Aussie Suzuki

The kinetochore is an essential structure for the faithful chromosome segregation during mitosis. The 16-protein complex known as the constitutive centromere-associated network (CCAN) localizes the centromere throughout the cell cycle and forms the basis of the kinetochore. Although cryo–electron microscopy has provided a structural model of an individual CCAN unit, how multiple CCAN units are organized within cells remains unclear. Here, using in silico and in vivo analyses, we demonstrate that multiple CCAN units form a spherical shell-like structure in interphase DT40 kinetochores. CENP-T and CENP-N localize to the CCAN shell, whereas the CENP-C cupin domain clusters at the center. Molecular dynamics simulations of CCAN assembly suggest that CENP-C–DNA interactions constrain the orientation of CCAN units, supporting a spherical shell-like organization via CENP-C oligomerization. Super-resolution imaging reveals the spherical shell-like structure at DT40 kinetochores, and CENP-C knockout or expression of a CENP-C DNA binding deficient mutant abolishes the structure, consistent with the simulation-derived model. These findings provide important insights into the spatial organization of functional kinetochores within cells.

Oxidative stress and genotoxic damage activate NF-κB signaling through intracellular pathways distinct from those initiated by membrane receptors. DNA damage selectively induces post-translational modifications at lysine residues 277 and 309 of the human ubiquitin-binding protein NEMO to promote NF-κB signaling, but the physiological importance of these modifications in vivo remains unclear. Here, we show that a newly developed mouse model (NEMO ^DK ) carrying germline arginine substitutions of the corresponding NEMO lysine residues exhibits B-cell-intrinsic defects in germinal center formation and anti-viral humoral responses. Mechanistically, we identify in NEMO ^DK B-cells a CD40-specific NF-κB signaling defect that is not linked to the well-characterized canonical or noncanonical NF-κB pathways. These B-cells fail to secure sustained NEMO monoubiquitination following CD40-induced ROS generation, which specifically reduces downstream RelA (p65) signaling required for the transcriptomic and epigenetic remodeling underlying homotypic B-cell aggregation, cell proliferation, class-switch recombination, and antibody-secreting cell generation. Our results establish a physiological role of murine NEMO K270 and K302 in linking CD40 engagement to B-cell responses through enabling sustained NEMO modification and RelA transcriptional activity.

Summary: The kinetochore is an essential structure for the faithful chromosome segregation during mitosis. The 16-protein complex known as the constitutive centromere-associated network (CCAN) localizes the kinetochore throughout the cell cycle and forms the basis of the kinetochore. Although a structural model of an individual CCAN unit has been proposed through cryo-electron microscopy analysis, little is known about how multiple CCAN units are organized within cells. Here, using in silico and in vivo analyses, we demonstrate that multiple CCAN units form a spherical, shell-like structure in interphase DT40 kinetochores. CENP-T and CENP-N localize to the CCAN shell, and the CENP-C cupin domain clusters at the center. Molecular dynamics simulations of CCAN assembly suggest that CENP-C contributes to the orientation of CCAN units, leading to the formation of a spherical shell-like structure via CENP-C oligomerization. Super-resolution imaging reveals the spherical shell-like structure at DT40 kinetochores, and knocking out CENP-C abolishes the structure, supporting the reliability of the predicted models. These findings provide important insights into the spatial organization of functional kinetochores within cells.

Expansion microscopy (ExM) is an innovative and cost-effective super-resolution imaging technique that enables nanoscale visualization of biological structures using conventional fluorescence microscopes. By physically enlarging biological specimens, ExM circumvents the diffraction limit and has become an indispensable tool in cell biology. Ongoing methodological advances have further enhanced its spatial resolution, labeling versatility, and compatibility with diverse sample types. However, ExM imaging is often hindered by sample drift during image acquisition, caused by subtle movements of the expanded hydrogel. This drift can distort three-dimensional reconstruction, compromising both visualization accuracy and quantitative analysis. To overcome this limitation, we developed 3D-Aligner, an advanced and user-friendly image analysis software that computationally corrects sample drift in fluorescence microscopy datasets, including but not limited to those acquired using ExM. The algorithm accurately determines drift trajectories across image stacks by detecting and matching stable background features, enabling nanometer-scale alignment to restore structural fidelity. We demonstrate that 3D-Aligner robustly corrects drift across ExM datasets with varying expansion factors and fluorescent labels. This protocol provides a comprehensive, step-by-step workflow for implementing drift correction in ExM datasets, ensuring reliable three-dimensional imaging and quantitative assessment.

The HIV type 1 (HIV-1) accessory proteins Virion Infectivity Factor (Vif) and Viral Protein R (Vpr) are essential for efficient virus replication in vivo. Vif mediates degradation of host restriction factor APOBEC3G, while Vpr modulates the host cell proteome in ways that promote virion infectivity and viral gene expression. In cycling cells including CD4+ T cells, Vif and Vpr also severely impact cell cycle progression for reasons that remain unknown. Here, we combined live-cell imaging with virological assays to define the relative impacts of Vif and Vpr on the cell cycle and single-cell virion production. We demonstrate that Vif and Vpr’s effects on the cell cycle are markedly distinct, with Vif arresting cells in mitosis, while Vpr causes a G2 phase delay followed by endoreplication and reversion of cells into a “pseudo-G1” cell state lacking G2 markers. When coexpressed, Vpr’s capacity to bypass mitosis acts to suppress cytotoxicity associated with Vif-mediated cell cycle arrest, with the two proteins cooperating to extend the infected cell’s time in interphase as much as fivefold. Vpr-driven endoreplication also caused duplication of transcriptionally active proviral genomes, yielding a correlative ~twofold increase in per-cell viral gene expression. Based on these findings, we propose that Vif and Vpr coordinately regulate the cell cycle to prolong the infected cell’s lifespan and maximize single-cell virion output.

The kinetochore, an intricate macromolecular protein complex located on chromosomes, plays a pivotal role in orchestrating chromosome segregation. It functions as a versatile platform for microtubule assembly, diligently monitors microtubule binding fidelity, and acts as a force coupler. Comprising over 100 distinct proteins, many of which exist in multiple copies, the kinetochore’s composition dynamically changes throughout the cell cycle, responding to specific timing and conditions. This dynamicity is important for establishing functional kinetochores, yet the regulatory mechanisms of these dynamics have largely remained elusive. In this study, we employed advanced quantitative immunofluorescence techniques to meticulously chart the dynamics of kinetochore protein levels across the cell cycle. These findings offer a comprehensive view of the dynamic landscape of kinetochore architecture, shedding light on the detailed mechanisms of microtubule interaction and the nuanced characteristics of kinetochore proteins. This study significantly advances our understanding of the molecular coordination underlying chromosome segregation.

Reverse expression patterns of CARM1 and MAP2K4 in breast cancer subtypes and CARM1-mediated MAP2K4 methylation potentiates the downstream JNK activation

Raw RPPA data, related to Figure 1

Methylation of MAP2K4 promotes MDA-MB-468 cell proliferation and migration