Vimentin intermediate filaments (VIFs) undergo active microtubule-dependent transport throughout the cell, with trajectories resembling those of motor-driven organelles, yet our recent work showed that only a small subset of VIFs lies close to microtubules. How VIFs engage the microtubule-based transport machinery, and whether additional transport routes contribute, remain unresolved. We hypothesized that VIFs move indirectly, by hitchhiking on motile organelles, and tested this using mitochondria. Live imaging showed mitochondria and adjacent VIFs moving together, and volume electron microscopy revealed VIFs closely following mitochondrial surfaces at sites distinct from microtubule contacts. Depleting the mitochondrial motor adaptor TRAK1 to inhibit anterograde mitochondrial transport shifted both mitochondria and VIFs toward the nucleus. Driving mitochondria toward the cell periphery with a tailless kinesin-1 in cells lacking endogenous kinesin-1, or toward the cell center with a minus-end-directed motor, redistributed a subset of VIFs in the same direction. Thus, VIF transport to a large extend is accomplished by hitchhiking on motile mitochondria.

This protocol describes a method for endogenous protein tagging in postmitotic primary neurons using homology-independent targeted integration (HITI), a CRISPR/Cas9-based approach that exploits non-homologous end joining (NHEJ). Unlike homology-directed repair (HDR), HITI does not require cell division, making it uniquely suited for postmitotic neurons. SpCas9 is delivered by a replication-incompetent simian immunodeficiency lentiviral vector (pLenti SIV), while the sgRNA, donor tag cassette, and a fluorescent plasma membrane marker are co-packaged in a single AAV vector. The two vectors are co-delivered at DIV5, and endogenous tagging is assessed at DIV15-23. The workflow is illustrated using Junctophilin-3 (JPH3) tagged with mEmerald at its endogenous locus. Airyscan microscopy imaging of endogenously tagged JPH3 at native expression levels revealed its localization at endoplasmic reticulum-plasma membrane (ER-PM) contact sites and uncovered a periodic distribution of ER-PM junctions along the dendritic arbor, enabling identification of a previously unrecognized ladder-like organization of the ER in neuronal dendrites. Genomic integration of the tag is validated at both 5' and 3' junctions by next-generation sequencing. The protocol can be adapted to other neuronal proteins by redesigning the guide RNA and donor sequence, and supports fluorescent proteins as well as epitope and self-labeling tags. The plasma membrane marker can also be replaced with other fluorescent reporters or markers for intracellular organelles to assess infection efficiency and verify subcellular localization of the protein of interest. The full workflow takes 5-6 weeks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Protocols included in this article: Basic Protocol 1: sgRNA design and HITI donor vector cloning Basic Protocol 2: Primary rat neuron culture and viral co-transduction Basic Protocol 3: Live-cell imaging of endogenously tagged proteins Support Protocol 1: Production of lentiviral and AAV particles Support Protocol 2: Validation of knock-in by next-generation sequencing (NGS).

Metabolic state is tightly linked to cell fate, and expression of the mitochondrial pyruvate carrier (MPC), which imports pyruvate into mitochondria, is a robust marker of it. The transcriptional regulators that set metabolic state, however, remain poorly defined. In the liver, β-catenin is one of the determinants of metabolic zonation, and activating CTNNB1 mutations are frequent in hepatocellular carcinoma. Here we show that β-catenin regulates metabolic state in liver cancer cells. An unbiased CRISPR screen that uses endogenous MPC1transcript abundance as the marker identifies CTNNB1. Activating β-catenin pharmacologically or genetically reduces MPC1 and MPC2 transcript and protein levels, and MPC expression is also low in β-catenin-driven liver tumors in vivo. Beyond the MPC, β-catenin activation impairs assembly of respiratory Complexes III and IV, reduces mitochondrial DNA content and respiratory capacity, elongates mitochondrial networks and remodels the lipidome. Stable isotope tracing shows reduced entry of glucose- and lactate-derived carbon into the TCA cycle alongside increased oxidation of palmitate and oleate. These findings identify β-catenin as a regulator of metabolic state that drives a fuel switch from carbohydrate to fatty acid oxidation in liver cancer cells.

Protein aggregate formation can cause cell damage and contribute to tissue pathologies including neurodegeneration. Protein aggregates are normally eliminated by cell-intrinsic mechanisms of lysosomal (autophagic) degradation. However, degradative capacity varies between cell types, raising the possibility that high-capacity cell types, such as macrophages, may contribute to aggregate disposal from the low-capacity cells. Here, we show that cellular protein aggregates are removed by macrophages via trogocytosis — a partial engulfment of the target cells. Lysosomal stress upon protein aggregate accumulation promotes cell surface expression of the transmembrane isoform of the macrophage growth factor CSF1 (tmCSF1), which is detected by macrophages through the CSF1 receptor to drive trogocytosis. Insufficient or impaired degradation of protein aggregates in lysosomes results in their transport along microtubules to the plasma membrane where they physically associate with tmCSF1. This enables macrophages to specifically trogocytose protein aggregates. These findings reveal a protein homeostasis system that eliminates protein aggregates in a non-cell-autonomous manner.

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.