Protein Segregation Between Dividing Hematopoietic Progenitor Cells in the Determination of the Symmetry/Asymmetry of Cell Division

Abstract

In the present study, we investigated how the symmetry/asymmetry of cell division in mitotic CD34(+) cells can be evaluated by determining the plane of cell division and the potential distribution of proteins between daughter cells. The orientation of the mitotic spindle is dependent upon the positioning of the centrosomes, which determine the plane of cell division and the sharing of proteins. If the functions of unequally shared proteins are relevant to the kinetics of cell division, they could determine whether the daughter cells undergo self-renewal or differentiation. The kinetic function of the proteins of interest was investigated using a colony-replating assay and carboxyfluorescein succinimidyl ester (CFSE) staining. We used Notch/Numb as a model system, since they have a role in balancing symmetric/asymmetric divisions. Mitotic cells were examined microscopically and centrosomal markers γ-tubulin/pericentrin were used with activated Notch-1 and Numb. We monitored the first crucial divisions by CFSE staining and found an inverse relationship between activated Notch and Numb expression, suggesting a reciprocal regulation. We suggest that the subpopulations expressing activated Notch or Numb have different cell fates. To determine the influence of Notch signaling on progenitor cell self-renewal, we used the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester (DAPT). DAPT influences self-renewal/differentiation outcome by affecting the frequency of symmetric renewal divisions without affecting the rate of divisions. Overall, the purpose of this study was to establish a cellular system for predicting the symmetry/asymmetry of hematopoietic progenitor divisions at the level of centrosomes and protein distribution and to investigate the influence of these proteins on progenitor cell kinetics.