Archaeal histone-based chromatin complexes are the evolutionary precursor of the eukaryotic nucleosomal landscape. Histones are encoded by most Archaea and are present in sufficient quantities in many heat-loving species to plausibly completely wrap and condense the genome(s). Establishing whether the in vivo archaeal histone-based chromatin structure(s) align to the repeating extended polymeric structures revealed with purified components in vitro remains an important goal to link the biological roles of archaeal and eukaryotic histone-based chromatin structures. Deploying advanced imaging and cryo-electron tomography techniques with the hyperthermophilic, histone-encoding archaeon Thermococcus kodakarensis, we reveal that the in vivo histone-based chromatin architecture changes dramatically during the growth cycle. While the cytoplasm contains a relatively uniform nucleoid in exponential growth, tightly packed, helical filaments that conform to archaeal histone-based chromatin observed in vitro emerge during the transition to stationary phase. The in vitro extended chromatin conformation of histone-based genome architecture observed in vivo establishes that archaeal information processing machinery can function on tightly packed and regularly organized histone-based chromatin structures. We also observe large, nucleic-acid rich, globular condensates directly alongside the helical filamentous chromatin conformation that may represent an additional conformation of archaeal chromatin. Our results suggest that alternative forms of archaeal chromatin may be achieved without obvious chromatin remodeling complexes or biologically significant levels of post-translational modifications to histone proteins, providing a plausible evolutionary driver for the emergence of more complex mechanisms that protect and regulate expression of eukaryotic genomes.

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed the consequences of checkpoint override, forcing nuclei through untimely cell cycle transitions, and highlight how cell-cell fusion experiments can be a powerful system to study how competing cytoplasmic states are integrated in a shared cytoplasm, such as in muscle, placenta formation and in cancer.

Small GTPases are generally viewed as organizers of organelle identity, with different families recruited to specific membranes by compartment-selective targeting mechanisms. Here, we identify a membrane state that reverses this relationship. Excessive peptide S-palmitoylation at Golgi generates multilamellar, filipin-poor whorls that recruit ARF, Rab, and Rho family GTPases normally associated with distinct cellular compartments. The whorls simultaneously excluded Golgi transmembrane residents, coat proteins, ER proteins, a GPI-anchored protein, and other palmitoylated proteins, demonstrating that they were selective membrane compartments rather than nonspecific protein aggregates. Preventing myristoylation of ARF6 or geranylgeranylation of Rab11a strongly reduced recruitment, whereas prenylation alone was insufficient, indicating recognition of a composite membrane-targeting code. Lowering cellular cholesterol promoted whorl formation, whereas cholesterol supplementation suppressed it. Chemically distinct lipid perturbations generated GTPase-positive Golgi whorls, suggesting convergence on a shared membrane-remodeling state. As whorls accumulated, endocytic recycling and anterograde trafficking declined, survival signaling decreased, and viability became poorly reversible. These findings show that lipid imbalance can redirect small GTPases across normal organelle boundaries, converting Golgi-derived membranes into selective sinks for trafficking regulators.

Proteins can adopt many distinct conformations, yet the sequence determinants governing which structural states are accessible remain poorly understood. Using fold-switching proteins and AlphaFold as an analytical lens, we identify an organizational principle in which access to alternative structural states-and in some cases the folded state-is governed by surprisingly few amino acids, termed gating residues. Gating generalizes to single-fold proteins, indicating that conformational accessibility is often hierarchically organized around a small number of disproportionately influential residues. AlphaFold has implicitly learned this principle but amplifies and sometimes misapplies it, concentrating conformational control onto too few or incorrect residues. Guided by this principle, targeted MSA editing recovered a conformation AlphaFold confidently mispredicted, suggesting a path toward more accurate prediction of alternative conformations and mutational effects.

Primary cilia are micron-scale cellular compartments with key roles in cell signaling and disease. Cilium assembly has been delineated as a multi-step process that is disrupted in ciliopathies, yet many features of cilia disassembly remain incompletely understood. Here, we review recent insights into cilia disassembly. In addition to discussing the longstanding link between cilia disassembly and the cell cycle, we consider new findings that broaden our understanding of the molecular mechanisms and physiological contexts that govern cilia disassembly. We also synthesize recent evidence that mutual antagonism between assembly and disassembly pathways governs ciliation state. Together, these studies reveal that both excessive and impaired disassembly can perturb ciliary signaling and function, with consequences for development, tissue homeostasis and tumorigenesis. Motivated by these advances, we propose a new ciliopathy subclassification - disassembly-related ciliopathies - to encompass diseases in which aberrant cilia disassembly contributes to pathogenesis. Continued exploration of the mechanisms controlling cilia disassembly will provide both fundamental biological insights and open new avenues for targeted treatment of diseases caused by ciliary dysfunction.

Terminally misfolded or damaged proteins in the endoplasmic reticulum (ER) are degraded by the proteasome in a process termed endoplasmic reticulum-associated degradation (ERAD). To reach the proteasome in the cytosol, cargo proteins must be dislocated across the ER lipid bilayer. Retrograde transport is performed by a set of membrane-spanning multi-protein complexes that are notoriously difficult to study. Despite decades of genetic and biochemical analysis, there is little consensus regarding its molecular mechanism. Here, we characterize the dynamic assembly of the mammalian Hrd1 complex, one of the most thoroughly studied mammalian dislocons, using in situ multicolor single-particle tracking. Surprisingly, quantitative dual-color tracking reveals that the majority of Hrd1 is assembled into stable homo-tetramers in situ. Using a herein developed single-molecule assay based on binding competition, we show that this tetramerization is driven by a short stretch (Hrd1479-530, HAF-H) within the cytosolic domain of Hrd1. Combining classical purification assays, quantitative imaging, and structural predictions, we demonstrate that Hrd1479-530 forms a highly stable tetrameric helix bundle via a conserved hydrophobic coiled-coil motif. While higher order assemblies of Hrd1 have been previously implicated, this work offers direct evidence of their role in dislocation, demonstrating that quantitative single-molecule imaging can yield information on species previously hidden from classical biochemistry.

