Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment

• DOI: 10.1016/j.yexcr.2023.113695
• Publication Date: 2023-06-30T15:03:06Z
• Authors: Vera Chvalova; Vignesh Venkadasu...; Zuzana Klimova; Jana Vojtova; Oldrich Benada; Ondrej Vanatko; Tomas Vomastek; Tomas Grousl
• Keywords: Mammals; Microscopy; Microscopy-based analysis; Microvilli; MDCK cells; Receptors, Cell Surface; Rat2 cells; Cell cycle; Dogs; rack1; Focal adhesions; GTP-Binding Proteins; Animals; Actin; Cell Division; Cell Proliferation

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.