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

Exp Cell Res. 2023 Sep 1;430(1):113695. doi: 10.1016/j.yexcr.2023.113695. Epub 2023 Jun 30.

Abstract

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.

Keywords: Actin; Cell cycle; Focal adhesions; MDCK cells; Microscopy-based analysis; Microvilli; RACK1; Rat2 cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Division
  • Cell Proliferation
  • Dogs
  • GTP-Binding Proteins* / genetics
  • Mammals / metabolism
  • Microscopy*
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism

Substances

  • GTP-Binding Proteins
  • Receptors, Cell Surface