Journal of cell science最新文献

Friedreich's ataxia (FRDA) is a neurodegenerative disorder characterized by severe neurological signs, affecting the peripheral and central nervous system, caused by reduced frataxin protein (FXN) levels. Although several studies have highlighted cellular dysfunctions in neurons, there is limited information on the effects of FXN depletion in astrocytes and on the potential non-cell autonomous mechanisms affecting neurons in FRDA. In this study, we generated a model of FRDA cerebellar astrocytes to unveil phenotypic alterations that might contribute to cerebellar atrophy. We treated primary cerebellar astrocytes with an RNA interference-based approach, to achieve a reduction of FXN comparable to that observed in individuals with FRDA. These FRDA-like astrocytes display some typical features of the disease, such as an increase of oxidative stress and a depletion of glutathione content. Moreover, FRDA-like astrocytes exhibit decreased Ca2+ responses to purinergic stimuli. Our findings shed light on cellular changes caused by FXN downregulation in cerebellar astrocytes, likely impairing their complex interaction with neurons. The potentially impaired ability to provide neuronal cells with glutathione or to release neuromodulators in a Ca2+-dependent manner could affect neuronal function, contributing to neurodegeneration.

Mutations can disrupt the native function of protein by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms for misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours - acting to stabilize a wide array of cytosolic proteins, including wild-type variants, and to specifically 'buffer' some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), which is essential for phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles upon PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome-lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH4+, which formed through protonation of NH3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study shows that PIKFYVE inhibition disrupts lysosomal homeostasis via ammonium accumulation.

Controlling organ size in plants is a complex biological process influenced by various factors, including gene expression, genome ploidy and environmental conditions. Despite its importance for plant growth and development, the mechanisms underlying organ size regulation remain unknown. Here, we investigated the role of RAN1, a member of the Ras-related nuclear GTPase family, in regulating pollen size. A RAN1 knockdown mutant (ran1-1) exhibited a significant reduction in pollen size, accompanied by impaired germination and reduced pollen tube growth. RAN1 mutation caused disruptions in actin filament organization, such as aberrant structure of actin collar due to the dysregulation of expression of actin-binding proteins. Furthermore, we identified that mutation of the transcription activator SHORT HYPOCOTYL UNDER BLUE1 (SHB1) showed similar but milder phenotypes in pollen compared to ran1-1. Genetic evidence suggested that SHB1 acts downstream of RAN1. Transient expression assays in leaves showed that SHB1 was largely retained in the cytoplasm of the ran1-1 mutant, potentially affecting the expression of actin-binding proteins. These findings highlight the pivotal role of RAN1 in modulating pollen size and development, providing valuable insights into cell size regulation.

Macrophages phagocytose, or eat, pathogens, dead cells and cancer cells. To activate phagocytosis, macrophages recognize 'eat me' signals like IgG and phosphatidylserine on the target cell surface. Macrophages must carefully adjust their phagocytic appetite to ignore non-specific or transient eat me signal exposure on healthy cells while still rapidly recognizing pathogens and debris. Depending on the context, macrophages can increase their appetite for phagocytosis, to prioritize an effective immune response, or decrease their appetite, to avoid damage to healthy tissue during homeostasis. In this Review, we discuss the biochemical and biophysical mechanisms that macrophages employ to increase or decrease their sensitivity or capacity for phagocytosis. We discuss evidence that macrophages tune their sensitivity via several mechanisms, including altering the balance of activating and inhibitory receptor expression, altering the availability of activating receptors, as well as influencing their clustering and mobility, and modulating inhibitory receptor location. We also highlight how membrane availability limits the capacity of macrophages for phagocytosis and discuss potential mechanisms to promote membrane recycling and increase phagocytic capacity. Overall, this Review highlights recent work detailing the molecular toolkit that macrophages use to alter their appetite.

