Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.
J Cell Sci. 2010 Nov 1;123(Pt 21):3693-702. doi: 10.1242/jcs.075275. Epub 2010 Oct 12.
Dishevelled-3 (Dvl3) is a multivalent scaffold protein that is essential to Wnt signaling during development. Although Dvl-based punctae have been visualized by fluorescence microscopy; the physical nature and dynamic character of the such complexes are enigmatic. We use steric-exclusion chromatography, affinity pull-downs, proteomics and fluorescence correlation microscopy to characterize supermolecular Dvl3-based complexes of totipotent mouse F9 cells. The molecular mass of the complexes ranges from that of homodimeric Dvl3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa. Addition of Wnt3a stimulates the formation of Dvl3-based complexes of greater molecular mass within 30 minutes. The presence of DKK1 and knockdown of Dishevelled proteins block formation of the 2 MDa Dvl3-based complexes and also block Wnt3a stimulation of the canonical pathway. Fluorescent correlation microscopy identified supermolecular Dvl3-based complexes with a molecular mass >30 MDa in live cells; these complexes were provoked to form structures with even greater molecular mass by Wnt3a. We establish for the first time the physical and functional nature of very large, supermolecular Dvl3-based complexes.
Dishevelled-3（Dvl3）是一种多价支架蛋白，对发育过程中的 Wnt 信号传导至关重要。尽管已经通过荧光显微镜观察到基于 Dvl 的小点；但此类复合物的物理性质和动态特征仍然是神秘的。我们使用空间排阻层析、亲和下拉、蛋白质组学和荧光相关显微镜来表征全能性小鼠 F9 细胞中超分子 Dvl3 基复合物。复合物的分子量范围从二聚体 Dvl3 到含有 0.4 至 2.0 MDa 超分子复合物的明确峰。添加 Wnt3a 可在 30 分钟内刺激形成更大分子量的基于 Dvl3 的复合物。DKK1 的存在和 Dishevelled 蛋白的敲低会阻止 2 MDa 基于 Dvl3 的复合物的形成，并阻止 Wnt3a 对经典途径的刺激。荧光相关显微镜在活细胞中鉴定出分子量大于 30 MDa 的超分子 Dvl3 基复合物；Wnt3a 可促使这些复合物形成具有更大分子量的结构。我们首次建立了非常大的、超分子 Dvl3 基复合物的物理和功能性质。
