Redick, M. A. | bioRxiv Science

bioRxiv2022

Single-cell dynamics of core pluripotency factors in human pluripotent stem cells

The human transcription factors OCT4, SOX2, and NANOG form a core signaling network critical for maintaining stem cell pluripotency and self-renewal potential. The spatiotemporal expression dynamics of these pluripotency factors throughout differentiation is unclear, limiting our understanding of stem cell fate decisions. Here, we combined CRISPR/Cas9-mediated gene editing with microraft array technology to generate human embryonic stem cell lines with endogenously tagged fluorophores for OCT4, SOX2, and NANOG. Fluorescence time-lapse imaging revealed that pluripotent stem cells show gastrulation-like patterning without direct chemical induction. Directed differentiation to the three primary germ layers--endoderm, mesoderm, and ectoderm--revealed distinct spatiotemporal patterns of SOX2 and NANOG expression in single cells. Finally, we captured dynamic changes in cell morphology during ectoderm differentiation corresponding to the formation of neural rosettes. This study provides a robust method for generating live-cell reporters in human stem cells and describes the single-cell dynamics of human pluripotency factors during differentiation.

developmental biology↗

bioRxiv2019

Autophagy regulates the localization and degradation of p16INK4a

The tumor suppressor protein p16INK4a (p16) is a well-established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the proteins intracellular localization and degradation. The autophagy-lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress-response proteins, but it is unclear whether p16 is involved in these pathways. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time-lapse fluorescence microscopy using an endogenous p16-mCherry reporter revealed that autophagy induced by genotoxic stress stimulates rapid p16 recruitment to acidic cytoplasmic vesicles. When vesicular acidification was inhibited by NH4Cl, nuclear p16 levels increased. Single-cell imaging revealed that p16 localizes to lysosomes upon stress, implicating the autophagy pathway as a regulator of p16 localization. Blocking autophagy with bafilomycin, chloroquine, or NH4Cl resulted in elevated p16 protein levels without increased transcription. Increased p16 coincided with accumulation of autophagosome chaperone p62/SQSTM1 (p62) and decreased levels of phosphorylated-Rb. Furthermore, chloroquine caused p16 aggregation within stalled vesicles containing autophagosome marker LC3, demonstrating that p16 is transported and degraded by the autophagy-lysosomal pathway. Knockdown of p62 resulted in delocalization of p16 aggregates to autophagosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age-related diseases.

cell biology↗