Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

Background

Glioma is a notoriously infiltrative tumour that often invades healthy brain tissue beyond surgical margins, which underscores the poor outcomes that are faced by patients with this disease¹. During infiltration, glioma cells encounter many different mechanical constraints which they must overcome. These mechanical challenges are detected and transduced through mechanosensitive proteins, a diverse group which includes mechanosensitive ion channels, integrins, and other cell-surface receptors. One class of proteins with emerging functions as mechanosensors are plexins, which are known to be important axon guidance molecules during nervous system development². Given that tumours frequently reactivate developmental programs, the authors of this preprint sought to understand how these programs contribute to glioma development and invasion. Using a comprehensive set of experiments, the authors demonstrate that Plexin-B2 is at the heart of a mechano-electrical signaling cascade that promotes brain tumour cell invasion.

Key Findings

Confined migration involves membrane internalization via endocytosis

To observe how glioma cells behave under confinement, the authors made microfluidic devices with narrow (3 microns) or wide (8 microns) constrictions. Surprisingly, the authors found that cells passing through narrow constrictions were faster and more persistent than cells passing through wide constrictions. Through fluorescent live-cell imaging, they observed f-actin assembly at the cell rear and surprisingly, active endocytosis at the cell front. These appeared to be linked processes, since treating glioma cells with pharmacological inhibitors of endocytosis also disrupted f-actin assembly at the cell rear.

Plexin-B2 function impacts endocytosis and membrane internalization of glioma cells

Next, the authors wondered what molecular players could underlie the relationship between endocytosis and f-actin assembly. They hypothesized that the axon guidance protein Plexin-B2 could be a potential candidate since it is upregulated in glioma and modulates tumour invasion via actomyosin contractility^{3, 4}. The authors found several striking phenotypes when they knocked out PLXNB2 using CRISPR (PB2KO) in glioma cells, including elongated cell processes, decreased cell stiffness, and lower plasma membrane tension compared to control. Next, the authors performed a dextran uptake assay, which measures active endocytosis. PB2KO cells had a lower overall number of endosomal puncta compared to control, suggesting impaired endocytosis. These results were also supported by membrane turnover assays, indicating that Plexin-B2 plays an important role in membrane internalization.

Plexin-B2-deficient glioma cells display compromised confined migration

Given the observed defects in membrane tension and endocytosis, the authors investigated whether Plexin-B2 affected confined cell migration. Using the same microfluidic devices, the authors found that PB2KO cells were slower, less persistent, and had longer stalling times compared to control. Furthermore, using new microfluidic devices in which cells could choose to migrate through 3 micron or 8 micron-wide tunnels connected by a central chamber, the authors observed that control cells were able to successfully pass through both narrow and wide tunnels, and had a propensity to change direction and explore alternative exit tunnels. In contrast, PB2KO cells had fewer successful passages and were unlikely to explore different exits while inside central chambers. Furthermore, unlike control cells traversing narrow tunnels, PB2KO cells did not exhibit polarized f-actin assembly at the cell rear and endocytosis at the cell front. Altogether, these results provide strong evidence that Plexin-B2 is a key regulator of confined migration.

Plexin-B2 coordinates mechano-electric coupling

Given the endocytic defects seen in PB2KO cells, the authors determined whether the lipid composition of the plasma membrane was affected. They first measured PIP2, a key membrane phospholipid and second messenger in many signalling pathways. Using a GFP-labeled probe that binds PIP2, the authors observed that while the majority of GFP signal was internalized in control cells, PB2KO cells retained PIP2 in the plasma membrane after 3 days of imaging. During live imaging, control cells appeared to accumulate polarized PIP2 signal at the cell front, whereas PB2KO cells did not. Since PIP2 is negatively charged, the authors wondered whether the electrical properties of the plasma membrane were affected. Using FluoVolt, a fluorescent voltage sensor, the authors found that control cells migrating through narrow tunnels displayed higher FluoVolt signal at the cell rear (indicating less negative charge), whereas PB2KO cells did not display this pattern. Furthermore, calcium imaging of migrating cells revealed an accumulation of Ca²⁺ at the cell rear, which was absent in PB2KO cells.

Flexible ring of Plexin-B2 extracellular domain is required for efficient confined migration

A previous study of Plexin-D1, a closely related protein, found that it acts as a sensor of blood flow in endothelial cells through the bending of its extracellular ring domain⁵. To determine whether Plexin-B2 is mechanosensitive, the authors introduced mutations into the flexible ring domain to generate locked mutants, and then expressed these variants in PB2KO glioma cells. Unlike the re-introduction of wildtype Plexin-B2, expressing locked mutants did not rescue the membrane internalization and migratory defects previously observed in PB2KO cells. Altogether, these experiments suggest that the ring domain is a key component of Plexin-B2’s function as a mechanosensitive coordinator of confined cell migration.

Above: During confined cell migration, Plexin-B2 coordinates endocytosis at the cellular front with actin fibre assembly at the cell rear via regulation of membrane tension and electrical charge. Schematic made using BioRender. 

Why I chose this preprint

I first learned about this story at the Society for Neuro-Oncology Annual Meeting in Vancouver, Canada in November 2023. The first author, Dr. Chrystian Junqueira Alves, presented a poster which included the main results reported in this preprint. I was amazed by the many novel findings of this study, such as the coordination between active endocytosis at the cell front with actin contraction at the cell rear, and the newly discovered mechanosensing function of Plexin-B2. This preprint highlights the importance of exploring non-canonical functions of well-established proteins, and I believe it’s a great demonstration of why cross-disciplinary collaborations are important for making new and unexpected discoveries.

Questions for the authors

1. Does Plexin-B2 influence collective cell migration as well? Are there any changes to the invasion pattern of glioma cells when Plexin-B2 is knocked out in vivo?
2. How might Plexin-B2 influence the lipid composition of the plasma membrane? Does it cooperate with lipid synthesis pathways?
3. Aside from compression, could Plexin-B2 also be sensitive towards other types of forces found in the brain such as fluid shear stress or tension?

 References

1. Mair, D.B., Ames, H.M., and Li, R. (2018). Mechanisms of invasion and motility of high-grade gliomas in the brain. Mol Biol Cell 29, 2509-2515. 10.1091/mbc.E18-02-0123.
2. Fournier, A.E., Nakamura, F., Kawamoto, S., Goshima, Y., Kalb, R.G., and Strittmatter, S.M. (2000). Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse. J Cell Biol 149, 411-422. 10.1083/jcb.149.2.411.
3. Shinoura, N., Shamraj, O.I., Hugenholz, H., Zhu, J.G., McBlack, P., Warnick, R., Tew, J.J., Wani, M.A., and Menon, A.G. (1995). Identification and partial sequence of a cDNA that is differentially expressed in human brain tumors. Cancer Lett 89, 215-221. 030438359503690X [pii].
4. Le, A.P., Huang, Y., Pingle, S.C., Kesari, S., Wang, H., Yong, R.L., Zou, H., and Friedel, R.H. (2015). Plexin-B2 promotes invasive growth of malignant glioma. Oncotarget 6, 7293-7304.10.18632/oncotarget.3421.
5. Mehta, V., Pang, K.L., Rozbesky, D., Nather, K., Keen, A., Lachowski, D., Kong, Y., Karia, D., Ameismeier, M., Huang, J., et al. (2020). The guidance receptor plexin D1 is a mechanosensor in endothelial cells. Nature 578, 290-295. 10.1038/s41586-020-1979-4.

Tags: brain cancer, cell migration, mechanotransduction

doi: https://doi.org/10.1242/prelights.36441

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