Molecular Mechanism of Autophagosome-Lysosome Fusion in Mammalian Cells.

In eukaryotes, targeting intracellular components for lysosomal degradation by autophagy represents a catabolic process that evolutionarily regulates cellular homeostasis. The successful completion of autophagy initiates the engulfment of cytoplasmic materials within double-membrane autophagosomes and subsequent delivery to autolysosomes for degradation by acidic proteases. The formation of autolysosomes relies on the precise fusion of autophagosomes with lysosomes. In recent decades, numerous studies have provided insights into the molecular regulation of autophagosome-lysosome fusion. In this review, an overview of the molecules that function in the fusion of autophagosomes with lysosomes is provided. Moreover, the molecular mechanism underlying how these functional molecules regulate autophagosome-lysosome fusion is summarized.

Recent advances of vacuolar protein-sorting 34 inhibitors targeting autophagy.

Autophagy is a ubiquitous pathological/physiological antioxidant cellular reaction in eukaryotic cells. Vacuolar protein sorting 34 (Vps34 or PIK3C3), which plays a crucial role in autophagy, has received much attention. As the only Class III phosphatidylinositol-3 kinase in mammals, Vps34 participates in vesicular transport, nutrient signaling and autophagy. Dysfunctionality of Vps34 induces carcinogenesis, and abnormal autophagy mediated by dysfunction of Vps34 is closely related to the pathological progression of various human diseases, which makes Vps34 a novel target for tumor immunotherapy. In this review, we summarize the molecular mechanisms underlying macroautophagy, and further discuss the structure-activity relationship of Vps34 inhibitors that have been reported in the past decade as well as their potential roles in anticancer immunotherapy to better understand the antitumor mechanism underlying the effects of these inhibitors.

Engineering cannabidiol synergistic carbon monoxide nanocomplexes to enhance cancer therapy via excessive autophagy.

Although carbon monoxide (CO)-based treatments have demonstrated the high cancer efficacy by promoting mitochondrial damage and core-region penetrating ability, the efficiency was often compromised by protective autophagy (mitophagy). Herein, cannabidiol (CBD) is integrated into biomimetic carbon monoxide nanocomplexes (HMPOC@M) to address this issue by inducing excessive autophagy. The biomimetic membrane not only prevents premature drugs leakage, but also prolongs blood circulation for tumor enrichment. After entering the acidic tumor microenvironment, carbon monoxide (CO) donors are stimulated by hydrogen oxide (H2O2) to disintegrate into CO and Mn2+. The comprehensive effect of CO/Mn2+ and CBD can induce ROS-mediated cell apoptosis. In addition, HMPOC@M-mediated excessive autophagy can promote cancer cell death by increasing autophagic flux via class III PI3K/BECN1 complex activation and blocking autolysosome degradation via LAMP1 downregulation. Furthermore, in vivo experiments showed that HMPOC@M+ laser strongly inhibited tumor growth and attenuated liver and lung metastases by downregulating VEGF and MMP9 proteins. This strategy may highlight the pro-death role of excessive autophagy in TNBC treatment, providing a novel yet versatile avenue to enhance the efficacy of CO treatments. Importantly, this work also indicated the applicability of CBD for triple-negative breast cancer (TNBC) therapy through excessive autophagy.

Assessment of Autophagy Markers Suggests Increased Activity Following LVAD Therapy.

Left ventricular reverse remodeling in heart failure is associated with improved clinical outcomes. However, the molecular features that drive this process are poorly defined. Left ventricular assist devices (LVADs) are the therapy associated with the greatest reverse remodeling and lead to partial myocardial recovery in most patients. In this study, we examined whether autophagy may be implicated in post-LVAD reverse remodeling. We found expression of key autophagy factors increased post-LVAD, while autophagic substrates decreased. Autolysosome numbers increased post-LVAD, further indicating increased autophagy. These findings support the conclusion that mechanical unloading activates autophagy, which may underly the reverse remodeling observed.

This is a public service announcement-BioRender has issued a recall of their autophagy template.

