Pappas, V. | bioRxiv Science

bioRxiv2026

Large RNA polymerase II condensates are promoter-centric assemblies associated with early stages of transcription at all expressed genes

Transcriptional condensates concentrate the machinery required for RNA polymerase II mediated transcription. These structures range from numerous small, short-lived species, to a handful of larger, stable assemblages. Large condensates have been implicated in driving potent transcription of several super-enhancer regulated genes, yet the underlying mechanisms and the range of their client genes remain unclear. Here, we developed a biochemical approach which combines density gradient centrifugation and affinity purification to partially purify large transcriptional condensates from nuclei, allowing systematic characterization of their nucleic acid components. We find that transcriptional condensate isolates engage thousands of gene promoters and harbor the nascent transcriptome, but do not stably co-purify with distal enhancers. Binding patterns of RNA polymerase II within condensates suggest these structures could facilitate promoter escape and promoter-proximal pause release. Together, our work supports a promoter-centric condensate organization and paves the way towards understanding the functional link between condensate architecture and nascent transcription.

molecular biology↗

bioRxiv2026

Cryo-electron tomography reveals paracellular claudin-15 pores at the tight junction

Tight junctions (TJs) are composed of anastomosing strands between epithelial cells. Members of the claudin family of proteins reside within TJ strands and either seal the paracellular space or assemble into charge and size-selective pathways. Functional studies suggest that claudin-mediated conductance pathways resemble traditional ion channels. However, such postulated pores have not been directly visualized. Using a model claudin deficient epithelium where exogenously introduced EGFP-CLDN15 is the only claudin family member expressed, our investigation sheds light on the arrangement and structure of the postulated claudin pores. Following correlative light and electron microscopical identification of TJs and cryo-electron tomography, we identified series of linearly distributed electron lucent features that locate between two closely apposed plasma membranes of adjacent cells. At these sites, the median spacing between adjacent features is 2.25 nm (IQR = 1.83), with a median 1.66 nm (IQR = 0.92) diameter. In contrast, such features were not observed in claudin deficient model epithelium with exogenous mCherry-ZO-1 expression. These findings agree with the postulated and extensively modeled claudin pores formed within the simple columnar epithelium. This provides the first direct evidence of paracellular pore organization and paves way for future biophysical investigation. SIGNIFICANCEBy combining correlative fluorescence imaging, FIB milling, and cryo-ET within an epithelial system restricted to a single claudin isoform, we were able to visualize repetitive, low-density pore features within CLDN15-containing tight junctions (TJs), structures not previously resolved in intact epithelia. These features were absent in claudin-negative controls and displayed placement and geometry consistent with CLDN15 X-ray crystallography and molecular dynamics models. Quantitative measurements of pore diameter, paracellular gap width, and pore spacing further support their assignment as CLDN15 pores. These findings establish a structurally validated platform for defining claudin pore ultrastructure and provide a foundation for future efforts to compare pore-forming and barrier-forming claudins, understand disease-associated junction remodeling, and guide therapeutic modulation of epithelial barrier function.

physiology↗

bioRxiv2022

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

biophysics↗