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Roth, Sophie Faith

University of British Columbia

In the nervous system, glial cells play an important role in ensheathing and protecting nerves from damage, however, the employed mechanisms to properly ensheath nerves are still unknown. In Drosophila melanogaster, the perineurial glia (PG) are a conserved class of glia that surround peripheral nerves and ensure proper ensheathment through interactions with the extracellular matrix (ECM). Glia-ECM interactions occur through focal adhesions (FAs) comprised of integrins and intracellular proteins including talin and vinculin. Loss of FAs leads to the loss of the perineurial glial sheath and disruption of nervous system function. Integrins also associate with the transmembrane protein Basigin (aka Neuroplastin) in the PG. Loss of Basigin leads to constriction across nerves causing deformation of the glial membrane, cytoskeleton and the overlying ECM. The loss of Basigin phenotype can be rescued by a loss of integrin, leading to the hypothesis that Basigin knockdown in the PG leads to increased integrin activation. We performed FLIM-FRET (Fluorescence Lifetime Imaging Microscopy - Förster Resonance Energy Transfer) using paired donor-acceptor sensors inserted in talin that vary in sensitivity to force to quantify changes in integrin activation. Compared to controls, we observed a significant decrease in the FLIM-FRET efficiency in Basigin knockdown animals. This result implies that the sensor is being stretched due to the lengthening of talin, likely because of increased integrin activation. However, the excitation lasers for both the donor and acceptor were turned on when collecting FLIM-FRET images, which led to a false positive FRET signal with donor-only controls and prompted the reinterpretation of results. We also observed a significant increase in the integrated density of vinculin in the PG when Basigin is reduced. Since vinculin binds and stabilizes talin in an extended state, an increase in vinculin recruitment suggests an increase in talin lengthening and thus an increase in integrin activation. Overall, these data provide further evidence that the phenotypes observed with the loss of Basigin are likely caused by an increase in integrin activation. These findings support the proposed role for Basigin as a negative regulator of integrin activation allowing for the modulation of glia-ECM adhesion and overall nerve morphology.

Text

2024-03-19

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87597

Zoology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/