Ultrasensitive two-photon probes for rapid detection of ß-galactosidase during fruit softening and cellular senescence.

Senescence is happening in every corner of the living organisms. ß-galactosidase (ß-gal) is one of the most important biomarkers during senescence in both plant and mammalian cells. Most ß-gal fluorescent probes were focused on bio-imaging, only a few probes were developed for the detection of ß-gal in fruit, and the probes that could detect ß-gal in both fruits and living cells were even less. Here, two ß-gal probes (TNap-ßGal and TBNap-ßGal) were synthesized, which can not only image the increase of ß-gal during both fruits softening and cellular senescence, but also prove that bananas are not suitable for storage in refrigerator and the subsequent accumulation of ß-gal still in lysosome of mammalian cells. In addition, TNap-ßGal was successfully applied to two-photon imaging of endogenous ß-gal in both hDPMSCs and tissues of human dental pulp for the first time.

HOPS-dependent lysosomal fusion controls Rab19 availability for ciliogenesis in polarized epithelial cells.

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering homotypic fusion and protein sorting (HOPS) complex disrupts this actin clearing and ciliogenesis, but it remains unclear how the ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body, and that this effect is specific to polarized epithelial cells. We also find that Rab19 functions in endolysosomal cargo trafficking in addition to having its previously identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion leads to the abnormal accumulation of Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin clearing and ciliogenesis in polarized epithelial cells.

Recent Development of Lysosome-Targeted Organic Fluorescent Probes for Reactive Oxygen Species.

Reactive oxygen species (ROS) are extremely important for various biological functions. Lysosome plays key roles in cellular metabolism and has been known as the stomach of cells. The abnormalities and malfunctioning of lysosomal function are associated with many diseases. Accordingly, the quantitative monitoring and real-time imaging of ROS in lysosomes are of great interest. In recent years, with the advancement of fluorescence imaging, fluorescent ROS probes have received considerable interest in the biomedical field. Thus far, considerable efforts have been undertaken to create synthetic fluorescent probes for sensing ROS in lysosomes; however, specific review articles on this topic are still lacking. This review provides a general introduction to fluorescence imaging technology, the sensing mechanisms of fluorescent probes, lysosomes, and design strategies for lysosome-targetable fluorescent ROS probes. In addition, the latest advancements in organic small-molecule fluorescent probes for ROS detection within lysosomes are discussed. Finally, the main challenges and future perspectives for developing effective lysosome-targetable fluorescent ROS probes for biomedical applications are presented.

pH-gated nanoparticles selectively regulate lysosomal function of tumour-associated macrophages for cancer immunotherapy.

Tumour-associated macrophages (TAMs), as one of the most abundant tumour-infiltrating immune cells, play a pivotal role in tumour antigen clearance and immune suppression. M2-like TAMs present a heightened lysosomal acidity and protease activity, limiting an effective antigen cross-presentation. How to selectively reprogram M2-like TAMs to reinvigorate anti-tumour immune responses is challenging. Here, we report a pH-gated nanoadjuvant (PGN) that selectively targets the lysosomes of M2-like TAMs in tumours rather than the corresponding organelles from macrophages in healthy tissues. Enabled by the PGN nanotechnology, M2-like TAMs are specifically switched to a M1-like phenotype with attenuated lysosomal acidity and cathepsin activity for improved antigen cross-presentation, thus eliciting adaptive immune response and sustained tumour regression in tumour-bearing female mice. Our findings provide insights into how to specifically regulate lysosomal function of TAMs for efficient cancer immunotherapy.

Sperm acrosomal released proteome reveals MDH and VDAC3 from mitochondria are involved in acrosome formation during spermatogenesis in Eriocheir sinensis.

