A central challenge in single-cell biology is understanding how molecular programs drive changes in cellular architecture that enable specialized function. A striking example of cellular remodeling is the differentiation of airway stem cells into the respiratory multiciliated epithelium, a protective tissue barrier that clears inhaled pathogens and particulate matter. Here, we present its first three-dimensional nanometer-scale reconstruction, revealing coordinated changes in cellular organization, organelle topology, and inter-organelle contacts during multiciliogenesis. We uncover a structural and functional association between motile cilia and mitochondria mediated by rootlets, striated cytoskeletal fibers that remain poorly characterized in human airway multiciliated cells. Rootlets connect to basal bodies through a multiprotein linker containing the uncharacterized rootletin/CROCC homolog CROCC2, oscillate at frequencies comparable to ciliary beating, promote basal body alignment, and when lost, reduce maximal mitochondrial respiratory capacity. Altogether, this work integrates structural, dynamic, and functional analyses to elucidate mechanisms underlying airway mucociliary defense.
Aaran Vijayakumaran, Christopher S. Godbehere, Analle Abuammar et al.· Cell Reports· 0 citations
2-cell like cells (2CLC) are a transiently cycling population of cells with totipotent-associated features. Although CTCF depletion induces 2CLC conversion in mouse ESC, whether this reprogramming is a consequence of disrupted higher-order chromatin organization or of CTCF-specific functions remained unclear. Here, we show that depletion of the cohesin release factor WAPL in ESC also promotes 2CLC reprogramming, which is increased by CTCF co-depletion. Single-cell RNA-seq/ATAC-seq analyses in CTCF/WAPL-depleted ESC revealed that chromatin accessibility precedes 2C-associated gene expression. Moreover, we identified ARID3A as a transcription factor that regulates the extent of 2CLC conversion following WAPL/CTCF depletion. Although WAPL or CTCF depletion induces distinct transcriptional changes in human ESC, these do not resemble transcriptional programs of early human embryogenesis, suggesting limited evolutionary conservation. Finally, we demonstrate that 2CLC conversion mediated by alterations in chromatin organization depends on the DPPA2/DUX axis and correlates with nucleolar integrity. Together, these findings establish a mechanistic link between higher-order chromatin organization and totipotency-like cell identity in mice. Higher-order chromatin organization has been implicated in the regulation of totipotency, but the mechanisms linking chromatin architecture to totipotent-like cell states remain poorly understood. This study shows that disruption of chromatin organization by degron-mediated CTCF and/or WAPL depletion promotes totipotent-like conversion through the DPPA2/DUX axis, accompanied by changes in chromatin accessibility and nucleolar integrity. WAPL depletion promotes 2CLC reprogramming in mouse ESC, and is enhanced by CTCF co-depletion. The transcription factor ARID3A limits the extent of 2CLC conversion following CTCF/WAPL-depletion. Increased chromatin accessibility precedes expression of 2C-associated transcripts following CTCF/WAPL depletion. 2CLC conversion induced by CTCF/WAPL depletion depends on the DPPA2/DUX axis and correlates with nucleolar integrity. WAPL or CTCF depletion in human ESC fails to activate transcriptional programs characteristic of early human embryogenesis, suggesting limited evolutionary conservation. WAPL depletion promotes 2CLC reprogramming in mouse ESC, and is enhanced by CTCF co-depletion. The transcription factor ARID3A limits the extent of 2CLC conversion following CTCF/WAPL-depletion. Increased chromatin accessibility precedes expression of 2C-associated transcripts following CTCF/WAPL depletion. 2CLC conversion induced by CTCF/WAPL depletion depends on the DPPA2/DUX axis and correlates with nucleolar integrity. WAPL or CTCF depletion in human ESC fails to activate transcriptional programs characteristic of early human embryogenesis, suggesting limited evolutionary conservation. Chromatin architectural defects trigger the DPPA2/DUX program and nucleolar remodeling to promote totipotent-like cell conversion in mouse embryonic stem cells.
Grace Carey, Maria Vega-Sendino, Desiree Tillo et al.· EMBO Journal· 2 citations