Toronto Western Research Institute, University of Toronto, 399 Bathurst Street, Toronto, ON M5T2S8, Canada.
J Neuroinflammation. 2012 Aug 8;9:190. doi: 10.1186/1742-2094-9-190.
To perform their functions during development and after central nervous system injury, the brain's immune cells (microglia) must migrate through dense neuropil and extracellular matrix (ECM), but it is not known how they degrade the ECM. In several cancer cell lines and peripheral cells, small multi-molecular complexes (invadopodia in cancer cells, podosomes in nontumor cells) can both adhere to and dissolve the ECM. Podosomes are tiny multi-molecular structures (0.4 to 1 μm) with a core, rich in F-actin and its regulatory molecules, surrounded by a ring containing adhesion and structural proteins.
Using rat microglia, we performed several functional assays: live cell imaging for chemokinesis, degradation of the ECM component, fibronectin, and chemotactic invasion through Matrigel™, a basement membrane type of ECM. Fluorescent markers were used with high-resolution microscopy to identify podosomes and their components.
The fan-shaped lamella at the leading edge of migrating microglia contained a large F-actin-rich superstructure composed of many tiny (<1 μm) punctae that were adjacent to the substrate, as expected for cell-matrix contact points. This superstructure (which we call a podonut) was restricted to cells with lamellae, and conversely almost every lamella contained a podonut. Each podonut comprised hundreds of podosomes, which could also be seen individually adjacent to the podonut. Microglial podosomes contained hallmark components of these structures previously seen in several cell types: the plaque protein talin in the ring, and F-actin and actin-related protein (Arp) 2 in the core. In microglia, podosomes were also enriched in phosphotyrosine residues and three tyrosine-kinase-regulated proteins: tyrosine kinase substrate with five Src homology 3 domains (Tks5), phosphorylated caveolin-1, and Nox1 (nicotinamide adenine dinucleotide phosphate oxidase 1). When microglia expressed podonuts, they were able to degrade the ECM components, fibronectin, and Matrigel™.
The discovery of functional podosomes in microglia has broad implications, because migration of these innate immune cells is crucial in the developing brain, after damage, and in disease states involving inflammation and matrix remodeling. Based on the roles of invadosomes in peripheral tissues, we propose that microglia use these complex structures to adhere to and degrade the ECM for efficient migration.
在中枢神经系统损伤后，大脑的免疫细胞（小胶质细胞）必须穿过密集的神经组织和细胞外基质（ECM）进行迁移，但目前尚不清楚它们如何降解 ECM。在一些癌细胞系和外周细胞中，小的多分子复合物（癌细胞中的侵袭伪足，非肿瘤细胞中的足突）既可以黏附又可以溶解 ECM。足突是一种微小的多分子结构（0.4 至 1μm），核心富含肌动蛋白及其调节分子，周围是一个包含黏附蛋白和结构蛋白的环。
我们使用大鼠小胶质细胞进行了几种功能测定：趋化性、ECM 成分纤维连接蛋白的降解以及通过基底膜类型的 ECM 基质胶进行趋化性侵袭的活细胞成像。使用荧光标记物和高分辨率显微镜来鉴定足突及其组成部分。
在迁移的小胶质细胞的前缘扇形薄片中，包含一个由许多微小的（<1μm）点状结构组成的富含肌动蛋白的超结构，这些点状结构与基质相邻，这与细胞-基质接触点一致。这个超结构（我们称之为足突）仅限于有薄片的细胞，相反，几乎每个薄片都包含一个足突。每个足突都包含数百个足突，也可以单独看到它们与足突相邻。小胶质细胞的足突包含先前在几种细胞类型中看到的这些结构的标志性成分：环中的斑块蛋白塔林，以及核心中的肌动蛋白和肌动蛋白相关蛋白（Arp）2。在小胶质细胞中，足突还富含磷酸酪氨酸残基和三种酪氨酸激酶调节蛋白：含有五个Src 同源 3 结构域的酪氨酸激酶底物（Tks5）、磷酸化的 caveolin-1 和 Nox1（烟酰胺腺嘌呤二核苷酸磷酸氧化酶 1）。当小胶质细胞表达足突时，它们能够降解 ECM 成分纤维连接蛋白和基质胶。
在小胶质细胞中发现功能性足突具有广泛的意义，因为这些固有免疫细胞的迁移对于发育中的大脑、损伤后以及涉及炎症和基质重塑的疾病状态至关重要。基于侵袭伪足在周围组织中的作用，我们提出小胶质细胞使用这些复杂结构来黏附和降解 ECM，以实现有效的迁移。
