HeLa cell expressing mcherry-H2B (red) stained with SiR-tubulin (green). Data collected by confocal imaging. Courtesy of Daniel Gerlich and Claudia Blaukopf, Institute of Molecular Biotechnology, Vienna.
Recently, Xenopus laevis eggs were used to study if homogenized egg cytoplasm can support spontaneous cellular re-assembly with restored macromolecular structures and functionality. Bright-field and fluorescent microscopy demonstrated that the cytoplasm of eggs arrested at interphase and homogenized can undergo self-assembly into sheets of 300-400 micron long, cell-like compartments in ~30 minutes. Successful self-assembly required formation of microtubules (MTs), but not F-actin, minus-end-directed cytoplasmic dynein motor activity, and adenosine triphosphate. MTs in the cell-like compartments appeared similar to the MTs of intact Xenopus embryos. Neither nuclei (chromatin) nor centrosomes were needed for self-assembly initiation. Genomic input was minimized, if not fully precluded, by inhibition of protein translation with cycloheximide. Introduction of demembranated sperm nuclei as a source of centrosomes and DNA enabled the cell-like compartments to undergo multiple cycles of mitosis if cycloheximide was excluded. Cytoskeleton’s SiR-actin and SiR-tubulin live cell imaging probes, along with green and far-red fluorescently-labeled tubulins (Cat.# CY-SC001, CY-SC002, TL488M, TL670M, respectively), revealed the dynamic re-organization, subcellular localization, and functional roles of MTs and F-actin in the formation of the cell-like compartments. The study confirms that cytoplasmic components of Xenopus egg extracts provide the necessary macromolecular complexes, spatial organization, and cell cycle function to initiate self-organization.
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