Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Max Perutz Laboratories, Department of Biochemistry and Cell Biology, University of Vienna, Vienna, Austria.
J Biomed Sci. 2024 Jan 23;31(1):14. doi: 10.1186/s12929-024-01002-z.
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.
水通道蛋白 4（AQP4）和中间丝（IF）蛋白在恶性脑胶质瘤（GBM）中的表达发生改变，但主要的 IF 细胞连接蛋白——网蛋白（PLEC）的表达及其对 GBM 迁移和侵袭的贡献尚不清楚。在这里，我们评估了网蛋白在影响质膜 AQP4 聚集体分布、迁移特性和星形胶质细胞细胞体积调节中的作用。
在人 GBM 中，使用公开可用的数据集分析神经胶质酸性蛋白（GFAP）、AQP4 和 PLEC 转录本的表达，并通过免疫组织化学确定 PLEC 与 AQP4 和 GFAP 的共定位。我们在野生型和网蛋白缺陷型原代和永生化小鼠星形胶质细胞、人星形胶质细胞和源自人恶性 GBM 的永久细胞系（U-251 MG 和 T98G）上进行了实验。通过定量实时 PCR 评估小鼠星形胶质细胞中 PLEC 同工型的表达。转染、免疫标记和共聚焦显微镜用于评估细胞骨架分布中 PLEC 诱导的变化、PLEC 及其同工型对质膜 AQP4 聚集体丰度和大小的影响，以及 PLEC 在质膜上的存在。通过 ELISA 测量细胞释放的 PLEC。通过划痕愈合测定和钙黄绿素标记分别评估永生化星形胶质细胞的迁移和细胞体积调节动力学。
在肿瘤脑组织样本中，基因表达和蛋白质定位水平上发现 PLEC 与 AQP4 之间存在正相关。网蛋白缺陷导致质膜 AQP4 聚集体的丰度和大小减少，并改变细胞骨架的分布和束状。星形胶质细胞主要表达 P1c、P1e 和 P1g 网蛋白同工型。与质膜 AQP4 聚集体相关的主要网蛋白同工型是 P1c，它也最显著地影响星形胶质细胞的迁移能力。在没有网蛋白的情况下，星形胶质细胞的集体迁移受损，周围细胞区域细胞质体积变化的动力学降低。GBM 细胞系中，质膜表面 PLEC 的丰度及其从细胞中的释放增加。
网蛋白影响导致 GBM 病理学的细胞特性。观察到细胞表面和释放的 PLEC 水平均增加，这代表了 GBM 诊断和治疗的潜在生物标志物和治疗靶点。
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