Journal of cell science最新文献

Therapy-induced senescence (TIS) in Glioblastoma (GBM) residual disease and escape from TIS account for resistance and recurrence, but the mechanism of TIS manifestation remains obscure. Here, we demonstrate that replication stress (RS) is critical for the induction of TIS in residual cells by employing an in-vitro GBM therapy-resistance cellular model. Interestingly, we found a 'bi-phasic' mode of DNA damage after radiation treatment and revealed that the second phase of DNA damage arises majorly in the S-phase of residual cells due to RS. Mechanistically, we show that persistent phosphorylated ATR is a safeguard for radiation resilience, while the other canonical RS molecules remain unaltered during the second phase of DNA damage. Importantly, RS precedes the induction of senescence, and ATR inhibition results in TIS reduction, leading to apoptosis. Moreover, ATR inhibition sensitized PARP-1 inhibitor-induced enhanced TIS-mediated resistance, leading to cell death. Our study demonstrates the crucial role of RS in TIS induction and maintenance in GBM residual cells, and targeting ATR alone or in combination with a PARPi will be an effective strategy to eliminate TIS for better treatment outcomes.

The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth, and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, membrane organization, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, cell adhesion and channel proteins, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane skeletal and protein organization and stability.

Tight junctions regulate epithelial barrier function and have been shown to be influenced by multiple classes of proteins. Apical integrins have been identified as potential regulators of epithelial barrier function, however, only indirect approaches have been used to measure integrin regulation of the epithelial barrier. Here, we used polymeric nanowires conjugated with anti-integrin β1 antibodies to specifically target apically localized integrins in either their closed or open conformation. Barrier regulation by apical integrins was found to be conformation specific. Nanowires targeting integrins in the closed conformation increased epithelial permeability and caused zonula occludens-1 (ZO-1) to change from a linear to a ruffled morphology. Claudin-2 and claudin-4 co-localized with ZO-1 and also was ruffled, however, claudin-1 and claudin-7 remained linear. Ruffling was myosin light chain (MLCK) and rho kinase (ROCK) dependent. Conversely, targeting integrins in the open conformation decreased epithelial permeability and made junctions more linearized. Anti-integrin β1 nanowires differentially affected actin and talin, depending on whether they contained activating or inhibitory antibodies. Thus, apical integrins can act as a conformation sensitive switch that regulates epithelial barrier function.

The FLO genes in S. cerevisiae are repressed by heterochromatin formation involving histone deacetylases, transcription factors, and non-coding RNAs. Here we report that mutations in processivity factor POL30 (PCNA) that show transient de-repression at the sub-telomeres and the mating-type loci, do not de-repress the FLO loci. However, deletions of the replisome-stability factors RRM3 and TOF1 along with pol30 mutations induce flocculation phenotypes. The phenotypes correlate with increased expression of reporter proteins driven by FLO11 promoter, the frequency of silent→active conversions of FLO11, and reduced expression of the regulatory lncRNAs ICR1 and PWR1. Alterations in the local replication landscape of FLO11 indicate a link between replication fork pausing and the stability of gene silencing. Analyses of these mutants at the sub-telomeres and HMLα show a similar de-repression phenotype and suggest transient instability of both active and silent states of FLO11. We conclude that RRM3 and TOF1 interact differentially with the pol30 mutations to promote transient de-repression or complete epigenetic conversions of FLO11. We suggest that interaction between POL30, RRM3, and TOF1 is essential to maintain epigenetic stability at the studied loci.

The cell cycle is a fundamental process essential for cell proliferation, differentiation and development. It consists of four major phases: G1, S, G2 and M. These phases collectively drive the reproductive cycle and are meticulously regulated by various proteins that play crucial roles in both the prevention and progression of cancer. Traditional methods for studying these functions, such as flow cytometry, require a substantial number of cells to ensure accuracy. In this study, we have developed a user-friendly immunofluorescence-based method for identifying cell cycle stages, providing single-cell resolution and precise identification of G1, early/mid S, late S, early/mid G2, late G2, and each sub-stage of the M phase using fluorescence microscopy called ImmunoCellCycle-ID. This method provides high-precision cell cycle identification and can serve as an alternative to, or in combination with, traditional flow cytometry to dissect detailed sub-stages of the cell cycle in a variety of cell lines.

