Molecular Biology of the Cell最新文献

Promyelocytic leukemia (PML) protein isoform II is a component of PML nuclear bodies (PML NBs) that also forms patches on nuclear lipid droplets (nLDs) and the inner nuclear membrane (INM). Here we tested whether different metabolic treatments that induce membrane curvature elastic stress (CES) in the INM, detected by recruitment of CTP:phosphocholine cytidylyltransferase α (CCTα) and a nuclear diacylglycerol (DAG) biosensor, are a precondition for PML-II membrane association. We found that treatment of U2OS cells with unsaturated 18-carbon fatty acids and DAG acyltransferase inhibitors caused the rapid formation of PML patches on the INM that coincided with DAG enrichment and the recruitment and stabilization of CCTα, all of which were reversed upon removal of the CES stimulus. PML patches were depleted of canonical PML NB-associated proteins, occurred at sites of lamin depletion, were specific for the PML-II isoform, and occurred in cells regardless of their capacity to assemble nLDs. Induction of INM curvature stress by knockout of the terminal enzymes of the CDP-choline pathway or lipid activators of CCTα also promoted PML patches as well as stabilization of CCTα on the INM. We conclude that CES in the INM promotes the reversible assembly of PML-II-dependent membrane-associated patches.

Paralogs engage in biological processes through both redundant and nonredundant functions. In the yeast Saccharomyces cerevisiae, approximately one-fifth of the genome consists of paralogs, with their encoded proteins involved in multiple pathways. However, the unique contributions of individual paralogs have remained poorly defined. Here, we undertook a systematic examination of eight paralog pairs in the glucose-sensing pathways, deleting each component and measuring the resulting changes in gene expression. To that end, we established a new transcription reporter system to monitor the response to glucose as well as to nonpreferred sugars in single cells. Focusing on the PKA catalytic subunits, comprised of the paralogs Tpk1 and Tpk3 as well as the isomorphic kinase Tpk2, we employed mass spectrometry to identify their contribution to cellular metabolism, used a GFP-based sensor to follow changes in cytosolic pH, and used BioID to identify unique and shared candidate binding partners. Our data reveal that paralogs in the glucose-sensing pathway contribute in multiple and unique ways to signal transduction, and establish potential mechanisms driving the preservation of these and other duplicated genes throughout long periods of evolution.

Syncytia provide a unique system in which to understand the mechanisms of cellular organization. Two dramatic features of syncytial cells are the number of nuclei and the positioning of nuclei within a shared cytoplasm. Whether the formation of the syncytia and the organization of the syncytia are linked is not known. We have characterized the subperineurial glial cells (SPG), which form the most restrictive layer of the Drosophila blood-brain barrier. We found that disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, Kinesin, or cytoplasmic Dynein affected both SPG cell development and general brain development. Specifically, the brains were smaller in each case, and the SPG cells were smaller when the LINC complex or cytoplasmic Dynein were disrupted. The number of nuclei per cell was increased when Kinesin was disrupted and decreased when cytoplasmic Dynein was disrupted. Finally, the positions of nuclei relative to their nearest neighbor were decreased when the expression of each gene was disrupted, and nuclei were closer to the cell edge when either Kinesin or cytoplasmic Dynein was disrupted. Together, these data illustrate that the formation of SPG cells and the organization of SPG cells are dependent on microtubule motors and the LINC complex.

We describe a noncanonical, membrane receptor-like regulation of the human copper transporter-1 (CTR1) in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on the physical properties of plasma membranes harboring these clusters that could favor endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain molecular dynamics (MD)-simulations and coupled interference reflection microscopy-total internal reflection fluorescence microscopy (TIRF) we demonstrated that CTR1 clusters induce positive membrane curvature, an increase in local membrane tension, and a decrease in local membrane fluctuation; alterations that favor the formation of endocytic pits. Clustering is facilitated by copper-sequestering methionine-rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adaptor protein AP-2. To summarize, we report a hitherto unknown "pre-endocytic" "receptor-like" phenomenon of ligand-induced clustering of a metal channel, which in turn regulates self-endocytosis by modulating membrane properties.

Puerto Rico Cancer Research Meeting (PRCRM) was conceived and established by four graduate students in response to the urgent need for specialized research forums on an Island that is geographically limited. PRCRM emphasizes collaborative efforts between local and national institutions, encouraging exchange of ideas that often lead to the development of new grant applications and research initiatives, empowering local investigators. Defining features of PRCRM are its commitment to inclusivity, the four graduate students completed their doctoral degrees and hold leadership positions at UCC, PHSU, NIH, Memorial Sloan Kettering, and, despite numerous challenges faced, the meeting ensures Puerto Ricans are represented.

