Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Nathalie Chênais,Catherine Labbé,Nathalie Morin,Hélène Rime,Thierry Lorca,Brigitte Guillet,Pierre-Yves Le Bail

doi:10.1038/s41598-019-39500-y

Abstract

Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Highlights

The interspecific efficiency of Xenopus egg extract to ensure the epigenetic remodeling of somatic cell chromatin in mammals makes it an ideal candidate to test on fish cells
No nuclear fluorescence was detected in the permeabilized cells if the import assay was carried out at 4 °C, even in the presence of Xenopus egg extract, indicating that the import is temperature dependent. These results indicate that the Xenopus egg extract and the temperature of 25 °C are essential to the success of nuclear import of nuclear localization signal (NLS)-proteins in the fin permeabilized cells
We demonstrated that fin somatic cells could be permeabilized by digitonin treatment and that this permeabilization allowed the penetration of exogenous proteins from Xenopus egg extract

Summary

Introduction

The interspecific efficiency of Xenopus egg extract to ensure the epigenetic remodeling of somatic cell chromatin in mammals makes it an ideal candidate to test on fish cells. Permeabilization methods include electro-permeabilization and permeabilization using pore-forming factors: bacterial toxins such as alpha-toxin or streptolysin O, or pore-forming detergents from plants such as digitonin These two latter molecules are often preferred because they allow the delivery of large molecules into the cytosol of permeabilized cells[3,4,7,8,9,10]: with digitonin and streptolysin O, passive incorporation of up to 100 kDa proteins was reported[17,18]. Before any study on the reprogramming of cultured cells by egg extract can be conducted, each stage of the treatment process must be validated, namely plasma membrane permeabilization, maintenance of nuclear import, plasma membrane resealing, and cell growth resumption in culture. The overall objective of the work was to provide a step-by-step demonstration of the capacity of fish fin cells to be successfully prepared for cell reprogramming using Xenopus egg extracts

Objectives

Methods

Results

Conclusion