The distribution of mitochondrial DNA-containing nucleoids is essential for mitochondrial function and genome inheritance; however, no known mechanisms can explain nucleoid segregation or their regular positioning. In this work, we found that mitochondria frequently undergo a reversible biophysical instability termed "pearling," transforming from a tubular into a regularly spaced beads morphology. Physiological pearling imposed a characteristic length scale and simultaneously mediated nucleoid disaggregation and established internucleoid distancing with high precision. Pearling onset was triggered by calcium influx, whereas the density of lamellar cristae invaginations modulated pearling prevalence and preserved nucleoid spacing following recovery. The dysregulation of mitochondrial calcium influx or inner membrane cristae integrity caused aberrant nucleoid clustering. Our results identify pearling as a mechanism governing nucleoid distribution and inheritance and offer insights into its regulation.

Messenger RNA (mRNA) transfection enables rapid, transient protein expression without nuclear entry, providing a powerful alternative to DNA or viral delivery in post-mitotic and otherwise difficult-to-transfect cells. Although in vitro transcribed (IVT) mRNAs have revolutionized therapeutic applications, their adoption in experimental biology remains limited by challenges in synthesis, variability across cell types, and concerns about cytotoxicity. Here, we define design principles that maximize IVT mRNA performance across diverse cellular and organismal systems. Through systematic comparison of capping strategies and base modifications, including N1-methyl-pseudouridine, 5-methylcytidine, and 5-methoxyuridine, we identify modifications that enhance translation while minimizing activation of cellular stress responses. Optimized transcripts drive robust protein expression within four hours, persist for up to one week, and support multiplexed expression of structurally and functionally distinct proteins in mammalian cells, including cancer cell lines, iPSC-derived systems, primary cells, and organoids, as well as in vivo in zebrafish embryos and in less genetically tractable models such as Danionella cerebrum and sea urchin embryos. To further expand accessibility for community use, we developed mRNAbow, a platform for generating low-toxicity mRNAs encoding organelle-targeted fluorescent proteins and biosensors for multiplex imaging, with corresponding plasmids made publicly available. Together, these advances establish a generalizable framework for IVT mRNA design and expand experimental access to synthetic mRNA technologies for dissecting cellular architecture and dynamics.

The endoplasmic reticulum (ER) is a highly interconnected membrane network that serves as a central site for protein synthesis and maturation. A crucial subset of ER-associated transcripts, termed secretome mRNAs, encode secretory, lumenal and integral membrane proteins, representing nearly one-third of human protein-coding genes. Unlike cytosolic mRNAs, secretome mRNAs undergo co-translational translocation, and thus require precise coordination between translation and protein insertion. Disruption of this process, such as through altered elongation rates, activates stress response pathways that impede cellular growth, raising the question of whether secretome translation is spatially organized to ensure fidelity. Here, using live-cell single-molecule imaging, we demonstrate that secretome mRNA translation is preferentially localized to ER junctions that are enriched with the structural protein lunapark and in close proximity to lysosomes. Lunapark depletion reduced ribosome density and translation efficiency of secretome mRNAs near lysosomes, an effect that was dependent on eIF2-mediated initiation and was reversed by the integrated stress response inhibitor ISRIB. Lysosome-associated translation was further modulated by nutrient status: amino acid deprivation enhanced lysosome-proximal translation, whereas lysosomal pH neutralization suppressed it. These findings identify a mechanism by which ER junctional proteins and lysosomal activity cooperatively pattern secretome mRNA translation, linking ER architecture and nutrient sensing to the production of secretory and membrane proteins.

The brain microvascular functions are strongly influenced by the local microenvironment and cellular organization. Intracellular organelles, including primary cilia and centrioles, play critical roles in sensing and transmitting environmental cues and maintaining vascular integrity. However, their distribution across the brain vasculature remains poorly understood. In this study, we utilized publicly available large-volume electron microscopy datasets encompassing the cerebral vasculature from pial arterioles through parenchymal capillaries to pial venules. We systematically analyzed the cellular organization and characterized the distribution of primary cilia and centrioles in the mouse and human brain microvasculature. We found primary cilia exclusively on human cortical endothelial cells (ECs), indicating inter-species differences between mouse and human. Primary cilia were frequently present on mural cells (MCs, smooth muscle cells or pericytes) surrounding venules and capillaries but rarely observed on arterioles in both mouse and human brains. These MC primary cilia exhibited heterogeneity in ciliogenesis, including cells with ciliary pockets, surface cilia, and a hybrid configuration we refer as a partial pocket. In the mouse brain, many MC primary cilia were closely ensheathed by astrocytic endfeet and occasionally extended between them to establish proximity to synapses, whereas all primary cilia in the human brain were confined within the basal lamina. Our analysis of cellular density revealed similar EC densities between arterioles and venules in mice, but not in human. EC centrioles were consistently positioned against the direction of blood flow relative to the nuclei, suggesting that they may serve as a structural marker for flow direction. Collectively, these findings provide a comprehensive characterization of primary cilia and centrioles, highlighting distinct interspecies differences between mouse and human brain microvasculature. The proximity to neural cells and gradient distribution of these subcellular structures suggest that they may act as antennae for sensing mechanical and chemical signals within the brain microvascular environment.