The vascular endothelium serves as a selectively permeable barrier, tightly regulating transendothelial transport of small molecules, macromolecules, and immune cells. In the context of cardiovascular and inflammatory pathologies, the integrity of this barrier is often markedly compromised, contributing to tissue dysfunction, multi-organ failure, and, ultimately, mortality. Therapeutic strategies aimed at mitigating endothelial barrier dysfunction primarily focus on enhancing the stability of endothelial cell-cell junctions. However, the precise molecular mechanisms governing junctional stability remain incompletely understood. In this study, we demonstrate that the N-terminal domain of the Rho guanine nucleotide exchange factor Trio (TrioN) enhances endothelial junctional stability by promoting the formation of tensile F-actin bundles at intercellular junctions, thereby inducing a linear junctional architecture and packaging of junctional proteins. This structural reorganization leads to an increase in endothelial barrier function. The small GTPase Rap1 is responsible for junctional tension, as depletion of Rap1 results in reduced junctional tension, loss of linearity, and increased permeability. In vivo TrioN gain-of-function experiments reveal enhanced local mechanical tension and a concomitant improvement in vascular barrier integrity within atherosclerotic vein grafts in murine models. These findings position TrioN as a promising therapeutic target for the restoration and reinforcement of endothelial barrier function in vascular disease.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by progressive degeneration of retinal photoreceptors. Traditional disease models suggest that defective repression of thioester protein C3 activity by complement factor H (CFH) is a major contributor to pathogenesis in AMD and a related disease, early-onset drusen maculopathy (EODM). Our previous study identified novel functions for human CFH and C. elegans CFH-1 in the maintenance of inversin compartment integrity in photoreceptors and mechanosensory neurons, indicating that CFH has a novel, evolutionarily conserved role in cilia compartment organization that is distinct from its established function in alternative complement pathway regulation. Here, we investigate the C. elegans thioester protein TEP-1, an ancestral relative of C3 and other members of the AMCOM family (C4, C5, CD109, and alpha-2-macroglobulin). TEP-1 localizes to select glial cell surfaces and regulates inversin compartment organization and intraflagellar transport (IFT) within the cilia of ensheathed sensory neurons. In addition to revealing a novel role for an AMCOM family member in sensory neuron structure and protein transport, the localization of C3 and CFH on human photoreceptors provides support for non-canonical models of AMD and EODM pathogenesis in which defects in cilia structure and protein transport contribute directly to the progressive photoreceptor dysfunction that characterizes these diseases.

During autophagy induction in Saccharomyces cerevisiae, over twenty autophagy-related (Atg) proteins localize to the site of autophagosome formation to generate the pre-autophagosomal structure (PAS), where phase-separated condensates of the Atg1 kinase complex serve as a scaffold for recruiting other Atg proteins. The lipid transfer protein Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and mediates lipid influx from the endoplasmic reticulum to the PAS for membrane expansion. In this study, we discover that the Atg2-Atg18 complex interacts with the Atg1 complex. This interaction involves the C-terminal regions of Atg2 and the Atg1 complex subunit Atg29, and is enhanced by Atg1-dependent phosphorylation of Atg29. This interaction, together with Atg18 binding to PI3P, promotes PAS localization of the Atg2-Atg18 complex. These findings provide new insight into PAS organization and highlight the Atg1 complex as a central hub coordinating Atg protein assembly during autophagosome formation.

Actin filaments and microtubules are fundamental components of the cytoskeleton in eukaryotic cells, orchestrating cellular processes such as intracellular transport, migration, and division. Traditionally studied as distinct entities, growing evidence highlights their intricate crosstalk, mediated by crosslinking proteins, motor proteins, and signaling pathways. Reconstituted in vitro systems provide a powerful framework for isolating and probing these interactions under controlled conditions, enabling direct connections between molecular coupling and higher-order cytoskeletal organization. Such approaches have revealed how actin-microtubule interactions shape network architecture, coordinate force transmission, and give rise to emergent mechanical behavior that cannot be inferred from either system alone. This review synthesizes mechanistic principles of actin-microtubule crosstalk revealed by reconstituted systems, spanning molecular interactions, network-scale organization, and mechanical feedback. These insights advance understanding of cytoskeletal coordination in cells and identify key challenges toward developing predictive frameworks that link molecular interactions to emergent cellular mechanics.

In yeast, early adaptation to hyperosmotic stress involves organelle-based mechanisms, including synthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) in the endolysosomal system. This low-level signaling lipid drives vacuolar fragmentation and activates the V-ATPase, which acidifies the vacuole and promotes salt sequestration. Under NaCl stress, PI(3,5)P₂ rapidly accumulates, triggering increased V-ATPase activity and vacuolar remodeling; these responses are impaired by deficient PI(3,5)P₂ synthesis. We visualized movements of a GFP fusion protein with the cytosolic domain of V-ATPase subunit Vph1 (Vph1NT-GFP) in a microfluidic system during salt stress. Upon NaCl addition, Vph1NT-GFP rapidly relocalizes to a region adjacent to the vacuole in a PI(3,5)P₂-dependent manner. The intensity and duration of this response depend on salt concentration, but the response is diminished by 30-45 min., even if salt is readded. Vph1NT-GFP returns to the same location upon repeated salt challenge, suggesting that PI(3,5)P₂ synthesis occurs at a localized domain/contact site that may be endosomal. When the high osmolarity glycerol pathway, which coordinates long-term transcriptional changes, is disrupted, Vph1NT-GFP recruitment is significantly extended. This underscores the integration of lipid signaling and transcriptional regulation in osmoadaptation. These findings suggest activation of endolysosomal targets by PI(3,5)P₂ synthesis provides immediate protection that primes cells for longer-term survival strategies. [Media: see text] [Media: see text] [Media: see text].