I have been surprised at the number of papers I edit where the schematic of macroautophagy/autophagy is wrong, and it is wrong in the same way. How could this happen? Did people get together and agree to misrepresent the morphological intermediates of autophagy? Not exactly.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Autophagy signaling in hypertrophied muscles of diabetic and control rats.

Autophagy plays a vital role in cell homeostasis by eliminating nonfunctional components and promoting cell survival. Here, we examined the levels of autophagy signaling proteins after 7 days of overload hypertrophy in the extensor digitorum longus (EDL) and soleus muscles of control and diabetic rats. We compared control and 3-day streptozotocin-induced diabetic rats, an experimental model for type 1 diabetes mellitus (T1DM). EDL muscles showed increased levels of basal autophagy signaling proteins. The diabetic state did not affect the extent of overload-induced hypertrophy or the levels of autophagy signaling proteins (p-ULK1, Beclin-1, Atg5, Atg12-5, Atg7, Atg3, LC3-I and II, and p62) in either muscle. The p-ULK-1, Beclin-1, and p62 protein expression levels were higher in the EDL muscle than in the soleus before the hypertrophic stimulus. On the contrary, the soleus muscle exhibited increased autophagic signaling after overload-induced hypertrophy, with increases in Beclin-1, Atg5, Atg12-5, Atg7, Atg3, and LC3-I expression in the control and diabetic groups, in addition to p-ULK-1 in the control groups. After hypertrophy, Beclin-1 and Atg5 levels increased in the EDL muscle of both groups, while p-ULK1 and LC3-I increased in the control group. In conclusion, the baseline EDL muscle exhibited higher autophagy than the soleus muscle. Although TDM1 promotes skeletal muscle mass loss and strength reduction, it did not significantly alter the extent of overload-induced hypertrophy and autophagy signaling proteins in EDL and soleus muscles, with the two groups exhibiting different patterns of autophagy activation.

Lipofuscin Granule Accumulation Requires Autophagy Activation.

Lipofuscins are oxidized lipid and protein complexes that accumulate during cellular senescence and tissue aging, regarded as markers for cellular oxidative damage, tissue aging, and certain aging-associated diseases. Therefore, understanding their cellular biological properties is crucial for effective treatment development. Through traditional microscopy, lipofuscins are readily observed as fluorescent granules thought to accumulate in lysosomes. However, lipofuscin granule formation and accumulation in senescent cells are poorly understood. Thus, this study examined lipofuscin accumulation in human fibroblasts exposed to various stressors. Our results substantiate that in glucose-starved or replicative senescence cells, where elevated oxidative stress levels activate autophagy, lipofuscins predominately appear as granules that co-localize with autolysosomes due to lysosomal acidity or impairment. Meanwhile, autophagosome formation is attenuated in cells experiencing oxidative stress induced by a doxorubicin pulse and chase, and lipofuscin fluorescence granules seldom manifest in the cytoplasm. As Torin-1 treatment activates autophagy, granular lipofuscins intensify and dominate, indicating that autophagy activation triggers their accumulation. Our results suggest that high oxidative stress activates autophagy but fails in lipofuscin removal, leaving an abundance of lipofuscin-filled impaired autolysosomes, referred to as residual bodies. Therefore, future endeavors in treating lipofuscin pathology-associated diseases and dysfunctions through autophagy activation demand meticulous consideration.

PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis.

