Astrocytes are numerous cells in the central nervous system responsible for various tasks, such as: transport of neurotransmitters, ions and protons, influence on neurogenesis, formation, maintenance and degradation of synapses, etc. In their proper functioning, the cytoskeleton plays various important roles; namely, it enables the internal transport of molecules, signalling, maintenance of mechanical properties, preservation of the cell shape and its movement. Plectin, an important binding protein of different cytoskeleton types, belongs to the plakin family. It is a very large protein (> 500 kDa), capable of cross-linking various elements of the cytoskeleton, such as actin fibres, intermediate filaments, microtubules, and linking them to the strategic intracellular locations, such as focal adhesions in the plasmalemma. It consists of an N- and C-terminal domain, which both contain different binding sites, and a central rod domain, which has the role of a spacer and enables oligomerization. In the master's thesis, we investigated the distribution and the role of plectin in focal adhesions of cultured mouse astrocytes. Here, we specifically focused on the isoform P1c, which is the most abundant isoform in astrocytes, and miniP1c, which is the isoform of P1c devoid of the central rod domain. The miniP1c isoform, due to its shorter transcript, could represent a potential application for gene therapy purposes in some plectinopathies. We show that, compared to P1c, the expression of a truncated miniP1c is higher. In both primary and immortalized astrocytes, the native plectin was distributed similarly as the introduced plectin P1c variants. We show that plectin is usually distributed in the form of a net in the perinuclear region, while towards the periphery of the cell it spreads in the form of fibrils, partly branching and partly maintaining straight fibres. We did not detect any differences in the localization of a P1c and miniP1c in the central part of the cells. We show that the lack of plectin in immortalized astrocytes significantly affects focal adhesions’ properties, such as their number, the size and fraction of the area they occupy. This phenotype was not rescued by forced expression of either P1c or mini P1c. In addition, we did not detect differences between localization of P1c and miniP1c in focal adhesions. Finally, we showed that in immortalized astrocytes, the actin fibre is similar in cells containing native plectin and in cells devoid of plectin that were transfected with P1c or miniP1c. Plectin P1c and miniP1c exhibited a high degree of colocalization with actin.
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