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2; also known as STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear. Gaining further insight into the function of DRAK2 in T cells will shed light on differentially regulated pathways in autoreactive and pathogen-specific T cells, which is crucial for improving autoimmune therapies. Here, we demonstrate that DRAK2 contributes to activation of myosin light chain (MLC2, encoded by Myl2) in both murine and human T cells. In the absence of Drak2, the amount of polymerized actin was decreased, suggesting that DRAK2 modulates actomyosin dynamics. We further show that myosin-dependent T cell functions, such as migration, T cell receptor microcluster accumulation, and conjugation to antigen presenting cells are decreased in the absence of Drak2. These findings reveal that DRAK2 plays an important role in regulating MLC activation within T cells.

Iron acquisition is crucial for plants. The abundance of IRON-REGULATED TRANSPORTER 1 (IRT1) is controlled through endomembrane trafficking, a process that requires small ARF-like GTPases. Only few components are known that are involved in the vesicular trafficking of specific cargo. Here, we report that the ARF-like GTPase TITAN 5 (TTN5) interacts with the large cytoplasmic variable region and protein-regulatory platform of IRT1. Heterozygous ttn5-1 plants can display a reduced activity of root iron reductase. This activity is needed for iron uptake via IRT1. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at places, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. TTN5 can also interact with peripheral membrane proteins that are components of the IRT1 regulation machinery, like the trafficking factor SNX1, the C2 domain protein EHB1 and the SEC14-GOLD protein PATL2. Hence, the link between iron acquisition and vesicular trafficking involving a small GTPase of the ARF family opens up the possibility to study the involvement of TTN5 in nutritional cell biology in the endomembrane system.

Specialized membrane and cortical protein regions are common features of cells and are utilized to isolate differential cellular functions. In Drosophila photoreceptors, the apical membrane domain is defined by two distinct morphological membranes: the rhabdomere microvilli and the stalk membrane. To define the apical cortical protein complexes, we performed proximity labeling screens utilizing the rhabdomeric-specific protein PIP82 as bait. We found that the PIP82 interactome is enriched for actin-binding and cytoskeleton proteins, as well as proteins for cellular trafficking. Analysis of one target, Bifocal, with PIP82 revealed two independent pathways for localization to the rhabdomeric membrane and an additional mechanism of crosstalk between the protein complexes of the rhabdomeric and stalk membranes. The loss of Bifocal, and enhancement in the Bifocal/PIP82 double mutant, results in the additional distribution of Crumbs, an apical stalk membrane protein, to the lateral basal photoreceptor membrane. This phenotype is recapitulated by the knockdown of the catalytic subunit of Protein Phosphatase 1, a known interactor of Bifocal. Taken together, these results expand our understanding of the molecular mechanisms underlying the generation of the two distinct photoreceptor apical domains.

To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called Iterative Bleaching Extends Multiplexity (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.

Cleavage of transmembrane segments on target proteins by the aspartyl intramembrane protease signal peptide peptidase (SPP) has been linked to immunity, viral infection and protein quality control. How SPP recognizes its various substrates and specifies their fate remains elusive. Here we identified the lanosterol demethylase CYP51A1 as an SPP substrate and show that SPP-catalyzed cleavage triggers CYP51A1 clearance by ER-associated degradation (ERAD). We observe that SPP targets only a fraction of CYP51A1 molecules and identified an amphipathic helix in the N-terminus as a key determinant for SPP recognition. SPP recognition is remarkably specific to CYP51A1 molecules with the amphipathic helix aberrantly inserted in the membrane with a type II orientation. Thus, our data are consistent with a role for SPP in topology surveillance, triggering the clearance of certain, potentially non-functional conformers.