Acrosome is inextricably related to membranous organelles. The origin of acrosome is still controversial, one reason is that limited articles were reported about the proteomic analysis of the acrosome. Mitochondrial proteins were found exist in the acrosome, nevertheless, only limited attention has been paid to the function of mitochondrial proteins in the acrosome formation. Eriocheir sinensis sperm has a large acrosome, which makes it an ideal model to study acrosome formation. Here, we firstly compared the rate of acrosome reaction induced by the calcium ionophore A23187 and ionomycin. The rate of acrosome reaction induced by ionomycin is higher (95.8%) than A23187 (58.7%). Morphological changes were observed using light, confocal and transmission electron microscopy. Further more, proteins released during the acrosome reaction as induced by ionomycin were collected for LC-MS/MS analysis. A total of 945 proteins, including malate dehydrogenase (MDH) and voltage-dependent anion channel 3 (VDAC3), were identified in the acrosomal released proteome. The number of proteins from mitochondria (17.57%) was higher compared with endoplasmic reituculum (1.59%) and lysosomes (1.8%). To investigate the functions of target mitochondrial proteins during spermatogenesis, poly-antibodies of MDH in E. sinensis were prepared. The characteristics, further analyzed using immunofluorescence, of two mitochondrial proteins during acrosome formation showed that MDH and VDAC3 were independently involved in the formation of acrosomal membrane. These findings illustrate the acrosomal released proteome and provide important data resource for understanding the relationship between mitochondria and the acrosome in Decapoda crustacean.

Ultrastructural characteristics of the accumulation of iron nanoparticles in the intestine of Cyprinus carpio (Linnaeus, 1758) under aquaculture.

During the development of nanotechnology, the production of many substances containing nanoparticles leads to the release of various nanoparticles into the environment, including the water ecosystem. The main goal of the current research was to study the ultrastructural characteristics of the entry and bioaccumulation of Fe3O4 nanoparticles in the small intestine of Cyprinus carpio (Linnaeus, 1758), as well as the pathomorphological changes in the fish organism. Two different doses (10 and 100 mg) of Fe3O4 nanoparticles were fed to fingerlings for 7 days and then intestinal samples were taken and studied. It was found that the extent of damages was boosted within the increment of nanoparticle concentration. The sequence and bioaccumulation of Fe3O4 nanoparticles in the small intestine of fish occurred as below: firstly, the nanoparticles passed into microvilli located in the apical part of enterocytes in the mucosa layer, from there into the cytoplasm of the epithelial cells, including cytoplasmatic organelles (nucleus, mitochondria, lysosomes, fat granules), and then into a lamina propria of the mucosa of the small intestine and passed into the endothelium of the blood vessels and to the erythrocytes of the vessels which located in the lumen. It was determined that although the nanoparticles were up to 30 nm in size, only particles with a maximum size of 20 nm could penetrate the intestinal wall. Thus, the release of Fe3O4 nanoparticles into the environment in high doses has a negative effect on the living ecosystem, including the body of fish living in the water.

Overcoming blood-brain barrier for targeted delivery of lysosome-targeting chimeras.

In a recent Chem article, Liu et al.1 introduced polydopamine-based lysosome-targeting chimeras (KPLYs). In in vitro cellular models, KPLYs adeptly cross the blood-brain barrier to target and eliminate ß-amyloid aggregates. They also reduce inflammation and modulate microglial activity.

TFEB.

Contreras and Puertollano introduce TFEB, a transcription factor that orchestrates cellular responses to stress via mechanisms including upregulation of lysosome biogenesis and autophagy.

Lysosome-related organelles promote stress and immune responses in C. elegans.

Lysosome-related organelles (LROs) play diverse roles and their dysfunction causes immunodeficiency. However, their primordial functions remain unclear. Here, we report that C. elegans LROs (gut granules) promote organismal defenses against various stresses. We find that toxic benzaldehyde exposure induces LRO autofluorescence, stimulates the expression of LRO-specific genes and enhances LRO transport capacity as well as increases tolerance to benzaldehyde, heat and oxidative stresses, while these responses are impaired in glo-1/Rab32 and pgp-2 ABC transporter LRO biogenesis mutants. Benzaldehyde upregulates glo-1- and pgp-2-dependent expression of heat shock, detoxification and antimicrobial effector genes, which requires daf-16/FOXO and/or pmk-1/p38MAPK. Finally, benzaldehyde preconditioning increases resistance against Pseudomonas aeruginosa PA14 in a glo-1- and pgp-2-dependent manner, and PA14 infection leads to the deposition of fluorescent metabolites in LROs and induction of LRO genes. Our study suggests that LROs may play a role in systemic responses to stresses and in pathogen resistance.

