Mechanisms of Hepatocyte Polarization and Apical Tube Formation

肝细胞极化和心尖管形成的机制

• 批准号: 10598034
• 负责人: Erfei Bi
• 金额: $ 38.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598034
• 关键词: Actin-Binding Protein; Actins; Adherens Junction; Affect; Apical; Architecture; Attenuated; Basic Science; Bile fluid; Binding; Biogenesis; Biology; Blood Circulation; Candidate Disease Gene; Cell model; Cells; Cholestasis; Cytokinesis; Cytoskeleton; Defect; Development; Drug Metabolic Detoxication; Epithelial Cells; Epithelium; Excretory function; Exocytosis; Filament; Gene Expression Profile; Genes; Geometry; Gold; Growth; Guanine Nucleotide Exchange Factors; Hepatic Cord; Hepatocyte; Human; In Vitro; Kinesin; Link; Liver; Liver diseases; Location; Mediating; Membrane; Metabolism; Microfilaments; Microtubules; Motor; Myosin Type V; Natural regeneration; Organ; Pathway interactions; Phosphatidylinositols; Play; Plus End of the Microtubule; Primary carcinoma of the liver cells; Process; Production; Proliferating; Public Health; Rattus; Recycling; Role; Scaffolding Protein; Serum Proteins; Side; Site; Small Interfering RNA; Surface; Testing; Tight Junctions; Time; Tube; Tubular formation; Vesicle; Visceral; cytohesin-1; daughter cell; gene discovery; in vivo; interdisciplinary approach; liver development; liver function; scaffold; spatiotemporal; trafficking; transcriptomics; vesicle transport

Project Summary/Abstract: The functions of the liver, including detoxification, synthesis of serum proteins, and bile production, critically depend on hepatocyte polarization and bile canaliculus (BC) formation. Defects in these processes are associated with serious liver diseases such as cholestasis and hepatocarcinoma. Using the rat hepatocyte line Can 10, the only known cells that can proliferate and form “tubular” BCs (tBCs) in vitro that resembles those in vivo, we discovered that hepatocyte polarization and “primordial” BC formation are linked to cytokinesis. This division-linked mechanism also applies to BC biogenesis during liver development. However, it remains a mystery how a primordial BC formed between two daughter cells at the division site is remodeled and grown into a tBC nestled between two rows of cells, as seen in the liver. We hypothesize that tBC formation involves spatiotemporal coordination of oriented divisions, with BC expansion driven by targeted exocytosis, elongation by pushing forces from both sides of the BC, and extension along the cell-cell contact through constant remodeling of the adherens and tight junctions. To identify the steps and key players involved, we employed two non-biased approaches. The first involves identification of genes from single-cell transcriptomic analysis of liver development that are induced or increased at stages of hepatocyte polarization and BC formation. The second involves identification of genes whose expressions are increased in the polarized Can 10 cells versus the unpolarized parental cells. These complementary approaches have led to the discovery of numerous genes that may play an important role in a process from the terminal stage of cytokinesis to tBC formation. 11 candidate genes were selected for a small siRNA-based screen, three (KIF21B, ABLIM3, and IPCEF1) were found to be required for tBC formation. None of these has been implicated previously in epithelial tube formation. In this application, we will use an interdisciplinary approach to test our hypotheses that the microtubule (MT) plus end-directed motor Kif21B and the actin-binding protein Ablim3 act in concert to drive BC expansion by controlling MT- and actin-mediated vesicle transport as well as adherens junction (AJ) assembly (Aim 1), and that the scaffold protein Ipcef1 promotes Arf6 activation by distinct cytohesins (GEFs) at distinct locations to control AJ remodeling, apical vesicle recycling, and actin-based protrusions to drive BC elongation (Aim 2). As the mechanisms of apical tube formation are highly conserved from worms to humans, the impact of our proposed study will likely reach far beyond the field of liver biology.

项目概要/摘要： 肝脏的功能，包括解毒，血清蛋白的合成和胆汁的产生， 依赖于肝细胞极化和胆小管（BC）的形成。这些过程中的缺陷是 与严重的肝脏疾病如胆汁淤积和肝癌相关。使用大鼠肝细胞 细胞系Can 10，唯一已知的可以在体外增殖和形成"管状" BC（tBC）的细胞， 在体内，我们发现肝细胞极化和"原始" BC形成与 胞质分裂这种分裂相关机制也适用于肝脏发育期间的BC生物合成。 然而，在分裂位点的两个子细胞之间形成的原始BC是如何形成的仍然是个谜。 重塑并生长成位于两排细胞之间的tBC，如在肝脏中所见。 我们假设，tBC的形成涉及时空协调的定向分工，与BC 由靶向胞吐作用驱动的扩张，由来自BC两侧的推力驱动的伸长，以及 通过粘附和紧密连接的不断重塑沿着细胞-细胞接触延伸。到 为了确定所涉及的步骤和主要参与者，我们采用了两种无偏见的方法。第一个涉及 从肝脏发育的单细胞转录组学分析中鉴定诱导或 在肝细胞极化和BC形成阶段增加。第二个问题是识别 与未极化的亲本细胞相比，在极化的Can 10细胞中表达增加的基因。 这些互补的方法已经导致了许多基因的发现，这些基因可能在基因组中起重要作用。 在从胞质分裂的终末阶段到tBC形成的过程中起作用。筛选出11个候选基因 对于基于siRNA的小规模筛选，发现tBC需要三个（KIF21B、ABLIM3和IPCEF1 阵这些都没有牵连以前在上皮管的形成。在本申请中，我们 我将使用跨学科的方法来测试我们的假设，微管（MT）加上末端导向 马达Kif21B和肌动蛋白结合蛋白Ablim3协同作用，通过控制MT- 和肌动蛋白介导的囊泡运输以及粘附连接（AJ）组装（Aim 1）， 支架蛋白Ipcef 1通过不同位置的不同细胞粘连蛋白（GEF）促进Arf 6活化，以控制 AJ重塑、顶端囊泡再循环和基于肌动蛋白的突起驱动BC延长（目的2）。为 从蠕虫到人类，顶管形成的机制是高度保守的， 拟议的研究可能会远远超出肝脏生物学领域。