Inter-organelle contacts enable crosstalk among organelles, facilitating the exchange of materials and coordination of cellular events. In this study, we demonstrated that, upon starvation, autolysosomes recruit Pi4KIIα (Phosphatidylinositol 4-kinase II α) to generate phosphatidylinositol-4-phosphate (PtdIns4P) on their surface and establish endoplasmic reticulum (ER)-autolysosome contacts through PtdIns4P binding proteins Osbp (Oxysterol binding protein) and cert (ceramide transfer protein). We found that the Sac1 (Sac1 phosphatase), Osbp, and cert proteins are required for the reduction of PtdIns4P on autolysosomes. Loss of any of these proteins leads to defective macroautophagy/autophagy and neurodegeneration. Osbp, cert, and Sac1 are required for ER-Golgi contacts in fed cells. Our data establishes a new mode of organelle contact formation - the ER-Golgi contact machinery can be reused by ER-autolysosome contacts by re-locating PtdIns4P from the Golgi apparatus to autolysosomes when faced with starvation.Abbreviations: Atg1: Autophagy-related 1; Atg8: Autophagy-related 8; Atg9: Autophagy-related 9; Atg12: Autophagy-related 12; cert: ceramide transfer protein; Cp1/CathL: cysteine proteinase-1; CTL: control; ER: endoplasmic reticulum; ERMCS: ER-mitochondria contact site; fwd: four wheel drive; GM130: Golgi matrix protein 130 kD; Osbp: Oxysterol binding protein; PG: phagophore; PtdIns4K: phosphatidylinositol 4-kinase; Pi4KIIα: Phosphatidylinositol 4-kinase II α; Pi4KIIIα: Phosphatidylinositol 4-kinase III α; PtdIns4P: phosphatidylinositol-4-phosphate; PR: photoreceptor cell; RT: room temperature; Sac1: Sac1 phosphatase; Stv: starvation; Syx17: Syntaxin 17; TEM: transmission electron microscopy; VAP: VAMP-associated protein.

Phagophore-lysosome/vacuole fusion in mutant yeast and mammalian cells.

Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and fusing with lysosomes/vacuoles, after which the materials are degraded for recycling purposes. Autophagy is associated with increased cell survival under different stress conditions. AP-lysosome/vacuole fusion is a critical step in autophagy. Some mutant cells can accumulate phagophores under autophagy-induction conditions. Autophagy is interrupted when accumulated phagophores cannot fuse with lysosomes/vacuoles, resulting in a significant decrease in cell survivability. However, phagophore-lysosome/vacuole fusion has been reported in related mammalian cells and yeast mutant cells. This observation indicates that it is possible to restore a partial autophagy process after interruption. Furthermore, these findings indicate that phagophore closure is not a prerequisite for its fusion with the lysosome/vacuole in the mutant cells. The phagophore-lysosome/vacuole fusion strategy can significantly rescue defective autophagy due to failed phagophore closure. This commentary discusses the fusion of phagophores and lysosomes/vacuoles and implications of such fusion events.Abbreviations: AB: autophagic body; AL: autolysosome; AP: autophagosome; ATG: autophagy related; EM: electron microscopy; ESCRT: endosomal sorting complex required for transport; ET: electron tomography; FIB: focus ion beam; IM: inner membrane; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; OM; outer membrane; STX17: syntaxin 17; TEM: transmission electron microscopy; TM: transmembrane domain; Vps: vacuolar protein sorting; WT: wild-type.

PSMB4 Degrades the Porcine Reproductive and Respiratory Syndrome Virus Nsp1α Protein via the Autolysosome Pathway and Induces the Production of Type I Interferon.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in pigs of all ages and reproductive failure in sows, resulting in great economic losses to the swine industry. In this work, we identified the interaction between PSMB4 and PRRSV Nsp1α by yeast two-hybrid screening. The PSMB4-Nsp1α interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and laser confocal experiments. The PCPα domain (amino acids 66 to 166) of Nsp1α and the C-terminal domain (amino acids 250 to 264) of PSMB4 were shown to be critical for the PSMB4-Nsp1α interaction. PSMB4 overexpression reduced PRRSV replication, whereas PSMB4 knockdown elicited opposing effects. Mechanistically, PSMB4 targeted K169 in Nsp1α for K63-linked ubiquitination and targeted Nsp1α for autolysosomal degradation by interacting with LC3 to enhance the activation of the lysosomal pathway. Meanwhile, we found that PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. In conclusion, our data revealed a new mechanism of PSMB4-mediated restriction of PRRSV replication, whereby PSMB4 was found to induce Nsp1α degradation and type I interferon expression, in order to impede the replication of PRRSV. IMPORTANCE In the swine industry, PRRSV is a continuous threat, and the current vaccines are not effective enough to block it. This study determined that PSMB4 plays an antiviral role against PRRSV. PSMB4 was found to interact with PRRSV Nsp1α, mediate K63-linked ubiquitination of Nsp1α at K169, and thus trigger its degradation via the lysosomal pathway. Additionally, PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. This study extends our understanding of the proteasome subunit PSMB4 against PRRSV replication and will contribute to the development of new antiviral strategies.

Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation.

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions. Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

The Pursuit of the "Inside" of the Amyloid Hypothesis-Is C99 a Promising Therapeutic Target for Alzheimer's Disease?

Aducanumab, co-developed by Eisai (Japan) and Biogen (U.S.), has received Food and Drug Administration approval for treating Alzheimer's disease (AD). In addition, its successor antibody, lecanemab, has been approved. These antibodies target the aggregated form of the small peptide, amyloid-ß (Aß), which accumulates in the patient brain. The "amyloid hypothesis" based therapy that places the aggregation and toxicity of Aß at the center of the etiology is about to be realized. However, the effects of immunotherapy are still limited, suggesting the need to reconsider this hypothesis. Aß is produced from a type-I transmembrane protein, Aß precursor protein (APP). One of the APP metabolites, the 99-amino acids C-terminal fragment (C99, also called ßCTF), is a direct precursor of Aß and accumulates in the AD patient's brain to demonstrate toxicity independent of Aß. Conventional drug discovery strategies have focused on Aß toxicity on the "outside" of the neuron, but C99 accumulation might explain the toxicity on the "inside" of the neuron, which was overlooked in the hypothesis. Furthermore, the common region of C99 and Aß is a promising target for multifunctional AD drugs. This review aimed to outline the nature, metabolism, and impact of C99 on AD pathogenesis and discuss whether it could be a therapeutic target complementing the amyloid hypothesis.

Niclosamide causes lysosome-dependent cell death in endometrial cancer cells and tumors.

Endometrial cancer is the most common female cancer showing continuous rise in its incidence and mortality rate. Despite the extensive research efforts in cancer therapeutics, still there is a lack of effective treatment options and the outcome is poor for patients with advanced and recurrent endometrial cancers. In this study, we aimed to evaluate the efficacy of niclosamide (NIC) against endometrial cancer. NIC is an FDA-approved anti-helminthic drug, which has been recently extensively studied as a potent anti-cancerous agent in several cancers. The anti-cancerous activity of NIC was analyzed in-vitro (ANC3A, Hec1B, and Ishikawa endometrial cancer cell lines) by cell viability-, soft agar-, invasion- and migration- assay. The action mechanism of NIC was demonstrated by western blot analysis and immune-fluorescence imaging and validated by specific inhibitors. The in-vivo efficacy of NIC was studied in the Ishikawa xenograft animal model. NIC effectively suppressed the viability (IC50<1 µM), colony formation ability, migration, and invasion of all endometrial cancer cells tested. We demonstrated that NIC inhibited AKT/mTOR signaling pathway and induced apoptosis and autophagy in endometrial cancer cells. Further study demonstrated that although NIC induced autophagosome formation, it inhibits autolysosome formation. In addition, we observed that NIC induced BAX co-localization with lysosome and inhibited Cathepsin B maturation from pro-cathepsin B, thereby inducing the lysosomal membrane permeability and release of hydrolytic enzymes from the lysosome to cytosol, which eventually contributed cell death. NIC also inhibited tumor weight and volume in the Ishikawa xenograft animal model without having any evidence of toxicity. Due to its potent anti-cancerous activity and safety profile, NIC seems to be a promising agent for human endometrial cancer therapeutics.

LYST deficiency impairs autophagic lysosome reformation in neurons and alters lysosome number and size.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

Catalpol inhibits hepatic stellate cell activation by reducing the formation and changing the contents of hepatocyte-derived extracellular vesicles.

