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.

项目摘要/摘要： 肝脏的功能，包括解毒、合成血清蛋白和胆汁生成，至关重要 取决于肝细胞极化和胆小管（BC）的形成。这些过程中存在的缺陷是 与严重的肝脏疾病如胆汁淤积和肝癌有关。使用大鼠肝细胞 Can 10 系是唯一已知的能够在体外增殖并形成“管状”BC (tBC) 的细胞，其类似于 在体内实验中，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过程中的BC生物发生。 然而，两个子细胞在分裂位点之间如何形成原始BC仍然是一个谜。 重塑并生长成位于两排细胞之间的 tBC，如肝脏中所见。 我们假设 tBC 的形成涉及定向分裂的时空协调，其中 BC 由定向胞吐作用驱动的扩张，由 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，沿着细胞与细胞接触延伸。到 为了确定所涉及的步骤和关键参与者，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分析中鉴定被诱导或 在肝细胞极化和BC形成阶段增加。第二个涉及识别 与未极化的亲本细胞相比，极化的 Can 10 细胞中表达增加的基因。 这些互补的方法导致了许多可能发挥重要作用的基因的发现。 胞质分裂末期到 tBC 形成过程中的作用。选出11个候选基因 对于基于 siRNA 的小型筛选，发现 tBC 需要三个（KIF21B、ABLIM3 和 IPCEF1） 形成。之前这些都没有涉及上皮管的形成。在这个应用程序中，我们 将使用跨学科方法来检验我们的假设，即微管（MT）加上末端定向 运动 Kif21B 和肌动蛋白结合蛋白 Ablim3 协同作用，通过控制 MT- 来驱动 BC 扩张 和肌动蛋白介导的囊泡运输以及粘附连接 (AJ) 组装（目标 1），并且 支架蛋白 Ipcef1 通过不同位置的不同细胞粘附素 (GEF) 促进 Arf6 激活 AJ 重塑、顶端囊泡回收和基于肌动蛋白的突起可驱动 BC 伸长（目标 2）。作为 从蠕虫到人类，顶管形成的机制是高度保守的，我们的影响 拟议的研究可能会远远超出肝脏生物学领域。