Journal of cell science最新文献

Long-distance transport in neurons relies on motor proteins that can move towards either the plus- or minus-end of microtubules. In axons, microtubules uniformly have a plus-end-out orientation, whereas dendrites of vertebrate neurons contain mixed polarity bundles: stable microtubules are typically minus-end-out, and dynamic microtubules are plus-end-out. This organization supports selective transport, yet how this dedicated microtubule organization is established is unclear. Here, we use single-molecule localization microscopy, expansion microscopy and live-cell imaging to examine how the microtubule cytoskeleton is reorganized during neuronal development in cultured rat hippocampal neurons. We find that early neurites contain mixed polarity microtubules, with stable microtubules initially mostly plus-end-out and often connected to centrioles. As neurons mature, these microtubules detach, slide and gradually reorient to become predominantly minus-end-out within the future dendrites. Moreover, prior to axon specification, neurons often have one or two minor neurites with an almost uniformly plus-end-out microtubule network. Our findings show how reorganization of stable microtubules underlies the establishment of the characteristic microtubule network in mature vertebrate neurons.

The meiotic spindle forms only around the chromosomes in oocytes, despite the exceptionally large volume of the cytoplasm. This spatial restriction is likely to be governed by local activation of key microtubule regulators around the chromosomes in oocytes, but the identities of these microtubule regulators and the mechanisms remain unclear. To address this, we developed a novel assay to visualise spatial regulation of spindle-associated proteins in Drosophila oocytes by inducing ectopic microtubule clusters. This assay identified several proteins including the TPX2 homologue Mei-38, which localised more strongly to microtubules near the chromosomes than away from them. In Mei-38, we identified a microtubule-binding domain containing a region that was also highly conserved in humans. The domain itself is regulated spatially, and contains a conserved serine and a nearby PP2A-B56-docking motif. A non-phosphorylatable mutation of this serine residue allowed the domain to localise to ectopic microtubules as well as spindle microtubules, whereas mutations in the PP2A-B56-docking motif greatly reduced the spindle localisation. As this phosphatase is concentrated at the kinetochores, it might act as a novel chromosomal signal spatially regulating spindle proteins within oocytes.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that plays important roles in cell proliferation, differentiation and migration. EGFR overexpression or mutation is a hallmark of some cancers, leading to hyperactivation of downstream signalling. Co-regulation between EGF-dependent EGFR signalling and extracellular matrix (ECM) adhesion occurs in both healthy and malignant cells. Increasing ECM stiffness can contribute to lung cancer progression and is sensed by integrins to promote proliferation and invasion. Emerging evidence suggests non-canonical roles for EGFR in mechano-sensing, but the molecular mechanisms and functional consequences remain unclear. Here, we demonstrate that EGFR is activated in human lung cancer cells upon early adhesion to ECM substrates with physiologically relevant stiffness (28 kPa versus 1.5 kPa), independently of canonical ligands and integrins. Mechano-induced EGFR activation correlates with and requires active Src and F-actin, and it is coupled to stiffness-dependent plasma membrane retention of EGFR within disordered lipid microdomains. Early stiffness-dependent EGFR activation is required for enhanced migration. These findings uncover a non-canonical role for EGFR in early adhesion related mechano-sensing with potential implications for treatment of lung cancer.

The ability of cells to stick to each other and form tissues is mediated by protein complexes at the plasma membrane, such as adherens junctions (AJs). Key aspects of AJ stability are the biomechanical properties of the constituent proteins and the forces generated by the associated actin cytoskeleton. This Review concisely overviews our current understanding of how these factors play out at different length scales. When actomyosin pulls on the cadherin-catenin complex, the molecular interactions within the complex lead to an increase in AJ stability. Transcellular E-cadherin clusters are dynamically maintained by constant turnover and recruitment of actin-binding proteins organises the internal actin cytoskeleton. Among these are actin polymerisers that sustain the actin network and provide the mechanical forces important for AJ integrity. Finally, the distribution of AJs around the cell periphery and the long-range organisation of the associated actin bundles could contribute to maintaining AJ stability across tissues. We conclude with a summary of recently developed biophysical tools useful for the study of AJ mechanics and a few open questions that we expect to see answered in the not-too-distant future.

Accurate cell segmentation is an essential step in the quantitative analysis of fluorescence microscopy images. Pre-trained deep learning models for automatic cell segmentation such as those offered by Cellpose perform well across a variety of biological datasets but might still introduce segmentation errors. Although training custom models can improve accuracy, it often requires programming expertise and significant time, limiting the accessibility of automatic cell segmentation for many wet lab researchers. To address this gap, we developed 'Toggle-Untoggle', a standalone desktop application that enables intuitive, code-free quality control of automated cell segmentation. Our tool integrates the latest Cellpose 'cyto3' model, known for its robust performance across diverse cell types, while also supporting the 'nuclei' model and user-specified custom models to provide flexibility for a range of segmentation tasks. Through a user-friendly graphical interface, users can interactively toggle individual segmented cells on or off, merge or draw cell masks, and export morphological features and cell outlines for downstream analysis. Here, we demonstrate the utility of Toggle-Untoggle in enabling accurate, efficient single-cell analysis on real-world fluorescence microscopy data, with no coding skills required.

