Astrocytes have many functions that maintain homeostasis in the central nervous system. An important connecting protein of the cytoskeleton elements in astrocytes is plectin, a multifunctional cytolinker protein from the plakin family. Since little is known about the influence of plectin on the dynamic properties of microtubules and the localization of tau protein in astrocytes, we wanted to verify this on immortalized mouse astrocytes, which express plectin (Plec+/+p53-/-) and which do not express plectin (Plec-/-p53-/-). Microtubule dynamics was monitored by live-cell confocal fluorescence microscopy, where the growing ends of microtubules were labeled with the protein Eb3 coupled to green fluorescent protein. We showed that microtubules polymerize faster in the absence of plectin. We also found that microtubules in the absence of plectin have a higher orientation coefficient of polymerization, more radially oriented growth relative to the cell centroid, and lower angular dispersion of growth. Plectin had no effect on the rate of initiation of growth of new microtubules and on the density of growing microtubules in cells. We found that microtubules in the absence of plectin are less fragmented, more overlapped, and have a higher total length normalized to the cell area. We found a relatively high level of tau protein expression in the form of aggregates in both Plec+/+p53-/- and Plec-/-p53-/- mouse astrocytes. We did not qualitatively detect that tau protein aggregates colocalized with microtubules. We showed that tau protein occupies a larger proportion of immortalized mouse astrocytes in the absence of plectin and that more tau protein is located in the peripheral part of the cells compared to astrocytes, which express plectin. We showed using STED superresolution microscopy that tau protein aggregates are denser in the absence of plectin. We attempted to rescue the aforementioned phenotype by lipofection of Plec-/-p53-/- astrocytes with a gene for the plectin isoform P1c. The transfection was successful, but there was no difference in the density and localization of tau protein aggregates between transfected and untransfected cells. We found that lipofection procedure alone significantly reduced the area and density of tau protein aggregates and changed their localization in both transfected and untransfected cells compared to mouse astrocytes in the experiment wihout lipofection. Our results suggest that plectin affects microtubule properties and tau protein distribution in immortalized mouse astrocytes. In the future, it would be necessary to investigate the mechanism of plectin's influence on the aforementioned properties and what are the reasons for tau protein to be found in the form of aggregates in immortalized mouse astrocytes. It would be necessary to study the influence of plectin on microtubule dynamics and tau protein distribution in primary astrocytes as well.
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