The Batten disease gene product CLN5 is the lysosomal bis(monoacylglycero)phosphate synthase.

Lysosomes critically rely on bis(monoacylglycero)phosphate (BMP) to stimulate lipid catabolism, cholesterol homeostasis, and lysosomal function. Alterations in BMP levels in monogenic and complex neurodegeneration suggest an essential function in human health. However, the site and mechanism responsible for BMP synthesis have been subject to debate for decades. Here, we report that the Batten disease gene product CLN5 is the elusive BMP synthase (BMPS). BMPS-deficient cells exhibited a massive accumulation of the BMP synthesis precursor lysophosphatidylglycerol (LPG), depletion of BMP species, and dysfunctional lipid metabolism. Mechanistically, we found that BMPS mediated synthesis through an energy-independent base exchange reaction between two LPG molecules with increased activity on BMP-laden vesicles. Our study elucidates BMP biosynthesis and reveals an anabolic function of late endosomes/lysosomes.

Lysosome-related genes: A new prognostic marker for lung adenocarcinoma.

Currently, a reliable early prognostic marker has not been identified for lung adenocarcinoma (LUAD), the most common malignancy. Recent studies demonstrated that lysosomal rupture is involved in cancer migration, progression, and immune microenvironment formation. We performed a bioinformatics analysis of lysosomal rupture to investigate whether lysosome-related genes (LRGs) are key in LUAD. The analysis identified 23 LRGs. Cytoscape visualization identified 10 core genes (CCNA2, DLGAP5, BUB1B, KIF2C, PBK, CDC20, NCAPG, ASPM, KIF4A, ANLN). With the 23 LRGs, we established a new risk scoring rule to classify patients with LUAD into high- and low-risk groups and verified the accuracy of the risk score by receiver operating characteristic curves and established a nomogram to evaluate clinical patients. Immunotherapy effectiveness between the high- and low-risk groups was evaluated based on the tumor mutational burden and analyses of immune cell infiltration and drug sensitivity. Pathway enrichment analysis revealed that lysosomes were closely associated with glucose metabolism, amino acid metabolism, and the immune response in patients with LUAD. Lysosomes are a likely new therapeutic target and provide new directions and ideas for treating and managing patients with LUAD.

A New Lysosome-Targeted NIR Fluorescent Probe for Specific Detection of Cysteine over Homocysteine and Glutathione.

In this paper, by modifying the thioxanthene-benzothiozolium fluorophore, BCy-Cys, a lysosome-targeted near-infrared (NIR) fluorescent probe was synthesized for the detection of cysteine (Cys) from homocysteine (Hcy)/glutathione (GSH). As expected, BCy-Cys exhibited high selectivity and high sensitivity for detection of Cys over Hcy/GSH, with an extremely low limit of detection at 0.31 µM, marked by obvious color changes. HRMS was conducted to confirm that the fluorescence intensity at 795 nm was significantly enhanced by the enhancement of intramolecular charge transfer (ICT). Importantly, BCy-Cys could be used to visualize both exogenous and endogenous lysosomal Cys, signifying its potential application in complex organismal systems.

A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons.

BACKGROUND: Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS. METHODS: In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). RESULTS: Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. CONCLUSIONS: Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS.

Codependencies of mTORC1 signaling and endolysosomal actin structures.

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.

Dendritic cell-based vaccine prepared with recombinant Lactococcus lactis enhances antigen cross-presentation and antitumor efficacy through ROS production.

