Plectin associates with focal adhesions and contributes to cytoskeletal organization and mechanical properties of astrocytes

Reactive astrogliosis, a hallmark of central nervous system pathologies, involves a spectrum of astrocyte responses, including morphological remodeling and the upregulation of intermediate filaments such as vimentin and glial fibrillary acidic protein (GFAP). Changes in astrocyte shape are driven by cytoskeletal dynamics and are important for interactions with the surrounding microenvironment. Focal adhesions (FAs), which serve as physical and signaling links between the cytoskeleton and the extracellular matrix, play a central role in these structural adaptations. Here, we identify plectin, a versatile cytoskeletal linker, as an important modulator of FA-associated processes in cultured mouse astrocytes. We demonstrate that plectin localizes to FAs in astrocytes, and its deficiency is associated with changes in their number, maturation, and turnover. Plectin also displays polarization within FAs, depending on their maturation state, and it contributes to the recruitment of key cytoskeletal elements, particularly vimentin, to FAs. In plectin-deficient astrocytes, the vimentin and GFAP network exhibits impaired connectivity, accompanied by altered viscoelastic properties of the cells. Compared with astrocytes maintained in serum-free neurobasal medium, astrocytes cultured in serum-containing medium, which resemble reactive astrocytes, exhibit elevated plectin levels along with an increased number and size of FAs, supporting the involvement of plectin in pathological conditions.<b>NEW & NOTEWORTHY</b> Plectin contributes to FA dynamics in astrocytes and exhibits spatial polarization within individual FAs as revealed by superresolution microscopy (SIM and STED). Atomic force microscopy demonstrated that plectin deficiency alters cell viscoelasticity, unveiling the role of plectin in the mechanical properties of astrocytes. Plectin expression, along with the FA protein vinculin, is upregulated in astrocytes cultured under serum-containing conditions-an in vitro model of reactive astrocytes-compared with serum-free, native-like conditions.