficient to organize actin filaments into tight linear bundles in vitro. Finally, our preliminary analysis of MISP knockout mice revealed a striking loss of rootlets and decrease in microvillar surface density. Based on our preliminary data, we propose the following CENTRAL HYPOTHESIS: At the apical surface of differentiating enterocytes, MISP organizes actin filaments generated by the IRTKS/EPS8 complex to form core bundles that support the protrusion of brush border microvilli. Using a unique combination of state-of-the-art light and electron microscopy technology and novel biological model systems, we will: (Aim 1) determine if MISP specifies sites of microvillar growth at the apical surface, (Aim 2) define the mechanism of MISP actin binding and bundling, (Aim 3) elucidate the function of MISP in enterocyte differentiation in vivo. We expect that completion of these Aims will lead to new paradigms for understanding intestinal epithelial morphogenesis.

概括 在差异化过程中，肠细胞建立了一个广泛的顶端微绒毛阵列，称为刷子边框 用于扩大可用于营养吸收的质膜表面积。单个微绒毛 结构很简单，由〜25个肌动蛋白丝的支撑芯束组成 表面包裹在膜上。除了充当营养摄取的唯一部位外，刷边框微绒毛 还为糖蛋白的锚定点提供了一个锚定点，并调节与腔微生物的相互作用。虽然 刷边框是肠道的主要功能接口，驱动及时的机制 直到最近，在肠细胞分化过程中的微绒毛形成一直尚不清楚。在我们的第一个资金中 时期，我们发现了几个因素，可以控制肌动蛋白丝聚合在微绒毛形成期间， 包括IRTKS/EPS8复合物。但是，建筑稳定的微壁也要求肌动蛋白丝是 组织成核心束，它们表现出足够高的弯曲刚度，以使顶表面变形。如何 新生的肠细胞协调肌动蛋白丝聚合的基本活动和在太空中捆绑 构建稳定微壁的时间仍然未知。在最近的初步研究中，我们使用了接近标签 在微绒毛组装过程中，鉴定irtks/eps8 uncta左右的蛋白质的方法；该屏幕LED 我们令人兴奋地发现有丝分裂主轴定位（MISP）是一种新的肌动蛋白丝捆绑蛋白 刷边框。 MISP沿着完整的隐窝villus轴表达，并在该轴上定位在顶部表面。靠近 超分辨率显微镜检查表明，MISP在核心上表现出极大的特异性富集 束根。在培养的细胞中，我们发现MISP稳定并伸长了根，并招募了其他 这些站点的规范肌动蛋白串。重要的是，我们发现纯化的MISP足以组织肌动蛋白 细丝在体外成紧密的线性束。最后，我们对MISP敲除小鼠的初步分析显示了 根部损失并降低微绒毛表面密度。根据我们的初步数据，我们建议 以下中心假设：在区分肠球细胞的顶端，MISP组织肌动蛋白 IRTKS/EPS8复合物生成的细丝形成了支撑刷子突出的核心束 边界微绒毛。使用最先进的光和电子显微镜技术的独特组合以及 新型生物学模型系统，我们将：（目标1）确定MISP是否指定了在 顶部表面（AIM 2）定义了MISP肌动蛋白结合和捆绑的机理，（AIM 3）阐明该功能 肠肠细胞分化的体内MISP。我们预计这些目标的完成将导致新的范式 理解肠上皮形态发生。

项目成果

Faster Mean-shift: GPU-accelerated clustering for cosine embedding-based cell segmentation and tracking.

• DOI: 10.1016/j.media.2021.102048
• 发表时间: 2021-07
• 期刊: MEDICAL IMAGE ANALYSIS
• 影响因子: 10.9
• 作者: Zhao, Mengyang;Jha, Aadarsh;Liu, Quan;Millis, Bryan A.;Mahadevan-Jansen, Anita;Lu, Le;Landman, Bennett A.;Tyska, Matthew J.;Huo, Yuankai
• 通讯作者: Huo, Yuankai