G protein-coupled receptor (GPCR) signalling pathways underlie numerous physiological processes, are implicated in many diseases and are major targets for therapeutics. There are more than 800 GPCRs, which together transduce a vast array of extracellular stimuli into a variety of intracellular signals via heterotrimeric G protein activation and multiple downstream effectors. A key challenge in cell biology research and the pharmaceutical industry is developing tools that enable the quantitative investigation of GPCR signalling pathways to gain mechanistic insights into the varied cellular functions and pharmacology of GPCRs. Recent progress in this area has been rapid and extensive. In this Review, we provide a critical overview of these new, state-of-the-art approaches to investigate GPCR signalling pathways. These include novel sensors, Förster or bioluminescence resonance energy transfer assays, libraries of tagged G proteins and transcriptional reporters. These approaches enable improved quantitative studies of different stages of GPCR signalling, including GPCR activation, G protein activation, second messenger (cAMP and Ca2+) signalling, β-arrestin recruitment and the internalisation and intracellular trafficking of GPCRs.

Rab7A has a key role in regulating membrane trafficking at late endosomes. By interacting with several different effectors, this small GTPase controls late endosome mobility, orchestrates fusion events between late endosomes and lysosomes, and participates in the formation of and regulates the fusion between autophagosomes and lysosomes. Rab7A is also responsible for the spatiotemporal recruitment of retromer, which is required for the endosome-to-trans-Golgi network retrieval of cargo receptors such as sortilin (SORT1) and CI-MPR (also known as IGF2R). Recently, several post-translational modifications have been shown to modulate Rab7A functions, including palmitoylation, ubiquitination and phosphorylation. Here, we show that phosphorylation of Rab7A at serine 72 is important to modulate its interaction with retromer, as the non-phosphorylatable Rab7AS72A mutant is not able to interact with and recruit retromer to late endosomes. We have previously shown that Rab7A palmitoylation is also required for efficient retromer recruitment. We found that palmitoylation of Rab7AS72A is reduced compared to that of the wild-type protein, suggesting an interplay between S72 phosphorylation and palmitoylation in regulating the Rab7A-retromer interaction. Finally, we identify NEK7 as a kinase required to phosphorylate Rab7A to promote retromer binding and recruitment.

The transparent ocular lens is essential for vision because it focuses light onto the retina. Despite recognition of 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, encoded by ANK2), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. The 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, which was evident from postnatal day 16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, cell adhesion proteins and channel proteins; loss and degradation of several membrane proteins [such as NrCAM. N-cadherin (CDH2) and aquaporin-0 (also known as MIP)]; 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, also known as TJP1) to change from a linear to a ruffled morphology. Claudin-2 and claudin-4 colocalized with ZO-1 and were also ruffled; however, claudin-1 and claudin-7 remained linear. Ruffling was dependent on myosin light chain kinases (MLCKs) and Rho kinases (ROCKs). Conversely, targeting integrins in the open conformation decreased epithelial permeability and made junctions more linearized. Anti-integrin β1 nanowires differentially affected actin and talin (analyzed using pan-talin antibodies), depending on whether they contained activating or inhibitory antibodies. Thus, apical integrins can act as a conformation-sensitive switch that regulates epithelial barrier function.

Animal genomes are packaged into chromatin, a highly dynamic macromolecular structure of DNA and histone proteins organised into nucleosomes. This accommodates packaging of lengthy genomic sequences within the physical confines of the nucleus while also enabling precise regulation of access to genetic information. However, histones existed before chromatin and have lesser-known functions beyond genome regulation. Most notably, histones are potent antimicrobial agents, and the release of chromatin to the extracellular space is a defence mechanism nearly as ancient and widespread as chromatin itself. Histone sequences have changed very little throughout evolution, suggesting the possibility that some of their 'non-canonical' functions are at play in parallel or in concert with their genome regulatory functions. In this Review, we take an evolutionary perspective of histone, nuclear chromatin and extracellular chromatin biology and describe the known extranuclear and extracellular functions of histones. We detail molecular mechanisms of chromatin release and extracellular chromatin sensing, and we discuss their roles in physiology and disease. Finally, we present evidence and give a perspective on the potential of extracellular histones to act as bioactive, cell modulatory factors.