Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain

Abstract

Microtubule-severing protein Spastin has been shown to co-localize with actin in migratory glioblastoma cells and is linked to glioblastomas’ migration and invasion capacity. However, the effectiveness of Spastin in glioblastoma migration and the molecular mechanism underpinning the orientation of Spastin towards actin filaments remain unknown. Here, we demonstrated that Spastin plays an active role in glioblastoma migration by showing a reduced migratory potential of T98G glioblastoma cells using real-time cell analysis (RTCA) in Spastin-depleted cells. Pull-down assays revealed that a cis–trans isomerase Pin1 interacts with Spastin through binding to the phosphorylated Pin1 recognition motifs in the microtubule-binding domain (MBD), and immunocytochemistry analysis showed that interaction with Pin1 directs Spastin to actin filaments in extended cell regions. Consequently, by utilizing RTCA, we proved that the migration and invasion capacity of T98G glioblastoma cells significantly increased with the overexpression of Spastin, of which the Pin1 recognition motifs in MBD are constitutively phosphorylated, while the overexpression of phospho-mutant form did not have a significant effect on migration and invasion of T98G glioblastoma cells. These findings demonstrate that Pin1 is a novel interaction partner of Spastin, and their interaction drives Spastin to actin filaments, allowing Spastin to contribute to the glioblastomas’ migration and invasion abilities.

miR96‐ and miR182‐driven regulation of cytoskeleton results in inhibition of glioblastoma motility

“…In contrast to spastin, palladin rescue dramatically restored the motility. The reason for spastin's incomplete rescue might be related to the recent finding by our lab that M87-spastin has the main role in glioblastoma motility and migration (Temizci et al, 2023), and although the plasmid used in the rescue experiments expresses both isoforms of spastin, due to modifications in the Kozak sequences, it expresses more M1-spastin than M87-spastin. It is possible that rescue with only M87-spastin expression could restore the motility completely.…”

“…Nonmammalian homologous Caenorhabditis elegans miR67 was used as a negative control during experiments to elucidate any nonspecific changes. Spastin over‐expression was performed with pcDNA3.1‐Spastin vector, which was already cloned in our lab and can produce both isoforms of spastin, M1 and M87 (Temizci et al, 2023). However, GBM cells endogenously produce M87‐spastin predominantly, while M1‐spastin is predominant in neurons (Temizci et al, 2023).…”

“…pcDNA3.1-Spastin vector, which was already cloned in our lab and can produce both isoforms of spastin, M1 and M87 (Temizci et al, 2023). However, GBM cells endogenously produce M87-spastin predominantly, while M1-spastin is predominant in neurons (Temizci et al, 2023).…”

“…It has been known that spastin induces neuronal side (Riano et al, 2009; Yu et al, 2008); moreover, its silencing causes loss of neuronal branching and appearance of only one long, unbranched axon (Yu et al, 2008). Additionally, depletion of spastin has been shown to result in elongated GBM cells with only one branch as well as a decrease in motility (Draberova et al, 2011; Temizci, Kucukvardar, & Karabay, 2023). Thus, direct or indirect regulation of spastin is linked to cellular motility through microtubule regulation.…”

“…miR96, miR182, and miR67 have no significant effect on cellular proliferation compared with mock transfection p > .05. miR182 were upregulated in U251 MG cells compared with normal human astrocytes. Interestingly, spastin and palladin which are regulated by miR96 and miR182 are also overexpressed in gliomas(Farran, 2021;Temizci et al, 2023). It might be the case that the exceeded expressions of spastin and palladin in gliomas might not be downregulated by the corresponding upregulation of miR96 and miR182 in gliomas.…”