Cellular functions of Ran GTPase

Ran GTPase 的细胞功能

• 批准号: 8349319
• 负责人: Petr Kalab
• 金额: $ 56.92万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349319
• 关键词: Address; Adenomatous Polyposis Coli; Attenuated; BRCA1 gene; Binding; Breast Cancer Cell; Cancer Etiology; Cancer cell line; Cell Cycle; Cell Nucleus; Cell division; Cell physiology; Cells; Chromatin; Chromosome Positioning; Chromosomes; Complex; Cultured Cells; Cytoplasm; Defect; Diffusion; Embryo; Employee Strikes; Energy-Generating Resources; Fibroblasts; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Goals; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; HMMR gene; Hela Cells; Human; Image; Importins; Interphase; Life; Malignant Neoplasms; Manuscripts; Measurement; Mediating; Meiosis; Methylation; Microscopy; Mitosis; Mitotic; Mitotic spindle; Molecular; Mus; N-terminal; NPM1 gene; Non-Malignant; Normal Cell; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Oocytes; Organism; Pathway interactions; Phosphorylation; Process; Prometaphase; Proteomics; Publications; Publishing; Relative (related person); Research; Role; Running; Serine; Somatic Cell; Somatic Mutation; TACC3 gene; Tissues; Up-Regulation; Writing; Xenopus laevis; adenoma; alpha Karyopherins; beta Karyopherins; cancer cell; cancer prevention; cancer therapy; cell type; egg; exportin 1 protein; falls; human STK6 protein; human tissue; imaging modality; improved; mRNA decapping; malignant breast neoplasm; nucleocytoplasmic transport; receptor; reconstitution; sensor; survivin; tissue culture; tissue/cell culture; tumor

Ran GTPase is a key regulator of macromolecular transport between nucleus and cytoplasm and has important role in several steps of cell division, including mitotic spindle assembly and nuclear envelope reformation at the exit from mitosis. Because RCC1, the guanine nucleotide exchange factor for Ran, binds to chromatin while RanGAP is cytoplasmic, the position of chromosomes is marked by the highest cellular concentration of RanGTP, the RanGTP gradient. Most, but not all, functions of Ran are mediated by its interactions with importin beta-related nuclear transport receptors (NTRs). Ran and NTRs functionally interact with nucleoporins (Nups) the components of NPCs. In interphase, step-wise RanGTP gradient across nuclear envelope provides direction and is also a source of energy for Ran-regulated transport of cargos carried by NTRs through the channels of nuclear pore complexes. In mitosis, diffusion limited RanGTP gradient induces localized release of spindle assembly factors (SAFs) from their inhibitory complexes with nuclear import receptors, importins. As a result, SAFs are preferably activated in mitotic cytoplasm surrounding chromosomes, providing essential spatial bias to mitotic spindle assembly. However, some SAFs are regulated by RanGTP in mitosis with no requirement for the existence of spatially resolved RanGTP gradient. Remarkably, most of the SAFs involved in Ran-regulated mitotic network are well known as cancer-related factors: TPX2, Aurora A, hTOG, HURP, BRCA1, RHAMM, NPM1, RASSF1a, TACC3/maskin, survivin, APC (adenoma polyposis coli) and others. In addition, more recently it was shown that upregulation of RanGTP gradient leads to transformation of NIH3T3 cells apparently through causing amplification of RanGTP-gradient dependent cytoplasmic decapping of mRNAs, which and thus inducing deregulated synthesis of growth promoting functions. In summary, multiple pieces of evidence suggest that potentially several different RanGTP gradient-regulated processes have an important role in cancer etiology. We are focusing on the role of Ran in mitotic spindle assembly and our goal is to elucidate differences, if any, in the contribution of Ran to mitosis in cancer cells vs. normal cells. Many of the Ran-regulated mitotic mechanisms of spindle assembly are highly conserved between different organisms. Thus, Ran-regulated SAFs) carry similar functions in Xenopus laevis meiotic/embryonic egg extracts, in meiotic mouse oocytes and in human tissue culture cells, suggesting their evolutionary conservation. For example, TPX2 activates Aurora A in HeLa cells and in X. laevis egg extracts. However, the relative contribution to spindle assembly and cell division is dramatically between different types of cells, such as in comparison of meiotic vs. somatic cells. We use two approaches in addressing these important questions: 1) Quantitative analysis of RanGTP gradient in mitotic normal and cancer cells 2) Proteomic and functional reconstitution analysis of Ran-regulated mitotic spindle assembly. In the first approach, in 2009/10 we developed improved FRET sensors for quantitative fluorescence lifetime imaging microscopy (FLIM) measurements of RanGTP gradient in live cells. Using these sensors, in the fall of 2010 we discovered that in striking contrast to HeLa, mitotic RanGTP gradient is virtually absent in normal primary human fibroblasts. This surprising finding was confirmed by two different FRET imaging methods, each with two different FRET sensors: RanGTP- binding sensor and sensor for RanGTP-regulated importin beta cargos. Moreover, we soon also determined that metastatic breast cancer MCF10CA1a cells displayed stronger RCC1 binding to chromatin and steeper mitotic RanGTP gradient than isogenic normal or non-malignant immortalized breast cancer cell lines. Because of the potentially high significance of this finding, in 2011 we focused most of the research in the lab on investigating the molecular mechanism underlying the differences in RanGTP gradient in normal vs. cancer cells. We found addition to increased concentration of Ran, the key factor responsible for steeper RanGTP gradients was increased RCC1 binding to chromatin. Live-cells fluorescence recovery after photobleaching (FRAP) measurements with RCC1-mCherry showed that throughout the cell cycle, the binding of RCC1 to chromatin was stronger in HeLa than in fibroblasts, required N-terminal methylation by NRMT and was supported by RCC1 phosphorylation on Serine 10. While NRMT depletion caused decreased RCC1 binding to chromatin and spindle defects in HeLa, the same treatment had no significant effect on spindle assembly in normal fibroblasts. Consistent with proposed Ran functions in mitotic spindle assembly, the absence of a steep mitotic RanGTP gradient in primary cells correlated with extended prometaphase. These findings suggest that RanGTP gradient and its role in mitotic spindle assembly are attenuated in normal somatic tissues by mechanisms including decreased RCC1 methylation. On the other hand, steep mitotic RanGTP gradient is a commonly expressed hallmark of rapidly dividing normal and cancer cells. We wrote a manuscript describing these findings and plan submitting it for publication within the next few weeks (aiming for October 1, 2011).