Hepatic stellate cell (HSC) activation is the central event in hepatic fibrosis. The cross-talk between HSCs and hepatocytes, which is mediated by extracellular vesicles (EVs), affects HSC activation. This study aimed to investigate whether Catalpol (CTP) attenuated hepatic fibrosis via modulating EVs. Mice were injected intraperitoneally with CCl4 for 4 weeks to induce hepatic fibrosis. They were gavaged with CTP daily. Mouse serum EVs were isolated and identified using nanoparticle tracking analysis and transmission electron microscopy. Mouse hepatocytes (AML12) and primary HSCs were used to investigate the cell-to-cell crosstalk. The autophagosome-autolysosome fusion was determined using the autophagic flux assay. Hepatic fibrosis was attenuated by CTP, with a decrease of the myofibroblast marker, alpha-smooth muscle actin. The CTP treatment lowered the serum EVs. The co-culture of HSCs and the EVs derived from the CTP-treated mice or hepatocytes reduced HSC proliferation and the expressions of ACTA2 and Col1a1. After the CCl4 treatment, the autophagosomes in AML12 cells were increased, while the autolysosomes were reduced. The decrease of autophagic cargo receptor SQSTM1 in the CTP group suggested that autophagic degradation was sustained. After inhibiting the endogenous Rac1-GTP of hepatocytes, the co-culture of EVs and HSCs reduced Rac1-GTP. The Rac1-GTP level in serum EVs from the CTP-treated mice was reduced in vivo. CTP inhibited autophagy in hepatocytes by reducing Rac1-GTP and thus affect the amount of Rac1-GTP in hepatocyte-derived EVs and the formation of EVs, which attenuated hepatic fibrosis via inhibiting HSC activation.

MALT-1 shortens lifespan by inhibiting autophagy in the intestine of C. elegans.

The caspase-like protease MALT1 promotes immune responses and oncogenesis in mammals by activating the transcription factor NF-κB. MALT1 is remarkably conserved from mammals to simple metazoans devoid of NF-κB homologs, like the nematode C. elegans. To discover more ancient, NF-κB -independent MALT1 functions, we analysed the phenotype of C. elegans upon silencing of MALT-1 expression systemically or in a tissue-specific manner. MALT-1 silencing in the intestine caused a significant increase in life span, whereas intestinal overexpression of MALT-1 shortened life expectancy. Interestingly, MALT-1-deficient animals showed higher constitutive levels of autophagy in the intestine, which were particularly evident in aged or starved nematodes. Silencing of the autophagy regulators ATG-13, BEC-1 or LGG-2, but not the TOR homolog LET-363, reversed lifespan extension caused by MALT-1 deficiency. These findings suggest that MALT-1 limits the lifespan of C. elegans by acting as an inhibitor of an early step of autophagy in the intestine.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

In human astrocytes neurotropic flaviviruses increase autophagy, yet their replication is autophagy-independent.

Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.

Optical Visualization of Red-GQDs' Organelles Distribution and Localization in Living Cells.

Recently, there has been a rapidly expanding interest in a new nanomaterial, graphene quantum dots (GQDs), owing to its profound potential in various advanced applications. At present, the study of GQDs mainly focuses on the new synthesis methods and surface modification. However, revealing the intracellular distribution of GQDs is currently not available, limiting in-depth understanding of its biological regulatory mechanism. To fill up this gap, the visualization study of red fluorescent graphene quantum dots (Red-GQDs) is helpful to clarify their subcellular distribution and metabolism in living cells system. Here, in this study, two-photon laser confocal microscopy was used to deeply analyze the uptake and subcellular distribution of Red-GQDs by HeLa cells at different concentrations and times through visual observation and discussed the effect of Red-GQDs on the metabolic of HeLa cells. The results indicated that Red-GQDs could be well-absorbed by HeLa cells and further revealed the differential distribution of Red-GQDs in different organelles (lysosomes and mitochondria) in a time-dependent manner. In addition, we confirmed that Red-GQDs significantly affect cell biological functions. Low concentrations of Red-GQDs are related to the autophagy pathway of cells, and high concentrations of Red-GQDs can induce ferroptosis in cells and promote the secretion of cellular exosomes. In the present study, the distribution and metabolic pathways of Red-GQDs in the subcellular structure of cells were characterized in detail through visual analysis, which can bring positive reference for the application of Red-GQDs in the future.