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1
Nakae, Isei, Tomoko Fujino, Tetsuo Kobayashi, Ayaka Sasaki, Yorifumi Kikko, Masamitsu Fukuyama, Keiko Gengyo-Ando, Shohei Mitani, Kenji Kontani und Toshiaki Katada. „The Arf-like GTPase Arl8 Mediates Delivery of Endocytosed Macromolecules to Lysosomes inCaenorhabditis elegans“. Molecular Biology of the Cell 21, Nr. 14 (15.07.2010): 2434–42. http://dx.doi.org/10.1091/mbc.e09-12-1010.
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Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.
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Trivedi, Purvi C., Jordan J. Bartlett und Thomas Pulinilkunnil. „Lysosomal Biology and Function: Modern View of Cellular Debris Bin“. Cells 9, Nr. 5 (04.05.2020): 1131. http://dx.doi.org/10.3390/cells9051131.
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Lysosomes are the main proteolytic compartments of mammalian cells comprising of a battery of hydrolases. Lysosomes dispose and recycle extracellular or intracellular macromolecules by fusing with endosomes or autophagosomes through specific waste clearance processes such as chaperone-mediated autophagy or microautophagy. The proteolytic end product is transported out of lysosomes via transporters or vesicular membrane trafficking. Recent studies have demonstrated lysosomes as a signaling node which sense, adapt and respond to changes in substrate metabolism to maintain cellular function. Lysosomal dysfunction not only influence pathways mediating membrane trafficking that culminate in the lysosome but also govern metabolic and signaling processes regulating protein sorting and targeting. In this review, we describe the current knowledge of lysosome in influencing sorting and nutrient signaling. We further present a mechanistic overview of intra-lysosomal processes, along with extra-lysosomal processes, governing lysosomal fusion and fission, exocytosis, positioning and membrane contact site formation. This review compiles existing knowledge in the field of lysosomal biology by describing various lysosomal events necessary to maintain cellular homeostasis facilitating development of therapies maintaining lysosomal function.
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Amick, Joseph, Arun Kumar Tharkeshwar, Catherine Amaya, und Shawn M. Ferguson. „WDR41 supports lysosomal response to changes in amino acid availability“. Molecular Biology of the Cell 29, Nr. 18 (September 2018): 2213–27. http://dx.doi.org/10.1091/mbc.e17-12-0703.
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C9orf72 mutations are a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. The C9orf72 protein undergoes regulated recruitment to lysosomes and has been broadly implicated in control of lysosome homeostasis. However, although evidence strongly supports an important function for C9orf72 at lysosomes, little is known about the lysosome recruitment mechanism. In this study, we identify an essential role for WDR41, a prominent C9orf72 interacting protein, in C9orf72 lysosome recruitment. Analysis of human WDR41 knockout cells revealed that WDR41 is required for localization of the protein complex containing C9orf72 and SMCR8 to lysosomes. Such lysosome localization increases in response to amino acid starvation but is not dependent on either mTORC1 inhibition or autophagy induction. Furthermore, WDR41 itself exhibits a parallel pattern of regulated association with lysosomes. This WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling as constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation. Collectively, this study reveals an essential role for WDR41 in supporting the regulated binding of C9orf72 to lysosomes and solidifies the requirement for a larger C9orf72 containing protein complex in coordinating lysosomal responses to changes in amino acid availability.
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Bonet-Ponce, Luis, Alexandra Beilina, Chad D. Williamson, Eric Lindberg, Jillian H. Kluss, Sara Saez-Atienzar, Natalie Landeck et al. „LRRK2 mediates tubulation and vesicle sorting from lysosomes“. Science Advances 6, Nr. 46 (November 2020): eabb2454. http://dx.doi.org/10.1126/sciadv.abb2454.
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Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson’s disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane–rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.
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Bakker, A. C., P. Webster, W. A. Jacob und N. W. Andrews. „Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts“. Journal of Cell Science 110, Nr. 18 (15.09.1997): 2227–38. http://dx.doi.org/10.1242/jcs.110.18.2227.
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Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and ss-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 micron. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 microM was sufficient to trigger formation of large lysosomes.
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Xu, Miao, Ke Liu, Manju Swaroop, Wei Sun, Seameen J. Dehdashti, John C. McKew und Wei Zheng. „A Phenotypic Compound Screening Assay for Lysosomal Storage Diseases“. Journal of Biomolecular Screening 19, Nr. 1 (27.08.2013): 168–75. http://dx.doi.org/10.1177/1087057113501197.
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The lysosome is a vital cellular organelle that primarily functions as a recycling center for breaking down unwanted macromolecules through a series of hydrolases. Functional deficiencies in lysosomal proteins due to genetic mutations have been found in more than 50 lysosomal storage diseases that exhibit characteristic lipid/macromolecule accumulation and enlarged lysosomes. Recently, the lysosome has emerged as a new therapeutic target for drug development for the treatment of lysosomal storage diseases. However, a suitable assay for compound screening against the diseased lysosomes is currently unavailable. We have developed a Lysotracker staining assay that measures the enlarged lysosomes in patient-derived cells using both fluorescence intensity readout and fluorescence microscopic measurement. This phenotypic assay has been tested in patient cells obtained from several lysosomal storage diseases and validated using a known compound, methyl-β-cyclodextrin, in primary fibroblast cells derived from Niemann Pick C disease patients. The results demonstrate that the Lysotracker assay can be used in compound screening for the identification of lead compounds that are capable of reducing enlarged lysosomes for drug development.