Introduction: Lactococcus lactis (L.L) is safe and can be used as vehicle. In this study, the immunoregulatory effect of L.L on dendritic cell (DC) activation and mechanism were investigated. The immune responses and antigen cross-presentation mechanism of DC-based vaccine prepared with OVA recombinant L.L were explored. Methods: Confocal microscopy and flow cytometry were used to analyze the mechanism of L.L promoting DC maturation, phagosome membrane rupture and antigen presentation. The antitumor effect of DC vaccine prepared with L.L-OVA was assessed in the B16-OVA tumor mouse model. Results: L.L significantly promoted DC maturation, which was partially dependent on TLR2 and downstream MAPK and NF-κB signaling pathways. L.L was internalized into DCs by endocytosis and did not co-localized with lysosome. OVA recombinant L.L enhanced antigen cross-presentation of DCs through the phagosome-to-cytosol pathway in a reactive oxygen species (ROS)- and proteasome-dependent manner. In mouse experiments, L.L increased the migration of DCs to draining lymph node and DC vaccine prepared with OVA recombinant L.L induced strong antigen-specific Th1 and cytotoxic T lymphocyte responses, which significantly inhibited B16-OVA tumor growth. Conclusion: This study demonstrated that recombinant L.L as an antigen delivery system prepared DC vaccine can enhance the antigen cross-presentation and antitumor efficacy.

Lysosome-targeted fluorescent probes: Design mechanism and biological applications.

As an integral organelle in the eukaryote, the lysosome is the degradation center and metabolic signal center in living cells, and partakes in significant physiological processes such as autophagy, cell death and cellular senescence. Fluorescent probe has become a favorite tool for studying organelles and their chemical microenvironments because of its high specificity and non-destructive merits. Over recent years, it has been reported that increasingly new lysosome-targeted probes play a major role in the diagnosis and monitor of diseases, in particular cancer and neurodegenerative diseases. In order to deepen the relevant research on lysosome, it is challenging and inevitability to design novel lysosomal targeting probes. This review first introduces the concepts of lysosome and its closely related biological activities, and then introduces the fluorescent probes for lysosome in detail according to different detection targets, including targeting mechanism, biological imaging, and application in diseases. Finally, we summarize the specific challenges and discuss the future development direction facing the current lysosome-targeted fluorescent probes. We hope that this review can help biologists grasp the application of fluorescent probes and broaden the research ideas of researchers targeting fluorescent probes so as to design more accurate and functional probes for application in diseases.

Lysosomal cholesterol overload in macrophages promotes liver fibrosis in a mouse model of NASH.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Crosstalk between autophagy and metabolic regulation of (CAR) T cells: therapeutic implications.

Despite chimeric antigen receptor (CAR) T cell therapy's extraordinary success in subsets of B-cell lymphoma and leukemia, various barriers restrict its application in solid tumors. This has prompted investigating new approaches for producing CAR T cells with superior therapeutic potential. Emerging insights into the barriers to CAR T cell clinical success indicate that autophagy shapes the immune response via reprogramming cellular metabolism and vice versa. Autophagy, a self-cannibalization process that includes destroying and recycling intracellular components in the lysosome, influences T cell biology, including development, survival, memory formation, and cellular metabolism. In this review, we will emphasize the critical role of autophagy in regulating and rewiring metabolic circuits in CAR T cells, as well as how the metabolic status of CAR T cells and the tumor microenvironment (TME) alter autophagy regulation in CAR T cells to restore functional competence in CAR Ts traversing solid TMEs.

A Homozygous MAN2B1 Missense Mutation in a Doberman Pinscher Dog with Neurodegeneration, Cytoplasmic Vacuoles, Autofluorescent Storage Granules, and an α-Mannosidase Deficiency.

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.

Tubular lysosome induction couples animal starvation to healthy aging.

Dietary restriction promotes longevity in several species via autophagy activation. However, changes to lysosomes underlying this effect remain unclear. Here using the nematode Caenorhabditis elegans, we show that the induction of autophagic tubular lysosomes (TLs), which occurs upon dietary restriction or mechanistic target of rapamycin inhibition, is a critical event linking reduced food intake to lifespan extension. We find that starvation induces TLs not only in affected individuals but also in well-fed descendants, and the presence of gut TLs in well-fed progeny is predictive of enhanced lifespan. Furthermore, we demonstrate that expression of Drosophila small VCP-interacting protein, a TL activator in flies, artificially induces TLs in well-fed worms and improves C. elegans health in old age. These findings identify TLs as a new class of lysosomes that couples starvation to healthy aging.