# Live cell optical super-resolution microscopy of dystroglycan mutants as a model for dystroglycanopathies in multiple cell lines

## TL;DR

This study uses advanced microscopy to examine how dystroglycan mutations affect protein localization in different cell types, offering insights into neuromuscular diseases.

## Contribution

The study introduces a novel approach using live cell super-resolution microscopy to analyze dystroglycan mutants across multiple cell lines.

## Key findings

- The C667F mutant is retained in the ER/Golgi, while T190M and wild-type DG localize to the plasma membrane in all three cell lines.
- The L84F mutant shows delayed trafficking in two cell lines but strong plasma membrane localization in HEK-293T cells.
- The I591D mutant accumulates at the plasma membrane in HEK-293T cells but is retained in the ER/Golgi in other cell lines.

## Abstract

Dystroglycan (DG) is an adhesion complex comprising two subunits, α-DG and β-DG, which interact non-covalently at the plasma membrane. As a component of the dystrophin-glycoprotein complex DGC, DG plays a crucial role in linking the cytoskeleton to the surrounding basement membranes. Rare primary point mutations in the DAG1 gene have been identified in patients with various forms of neuromuscular dystrophy, ranging in phenotype from mild to severe.

To gain a deeper understanding of the molecular mechanisms underlying these pathologies, we have designed a series of chimeric GFP-tagged full-length α/β-DG constructs and expressed them in three different cell lines (U-2OS, HEK-293T and C2C12). Wild-type DG constructs were compared to their counterparts carrying pathologic missense mutations previously described in patients, namely, L84F, T190M and C667F and with the mutant I591D, i.e., the topological equivalent of V567D identified in zebrafish.

Live super-resolution fluorescence microscopy showed that the C667F mutant is retained within the ER/Golgi while the T190M and wild-type proteins are correctly localized to the plasma membrane in all 3 cell lines. The L84F mutant exhibits a delay in trafficking to the plasma membrane in two of the cell lines, while localizing strongly at the plasma membrane in the high-expression HEK-293T cells. Similarly, the I591D mutant accumulated at the plasma membrane in the HEK-293T cells, in contrast to the clear retention in the endoplasmic reticulum/Golgi apparatus observed in U-2OS and C2C12 cells.

Our data demonstrate the importance of using a range of different cell lines for a comprehensive study of DG mutants or variants by live cell optical super-resolution microscopy.