The nucleus-vacuole junction (NVJ) in Saccharomyces cerevisiae is a multifunctional contact site between the membrane of the nuclear endoplasmic reticulum (ER) and the vacuole that has diverse roles in lipid metabolism, transfer and storage. Adaptation of NVJ functions to metabolic cues is mediated by a striking remodeling of the size and the proteome of the contact site, but the extent and the molecular determinants of this plasticity are not fully understood. Using microscopy-based screens, we monitored NVJ remodeling in response to glucose availability. We identified Pex31, Nsg1, Nsg2, Shr5, and Tcb1 as NVJ residents. Glucose starvation typically results in an expansion of the NVJ size and proteome. Pex31 shows an atypical behavior, being specifically enriched at the NVJ in high-glucose conditions. Loss of Pex31 uncouples NVJ remodeling from glucose availability, resulting in recruitment of glucose starvation-specific residents and NVJ expansion at glucose-replete conditions. Moreover, PEX31 deletion results in alterations of sterol ester storage and a remodeling of vacuolar membranes that phenocopy glucose starvation responses. We conclude that Pex31 has a role in metabolic adaptation of the NVJ.

Fanconi anemia is a rare genetic disease caused by the loss of function of one of the 23 associated genes and is characterized by bone marrow failure, cancer predisposition and developmental defects. The proteins encoded by these genes (FANC proteins) mainly function in DNA damage response and repair. Although FANC deficiency has multiple effects on the regulation of lipid metabolism, the molecular function of FANC proteins in the context of Fanconi anemia pathology remains unclear. In the present study, we demonstrate that FANCD2, a key component of FANC proteins, interacts with factors involved in fatty acid biosynthesis or sphingolipid metabolism and that FANCD2 deficiency downregulates the cellular levels of fatty acids. Moreover, a portion of FANCD2 is localized to nuclear lipid droplets in response to oleic acid (OA) treatment. These subcellular dynamics are independent of FANCD2 monoubiquitylation, which is essential for the DNA damage response. Collectively, these findings demonstrate that FANCD2 responds to not only DNA damage but also OA exposure, providing insights into the pathogenesis of lipid dysregulation in Fanconi anemia.

Growth signals and immune responses in cancer typically originate in the same compartments. In early stages of tumor development, inflammatory cells trigger responses against growing cancers. At a molecular level, it is unclear how the innate immune system recognizes tumorigenesis. At later stages, cancer cells resist cell death and evade immune detection, thereby suppressing anti-tumor responses and promoting cancer hallmarks. Often, chronic inflammatory responses become tumor friendly and incline towards tumorigenesis disturbing metabolic signaling, thereby rewiring nutritional supply for cancer growth. The precise connecting link between cancer, nutrition and metabolism remains unclear. Drosophila provides an ideal platform to explore the links between hyperactive immune signaling, defective fat metabolism and pseudotumor formation. Therefore, we examined the effects of methotrexate on these pathophysiological processes in larvae with hyperactive Toll/NF-κB pathway. We determined that both chemical (methotrexate) and genetic [rescue of Ubc9-/- mutants by introducing a wild-type copy of Cactus (negative regulator of the Toll pathway)] interventions alleviated abnormalities associated with Toll/NF-κB hyperactivity and its influence on insulin signaling. Our study underscores drug repurposing studies and provides insights into how immune-metabolic crosstalk rewires inflammation-driven tumorigenesis.

Sphingolipids are essential for cell membrane structure and the regulation of organelle functions. Sphingolipid synthesis requires the coordinated activity of multiple organelles, including the endoplasmic reticulum, Golgi, lysosomes and mitochondria, which are connected via membrane contact sites. Metabolic remodeling of sphingolipid pathways is observed in aging and numerous age-related disorders. However, numerous studies have highlighted the complex and species-specific roles of sphingolipid metabolism in aging. In budding yeast, inhibition of sphingolipid synthesis extends lifespan by a mechanism that is poorly understood. Recent findings suggest that inhibition of sphingolipid synthesis in cells mimics methionine restriction, a condition known to extend lifespan across different experimental models. However, how sphingolipid remodeling alters cellular methionine levels, and whether this directly influences aging, remains unclear. In this Review, we explore the roles of sphingolipids in organelle function, highlighting their metabolic connections to methionine restriction and aging.