RAN GTP酶是核质间大分子运输的关键调节因子，在细胞分裂的几个步骤中发挥重要作用，包括有丝分裂纺锤体组装和有丝分裂出口核膜重塑。由于RCC1是RAN的鸟嘌呤核苷酸交换因子，与染色质结合，而RanGAP是细胞质的，染色体的位置由RanGTP的最高细胞浓度，RanGTP梯度来标记。RAN的大部分(但不是全部)功能是通过与Importinβ相关的核运输受体(NTRs)的相互作用来实现的。RAN和NTRs在功能上与核孔蛋白(NUP)相互作用，这些核孔蛋白是NPC的组成部分。在间期，跨核膜的逐步RanGTP梯度提供了方向，也是RAN调节的NTRs通过核孔复合体通道运输货物的能量来源。在有丝分裂中，扩散受限的RanGTP梯度诱导纺锤体组装因子(SAF)从其与核输入受体Importins的抑制复合体中局部释放。因此，SAF最好在染色体周围的有丝分裂细胞质中被激活，为有丝分裂纺锤体的组装提供必要的空间偏向。然而，一些SAF在有丝分裂过程中受到RanGTP的调控，而不需要存在空间分辨的RanGTP梯度。值得注意的是，大多数参与RAN调控的有丝分裂网络的SAF都是众所周知的癌症相关因子：TPX2、Aurora A、hTOG、HURP、BRCA1、RHAMM、NPM1、RASSF1a、TACC3/Maskin、Survivin、APC(结肠腺瘤息肉病)等。此外，最近的研究表明，RanGTP梯度的上调明显导致NIH3T3细胞的转化，这是通过扩增依赖RanGTP梯度的细胞质去核糖核酸，从而诱导促进生长功能的非调控合成而实现的。综上所述，多项证据表明，几种不同的RanGTP梯度调节过程在癌症病因学中具有重要作用。我们的重点是RAN在有丝分裂纺锤体组装中的作用，我们的目标是阐明RAN对癌细胞和正常细胞有丝分裂的贡献是否存在差异。纺锤体组装的许多RAN调节的有丝分裂机制在不同的生物之间高度保守。因此，RAN调控的SAF)在非洲爪哇减数分裂/胚胎卵子提取物、小鼠减数分裂卵母细胞和人类组织培养细胞中具有相似的功能，表明它们在进化上是保守的。例如，TPX2激活HeLa细胞和莱维氏X.laevis卵子提取物中的Aurora A。然而，不同类型的细胞对纺锤体组装和细胞分裂的相对贡献是显著的，例如减数分裂细胞与体细胞的比较。我们使用两种方法来解决这些重要问题：1)有丝分裂正常细胞和癌细胞RanGTP梯度的定量分析；2)RAN调节的有丝分裂纺锤体组装的蛋白质和功能重组分析。在第一种方法中，我们在2009/10年度开发了改进的FRET传感器，用于定量荧光寿命成像显微镜(FLIM)测量活细胞中的RanGTP梯度。利用这些传感器，我们在2010年秋天发现，与HeLa形成鲜明对比的是，在正常的原代人成纤维细胞中，几乎没有有丝分裂的RanGTP梯度。这一令人惊讶的发现得到了两种不同的FRET成像方法的证实，每种方法都有两种不同的FRET传感器：RanGTP结合传感器和RanGTP调节的Importin beta货物的传感器。此外，我们很快还发现，转移性乳腺癌MCF10CA1a细胞与染色质的RCC1结合更强，有丝分裂RanGTP梯度更大，比同基因正常或非恶性永生化的乳腺癌细胞系更强。由于这一发现潜在的高度重要性，2011年，我们将实验室的大部分研究集中在研究正常细胞和癌细胞RanGTP梯度差异的分子机制上。我们发现，除了RAN浓度的增加外，导致RanGTP梯度变陡的关键因素是RCC1与染色质的结合增加。RCC1-mCherry的活细胞荧光恢复(FRAP)检测表明，在整个细胞周期中，HeLa中RCC1与染色质的结合比成纤维细胞强，需要N端甲基化，并得到丝氨酸10上RCC1磷酸化的支持。虽然NRMT缺失会导致HeLa中RCC1与染色质的结合减少和纺锤体缺陷，但同样的处理对正常成纤维细胞的纺锤体组装没有显著影响。与已提出的RAN在有丝分裂纺锤体组装中的功能一致，原代细胞中缺乏陡峭的有丝分裂RanGTP梯度与延长的前中期相相关。这些发现表明，在正常的体细胞组织中，RanGTP梯度及其在有丝分裂纺锤体组装中的作用受到包括RCC1甲基化减少在内的机制的减弱。另一方面，陡峭的有丝分裂RanGTP梯度是正常细胞和癌细胞快速分裂的常见标志。我们写了一份手稿，描述了这些发现，并计划在接下来的几周内提交出版(目标是2011年10月1日)。