Mechanisms of Hepatocyte Polarization and Apical Tube Formation

项目摘要/摘要： 肝脏的功能，包括解毒、血清蛋白合成和胆汁产生，至关重要 取决于肝细胞极化和胆小管(BC)的形成。这些过程中的缺陷是 与严重的肝病有关，如胆汁淤积症和肝癌。利用大鼠肝细胞 LINCAN 10，唯一已知的在体外可以增殖并形成类似于 在体内，我们发现肝细胞极化和原始BC的形成与 胞质分裂。这种分裂相关的机制也适用于肝脏发育过程中的BC生物发生。 然而，如何在分裂地点的两个子细胞之间形成原始BC仍然是一个谜。 重塑并生长成嵌在两排细胞之间的TBC，如在肝脏中所见。 我们假设TBC的形成涉及定向分区的时空协调，BC 由靶向胞吐驱动的扩张，由BC两侧的推力驱动的伸长，以及 通过贴壁和紧密连接的不断重塑，沿着细胞-细胞接触延伸。至 为了确定所涉及的步骤和关键参与者，我们采用了两种不带偏见的方法。第一个涉及到 从肝脏发育的单细胞转录分析中鉴定诱导或诱导的基因 在肝细胞极化期和BC形成期增加。第二个问题涉及识别 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。 这些互补的方法导致了许多基因的发现，这些基因可能在 在从胞质分裂末期到TBC形成的过程中发挥作用。筛选出11个候选基因 对于基于小siRNA的筛查，发现TBC需要三个(KIF21B、ABLIM3和IPCEF1) 队形。这些都不是以前涉及上皮管形成的因素。在此应用程序中，我们 将使用跨学科的方法来测试我们的假设微管(MT)加末端定向 马达Kif21B和肌动蛋白结合蛋白Ablim3协同作用，通过控制MT-3来驱动BC的扩张 和肌动蛋白介导的囊泡运输以及黏附连接(AJ)组装(目标1)，以及 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、心尖小泡循环和以肌动蛋白为基础的突起以驱动BC延长(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 拟议中的研究可能会远远超出肝脏生物学领域。

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.