水通道蛋白 4 (AQP4) 和中间丝 (IF) 蛋白的表达在恶性胶质母细胞瘤 (GBM) 中发生改变，但主要的基于 IF 的细胞接头、凝集素 (PLEC) 的表达及其对 GBM 迁移和侵袭的贡献，未知。在这里，我们评估了凝集素在影响星形胶质细胞中质膜 AQP4 聚集体的分布、迁移特性和细胞体积调节方面的贡献。在人 GBM 中，使用以下方法分析了胶质纤维酸性蛋白 (GFAP)、AQP4 和 PLEC 转录本的表达公开可用的数据集，并通过免疫组织化学确定 PLEC 与 AQP4 和 GFAP 的共定位。我们对野生型和缺乏凝集素的原代和永生化小鼠星形胶质细胞、人星形胶质细胞以及源自人类恶性 GBM 的永久细胞系（U-251 MG 和 T98G）进行了实验。通过定量实时 PCR 评估小鼠星形胶质细胞中凝集素亚型的表达。使用转染、免疫标记和共聚焦显微镜来评估凝集素诱导的细胞骨架分布的改变、凝集素及其亚型对质膜 AQP4 聚集体的丰度和大小的影响，以及质膜上凝集素的存在。通过 ELISA 测量细胞中凝集素的释放。分别通过伤口愈合实验和钙黄绿素标记来评估永生化星形胶质细胞的迁移和细胞体积调节的动态。在肿瘤脑样本中，plectin和AQP4在基因表达和蛋白质定位水平上存在正相关性。凝集素的缺乏导致质膜 AQP4 聚集体的丰度和大小减少，并改变细胞骨架的分布和成束。星形胶质细胞主要表达 P1c、P1e 和 P1g 凝集素亚型。与质膜 AQP4 聚集体相关的主要凝集素亚型是 P1c，它也对星形胶质细胞的迁移率影响最显着。在缺乏凝集素的情况下，星形胶质细胞的集体迁移受到损害，外周细胞区域细胞质体积变化的动态性降低。在 GBM 细胞系中，质膜表面的凝集素丰度及其从细胞中的释放增加。凝集素影响有助于 GBM 病理学的细胞特性。观察到的细胞表面和释放的凝集素水平的增加代表了 GBM 诊断和治疗中的潜在生物标志物和治疗靶标。© 2024。作者。

The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes.In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively.A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin's abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines.Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.© 2024. The Author(s).