Journal of cell science最新文献

Lysosomes are the main digestive organelles and serve as a signaling hub linking environmental cues to cellular metabolism. Through these functions, lysosomes play a crucial role in maintaining cellular and organismal homeostasis. However, how lysosomal homeostasis itself is maintained is not well understood. Lysosomes are frequently damaged by a variety of substances, including crystals, silica, lipids, bacteria, toxins, amyloid proteins and reactive oxygen species. When lysosomes are damaged, their acidic contents leak out, leading to oxidative stress, inflammation and cell death. Damaged lysosomes are thus harmful to cells, and to restore lysosomal function after damage, cells have developed several defense mechanisms, collectively called the lysosomal damage response (or endo-lysosomal damage response). Recent studies have shown that this response is composed of three main pathways depending on the degree and duration of damage - repair, removal of the damaged lysosomes, and lysosomal biogenesis and regeneration. Growing evidence suggest that the failure and/or dysregulation of this response is implicated in aging and several diseases, including neurodegenerative diseases and kidney disease. In light of the rapid growth of this field, this Review summarizes our current knowledge of the lysosomal damage response, its significance in aging and diseases, and future perspectives.

Adeno-associated virus (AAV) is a widely used vector for gene delivery, yet the host intracellular trafficking barriers often limit its therapeutic efficacy. Here, we identify microtubule detyrosination - a microtubule post-translational modification - as a key regulator of AAV2 endo-lysosomal processing. Using super-resolution microscopy (SIM/STORM), we show that upon AAV2 endocytosis, the host upregulates microtubule detyrosination via the GSK3β-CLASP2 signaling axis. Single-particle tracking of recombinant virus reveals that detyrosinated microtubules form a physical and functional barrier, restricting AAV2 motility and promoting lysosomal trapping. Restoring microtubule tyrosination via tubulin-tyrosine ligase overexpression or pharmacological inhibition of detyrosination with parthenolide, enhances endosomal escape and perinuclear accumulation of AAV2, translating to improved gene delivery in host cells. Notably, a clinically relevant pro-drug of parthenolide (DMAPT) also displayed a similar trend of enhancing AAV2-driven coagulation factor IX expression in hemophilia B mouse models. Our findings uncover a host mechanism that reshapes the microtubule landscape to restrict AAV2 trafficking and identify microtubule detyrosination as a novel druggable target to improve AAV-based gene therapy.

Living cells contain dynamic structures that constantly change shape, merge together, split apart and travel in coordinated patterns, much like flocks of birds or schools of fish. Quantifying these complex, collective behaviours can be challenging, as most available tools are designed to follow discrete objects rather than distributed, shape-shifting pixel-level activity patterns. We developed ARCOS.px, a freely available software tool with a user-friendly graphical interface, to automatically identify and track these coordinated dynamic cellular events in time-lapse microscopy movies. The software works by taking semantically segmented binary images, in which pixels are classified as 'active' or 'inactive'. It then uses spatial clustering to group pixels into distinct coordinated events, and tracks how these events evolve over time. We tested our method by tracking cellular structures involved in cell movement and signalling in REF52 cells. Our analyses revealed how different drugs affect the behaviour of these structures and uncovered the timing relationships between different cellular components during wave-like spreading events. ARCOS.px fills a gap in current image analysis tools by enabling researchers to quantify coordinated intracellular phenomena, which was previously difficult to achieve.

Hair cells in the utricle and saccule form two groups with oppositely oriented stereociliary bundles that enable detection of broad ranges of motion. These groups are aligned along a common polarity axis established by core planar cell polarity (PCP) proteins, which individual cells interpret differently to generate opposing bundle orientations. EMX2, GPR156 and STK32A determine how these groups integrate PCP signaling during this process. We tested functional interactions between these factors using genetic epistasis experiments and evaluating hair cells in mice with combined mutations in Gpr156 and Stk32a or Emx2 and Stk32a. We show in the utricle that: (1) GPR156 functions to reverse stereociliary bundles relative to the PCP axis but can be blocked by STK32A; and (2) EMX2 establishes the boundary between the two groups by repressing Stk32a transcription. We further demonstrate that these factors have similar functional relationships in the cochlea, despite the absence of polarity reversal in that tissue. Together, these phenotypes support a mechanism whereby EMX2 regulates Stk32a transcription, thereby allowing GPR156 to reverse the orientation of stereociliary bundles in one group of hair cells.

Individual epithelial cells that express oncogenes are often extruded from monolayers of wild-type cells, but the extrusion mechanism is not fully understood. We examined extrusion of mammary epithelial cells caused by induction of oncogenic Ras(Q61L). Ras-dependent extrusion requires phosphorylation of ERK (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) but not activation of AKT kinases. Unexpectedly, however, extrusion was suppressed by erlotinib, an inhibitor of epidermal growth factor receptor (EGFR), and by deletion of EGFR. In pancreatic and lung cancers, EGFR is required for full activation of Ras. However, EGFR inhibition or deletion had no impact on Ras(Q61L)-GTP levels or ERK phosphorylation. EGFR expression was not required in surrounding wild-type cells but was needed by the Ras(Q61L) cells for extrusion, yet deletion of the Ras guanine-nucleotide-exchange factors SOS1 and SOS2 (SOS1/2) did not block extrusion. Moreover, expression of a constitutively active MEK instead of Ras was sufficient to drive extrusion, and EGFR inhibition in these cells reduced extrusion. Notably, expression of Ras triggered internalization of E-cadherin (CDH1), which was partially blocked by EGFR inhibition. Together, these data demonstrate an unanticipated requirement for non-canonical EGFR signaling in cancer cell extrusion, which might act in part by promoting E-cadherin endocytosis.

