An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction.
Eur J Cell Biol
; 98(5-8): 151045, 2019 Dec.
Article
de En
| MEDLINE
| ID: mdl-31563281
ABSTRACT
Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600â¯g and 35⯰C for 60â¯min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Noyau de la cellule
/
Colchicine
/
Cytoplasme
/
Matrice extracellulaire
Limites:
Humans
Langue:
En
Journal:
Eur J Cell Biol
Année:
2019
Type de document:
Article
Pays d'affiliation:
Chine