Biology subjects

Huebner, B.

Publications and source records attributed to Huebner, B..

3 recordsLinked to original sources

bioRxiv2026

Native in situ architecture of the human inactive X chromosome revealed by correlative light and electron microscopy

Knowledge of the nuclear organization of specific chromatin domains at the ultrastructural level remains limited. To address this, we employed correlative light and electron microscopy (CLEM) approaches to investigate the inactive X chromosome (Xi) in female human RPE1cells. Tandem fusion of macroH2A (mH2A), a histone variant enriched on the Xi, to eGFP and APEX2 enabled direct labeling and identification of the Xi by light microscopy, transmission electron microscopy, and electron tomography using conventional CLEM. The eGFP tag further allowed us to visualize the Xi at single nucleosome resolution under close-to-native conditions using cryo-CLEM. We found that, although heterochromatin domains of the Xi are substantially larger than those of autosomal regions, their chromatin density was not significantly different. Furthermore, our data reveal the organization of the interchromatin compartment (IC) within the Xi, characterized by a network of larger lacunae and smaller channels. Overall, we provide an ultrastructural characterization of a specific heterochromatin domain at unprecedented resolution, advancing our understanding of 3D chromatin organization and nuclear architecture in situ.

cell biology↗

bioRxiv2025

Homer condensates orchestrate YAP-Wnt signaling crosstalk downstream of the Crumbs polarity complex

The Hippo pathway governs cell growth, proliferation, and differentiation and is frequently deregulated in cancer. YAP, the central transcriptional co-activator of the Hippo pathway, is suppressed by diverse upstream signals including cell density and polarity. YAP also functionally interacts with the Wnt/{beta}-catenin pathway, yet how polarity cues coordinate this pathway crosstalk remains poorly understood. Here, we demonstrate that Homer scaffolding proteins couple the Crumbs polarity complex to the coordinated regulation of YAP and Wnt signaling. Homers directly interact, via their EVH1 domains, with the Crumbs component PATJ and the NDR kinase scaffold Furry-like (FRYL). Functionally, Homers antagonize FRYL to promote YAP activation while cooperating with FRYL to enhance Wnt/{beta}-catenin signaling, revealing pathway-selective regulation. PATJ, in contrast, acts upstream to restrain Homer-driven YAP signaling. Interestingly, in non-polarized epithelial and colorectal cancer cells, Homers form cytoplasmic biomolecular condensates whose assembly and material properties are differentially modulated by PATJ and FRYL. Whereas FRYL promotes spherical, liquid-like Homer condensates, PATJ drives the formation of irregular, network-like assemblies, thereby altering condensate topology and signaling output. Collectively, our findings establish Homer-driven phase separation as a tunable signaling mechanism that integrates polarity cues with YAP-Wnt pathway coordination and transcriptional output. SignificanceThe Hippo/YAP and Wnt signaling pathways play important roles in development and are frequently deregulated in cancer. Both pathways respond to epithelial cell architecture, but how polarity cues coordinate their crosstalk remains poorly understood. Here, we identify Homer proteins as phase-separating scaffolds that link apico-basal polarity to YAP-Wnt pathway integration. We show that the material properties of Homer condensates are tunable and depend on protein abundance and binding partners, providing a mechanism by which polarity cues can shape signaling output through changes in phase behavior. Our findings establish Homer condensates as a polarity-sensitive signaling hub that modulates YAP transcriptional programs and coordinates YAP-Wnt pathway crosstalk.

cell biology↗

bioRxiv2025

Bulk exocytosis of large intracellular apical precursor organelles establishes apical domain identity during de novo lumen formation

The formation of a microvilli-rich lumen is a key event in epithelial polarity development and tissue morphogenesis. During de novo lumenogenesis, epithelial cells establish luminal identity by directing apical cargo to an apical membrane initiation site (AMIS). Although this process has been widely studied, the mechanisms governing AMIS formation and its progression into a luminal precursor remain poorly understood. Here we combined quantitative light and electron microscopy with proximity proteomics to investigate the mechanistic basis of lumen initiation in MDCK-II cells. Contrary to prevailing models, we find that apical cargo is delivered to the AMIS in large intracellular apical precursor organelles, termed vacuolar apical compartments (VACs). VACs possess a preassembled microvilli-rich cortex and undergo exocytic fusion at the AMIS to generate a nascent lumen. Moreover, lumen initiation is tightly coordinated with the assembly and rearrangement of apical cell-cell junctions and requires the Crumbs complex protein PatJ, which controls the architecture of the apical-lateral border and connects the tight junction to the apical cortex. Together, our results identify PatJ as a critical organizer of the apical-lateral interface and indicate that VACs act as specialized transport organelles that deliver a preassembled apical cortex to the AMIS, enabling rapid and efficient lumen initiation.

cell biology↗