Lysosome-related organelles (LROs) are a heterogeneous family of organelles found in many cell types, whose similarities to lysosomes include acidification by vacuolar-type proton ATPases (V-ATPases). However, some organelles with hallmarks of LROs are nonetheless non-acidic. Here, we investigate this phenomenon using the ciliate Tetrahymena thermophila, which has secretory LROs called mucocysts. Using three approaches, we show that mature mucocysts, poised for exocytosis, are non-acidic. However, mucocysts forming in the cytoplasm are acidic, and a specific V-ATPase a-subunit is present and indispensable for mucocyst biogenesis. In the absence of this subunit, cells show defects in at least two features of mucocyst formation, namely heterotypic vesicle fusion of mucocyst precursors and proprotein processing. The stage specificity of acidification can be explained by our finding that several other canonical V-ATPase subunits are present in the forming mucocysts but not in mature mucocysts. Based on our data, we argue that a specific V-ATPase complex is targeted to newly forming, immature mucocysts and subsequently disassembles at a later stage in the maturation pathway.

Hepatocytes, the parenchymal cells of the liver, exhibit a unique epithelial polarity phenotype in which their bile canaliculi-forming luminal domains and underlying F-actin-linked cell-cell adhesion belt organize not parallel but perpendicular to their basal extracellular matrix (ECM)-contacting domains. Hepatocytes also differ from other epithelia in that they form two basal domains on opposite sites, face only a sparse ECM and express mesenchymal rather than epithelial-typical integrins. What role these hepatocyte-specific cell-ECM interactions play in the establishment and maintenance of the unique hepatocyte polarity phenotype is unknown. We report that in primary rat hepatocyte cultures, development and maintenance of a bile canaliculi network requires the repression of contractile substrate-parallel cell-cell adhesions near matrix-contacting sites. This occurs only when cells contact ECM at two sites; it requires the integrin α1β1, and on rigid matrix, additionally an αV-integrin. We furthermore found that low matrix rigidity, as characteristic of the healthy liver, favors bile canaliculi formation, which becomes independent of p120 catenin-dependent adherens junctions. Our findings thus link the unique hepatocyte polarity phenotype to adherens junction formation downstream of their unique ECM and integrin makeup.

Increased aerobic glycolysis and increased cell motility are hallmarks of metastatic cancer. Migrating cancer cells are highly polarized, suggesting that glycolytic enzymes could be spatially regulated. Here, we investigated the role of the liver isoform of the 'gatekeeper' glycolytic enzyme phosphofructokinase-1 (PFKL) in breast cancer cell migration. Depletion of PFKL significantly decreased migration velocity and directional sensing. We have observed the localization of PFKL to lamellipodia of migrating breast cancer cells, where it colocalized with hexokinase-2 and pyruvate kinase M2. We then investigated the functional requirements of PFKL for directional migration. First, we found that expression of catalytically inactive PFKL or indirect pharmacological inhibition of PFKL activity significantly decreased directional migration. Second, we discovered that disrupting PFKL filament formation by expression of a filament-incompetent mutant decreased PFKL recruitment to lamellipodia and directional sensing, without altering migration velocity. These findings indicate that both catalytic activity and subcellular localization are required for directional migration in breast cancer cells. These results suggest a novel function of PFKL filaments in cells and provide insight into the function of compartmentalized glycolysis in the cytoplasm.

In the colon, the single-layered epithelium forms crypts that extend into the mucosa and are surrounded by a fibroblast network essential for extracellular matrix (ECM) production, remodeling and epithelial support. Fibroblasts are heterogeneous, but inconsistent nomenclature and lack of markers have hindered their classification. Using single-cell RNA sequencing (scRNA-seq), we identified six distinct fibroblast subpopulations in mouse colonic mucosa, each with unique molecular profiles and specialized functions. Some fibroblasts focus on ECM production and remodeling, whereas others show high contractility. Certain subsets of the fibroblasts secrete cytokines promoting epithelial differentiation or stem cell niche maintenance. Spatial mapping revealed their organization within the mucosa, and trajectory analysis suggested distinct differentiation pathways. Cell cycle scoring confirmed that fibroblasts remain largely non-proliferative under homeostasis. By integrating our dataset with published ones, we identify conserved fibroblast populations and propose a standardized nomenclature for intestinal fibroblasts. This framework enhances communication and understanding of fibroblast diversity and their roles in gut homeostasis and disease.

Mahogunin ring finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitylation and degradation. Although MGRN1 is known to regulate the Hedgehog signaling effector Smoothened (SMO) via the transmembrane adapter multiple epidermal growth factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify attractin (ATRN) and attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN interacts with the RING domain of MGRN1. Functional assays suggest that ATRN and ATRNL1 work with MGRN1 to promote the ubiquitylation and degradation of the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 or ATRN leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, resulting in enhanced eumelanin production. These findings expand the known repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to facilitate substrate receptor specificity.