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
基本信息
- 批准号:10521921
- 负责人:
- 金额:$ 44.79万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ActinsActivities of Daily LivingApicalArchitectureAreaBindingBiological ModelsBrush BorderBundlingCell Differentiation processCell LineCell membraneComplexCultured CellsDataDefectEPS8 geneElectron MicroscopyEnterocytesEpithelialEpithelial CellsEventExhibitsFilamentFingersFundingGlycocalyxGrowthImageImaging technologyIn VitroIndividualInternetIntestinesKnockout MiceLabelLeadMapsMembraneMicrobeMicrofilamentsMicroscopyMitotic spindleMolecularMorphogenesisNutrientPathologyPathway interactionsPhysiologyPositioning AttributePropertyProteinsResolutionRoleSiteSmall IntestinesSpecific qualifier valueStructureSurfaceTechnologyTestingTimeTissuesVillusapical membranebasecellular microvillusdensityhuman diseasein vivoinsightintestinal epitheliumlight microscopymalformationmechanical forcemouse modelnovelnutrient absorptionpathogenpolymerizationreconstitutionrecruitsoluteuptake
项目摘要
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
在分化过程中,肠细胞会形成广泛的微绒毛顶端阵列,称为刷状缘,
用于扩大可用于营养吸收的质膜表面积。单个微绒毛
结构简单,由从顶端突出的约 25 根肌动蛋白丝组成的支撑核心束组成
表面包裹着薄膜。除了作为营养吸收的唯一场所外,刷状缘微绒毛
还为糖萼提供锚定点并调节与腔内微生物的相互作用。虽然
刷状缘是肠道的主要功能界面,是驱动及时肠道的机制
直到最近,肠上皮细胞分化过程中微绒毛的形成仍不清楚。在我们的第一次融资期间
在此期间,我们发现了在微绒毛形成过程中控制肌动蛋白丝聚合的几个因素,
包括 IRTKS/EPS8 复合体。然而,构建稳定的微绒毛还需要肌动蛋白丝
组织成芯束,其弯曲刚度足够高,足以使顶面变形。如何
新生肠细胞协调肌动蛋白丝在空间聚合和成束的基本活动
建立稳定的微绒毛的时间仍然未知。在最近的初步研究中,我们使用了邻近标记
在微绒毛组装过程中识别 IRTKS/EPS8 点约 20 nm 范围内的蛋白质的方法;这个屏幕导致
我们激动人心地发现有丝分裂纺锤体定位 (MISP) 作为一种新的肌动蛋白丝束蛋白
刷边框。 MISP 沿完整的隐窝绒毛轴表达,定位于顶端表面。更近一些
超分辨率显微镜检查表明,MISP 在核心上表现出惊人的特异性富集
束根。在培养细胞中,我们发现 MISP 可以稳定并延长细根,并招募其他细胞
这些位点的规范肌动蛋白捆绑器。重要的是,我们发现纯化的 MISP 足以组织肌动蛋白
丝在体外形成紧密的线性束。最后,我们对 MISP 敲除小鼠的初步分析揭示了
细根明显减少,微绒毛表面密度降低。根据我们的初步数据,我们建议
以下中心假设:在分化的肠上皮细胞的顶端表面,MISP 组织肌动蛋白
IRTKS/EPS8复合物产生的细丝形成支撑刷子突出的核心束
边界微绒毛。采用最先进的光学和电子显微镜技术的独特组合
新颖的生物模型系统,我们将:(目标 1)确定 MISP 是否指定微绒毛生长位点
顶端表面,(目标 2)定义 MISP 肌动蛋白结合和捆绑的机制,(目标 3)阐明功能
MISP 在体内肠细胞分化中的作用。我们期望这些目标的完成将带来新的范式
用于了解肠上皮形态发生。
项目成果
期刊论文数量(0)
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MATTHEW J TYSKA其他文献
MATTHEW J TYSKA的其他文献
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{{ truncateString('MATTHEW J TYSKA', 18)}}的其他基金
Acquisition of a Focused Ion Beam Scanning Electron Microscope with cryo-stage
配备冷冻台的聚焦离子束扫描电子显微镜
- 批准号:
10415675 - 财政年份:2022
- 资助金额:
$ 44.79万 - 项目类别:
Myosin-2 function in the enterocyte terminal web
肌球蛋白 2 在肠细胞终末网中的功能
- 批准号:
10370436 - 财政年份:2021
- 资助金额:
$ 44.79万 - 项目类别:
Myosin-2 function in the enterocyte terminal web
肌球蛋白 2 在肠细胞终末网中的功能
- 批准号:
10578826 - 财政年份:2021
- 资助金额:
$ 44.79万 - 项目类别:
Myosin-2 function in the enterocyte terminal web
肌球蛋白 2 在肠细胞终末网中的功能
- 批准号:
10211464 - 财政年份:2021
- 资助金额:
$ 44.79万 - 项目类别:
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 - 财政年份:2017
- 资助金额:
$ 44.79万 - 项目类别:
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