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Cuervo, A. M., E. Knecht, S. R. Terlecky und J. F. Dice. „Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation“. American Journal of Physiology-Cell Physiology 269, Nr. 5 (01.11.1995): C1200—C1208. http://dx.doi.org/10.1152/ajpcell.1995.269.5.c1200.
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Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.
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Liu, Ji, Wennan Lu, Sonia Guha, Gabriel C. Baltazar, Erin E. Coffey, Alan M. Laties, Ronald C. Rubenstein, William W. Reenstra und Claire H. Mitchell. „Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells“. American Journal of Physiology-Cell Physiology 303, Nr. 2 (15.07.2012): C160—C169. http://dx.doi.org/10.1152/ajpcell.00278.2011.
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The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTRinh-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4−/− mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.
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Alquier, C., P. Guenin, Y. Munari-Silem, C. Audebet und B. Rousset. „Isolation of pig thyroid lysosomes. Biochemical and morphological characterization“. Biochemical Journal 232, Nr. 2 (01.12.1985): 529–37. http://dx.doi.org/10.1042/bj2320529.
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Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, β-galactosidase and β-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.
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Zeng, Wenping, Canjun Li, Ruikun Wu, Xingguo Yang, Qingyan Wang, Bingqian Lin, Yanan Wei et al. „Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta“. PLOS Biology 22, Nr. 4 (23.04.2024): e3002591. http://dx.doi.org/10.1371/journal.pbio.3002591.
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Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aβ clearance in an autophagy-dependent manner in cellular models, and alleviates Aβ-induced paralysis in the Caenorhabditis elegans model of Alzheimer’s disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.
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Peng, Wesley, Yvette C. Wong und Dimitri Krainc. „Mitochondria-lysosome contacts regulate mitochondrial Ca2+dynamics via lysosomal TRPML1“. Proceedings of the National Academy of Sciences 117, Nr. 32 (23.07.2020): 19266–75. http://dx.doi.org/10.1073/pnas.2003236117.
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Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria–lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria–lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria–lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria–lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria–lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria–lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.
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Tan, Sin-Lih, Muruj Barri, Peace Atakpa-Adaji, Colin W. Taylor, Ewan St. John Smith und Ruth D. Murrell-Lagnado. „P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors“. International Journal of Molecular Sciences 22, Nr. 19 (28.09.2021): 10492. http://dx.doi.org/10.3390/ijms221910492.
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The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.
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Wang, Meng. „LYSOSOMAL SIGNALS IN LONGEVITY REGULATION ACROSS THE SCALE“. Innovation in Aging 7, Supplement_1 (01.12.2023): 340. http://dx.doi.org/10.1093/geroni/igad104.1133.
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Abstract Lysosomes are key organelles in the cell that constitute an acidic subcellular environment and contain approximately 60 different types of hydrolytic enzymes. With the aid of these acidic hydrolytic enzymes, lysosomes are highly metabolically active and can digest various macromolecules delivered through endocytosis, phagocytosis, and autophagy. Moreover, lysosomes function as a “signaling hub” that integrates metabolic inputs, organelle interaction, and longevity control. Our studies discovered a pro-longevity lysosomal acidic lipase that activates a lysosome-to-nucleus retrograde lipid signaling pathway in Caenorhabditis elegans, and in turn revealed the critical role of this lysosomal lipid signaling in promoting oxidative stress tolerance and lipid catabolism through tuning mitochondrial activities. Furthermore, we discovered that this lysosomal lipase induces the release of lipid messengers from peripheral metabolic tissues, which act on neurons and lead to the up-regulation of the neuropeptide signaling pathway by activating a nuclear hormone receptor. More recently, lysosome-specific proteomic profiling has revealed that AMPK is specifically recruited to the lysosome upon lysosomal lipolysis and mediates the longevity effect, suggesting a previously unknown interaction between AMPK and lysosomal lipid signaling. Together, these findings highlight the crucial role of lysosomal signals in actively coordinating organelle crosstalk and tissue communication to improve longevity and promote healthy aging.
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Hernandez, Grace A., Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias und Rushika M. Perera. „Abstract B052: Defining the lysosome proteome during tumor evolution“. Cancer Research 84, Nr. 2_Supplement (16.01.2024): B052. http://dx.doi.org/10.1158/1538-7445.panca2023-b052.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.
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Pierga, Alexandre, Raphaël Matusiak, Margaux Cauhapé, Julien Branchu, Lydia Danglot, Maxime Boutry und Frédéric Darios. „Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1“. PLOS Biology 21, Nr. 10 (23.10.2023): e3002337. http://dx.doi.org/10.1371/journal.pbio.3002337.
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The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.
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Hipolito, Victoria E. B., Jacqueline A. Diaz, Kristofferson V. Tandoc, Amra Saric, Ivan Toposiviric und Roberto J. Botelho. „mTOR induces lysosome expansion by selective translation of lysosomal transcripts during phagocyte activation“. Journal of Immunology 200, Nr. 1_Supplement (01.05.2018): 170.19. http://dx.doi.org/10.4049/jimmunol.200.supp.170.19.
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Abstract The molecular mechanisms that govern and adapt organelle number, size, morphology and activities to suit the needs of many cell types and the conditions that a cell may encounter remain poorly defined. Lysosomes are organelles that degrade cargo from a variety of routes including endocytosis, phagocytosis and autophagy. Lysosomes have emerged as a signalling platform that senses and couples stress signals such as nutrient deprivation to regulatory kinase hubs like mTOR and AMPK to modulate metabolic activity. For phagocytes and antigen-presenting cells like macrophages and dendritic cells, lysosomes are a kingpin organelle since they are essential to kill and process pathogens, and present antigens. During phagocyte activation, lysosomes undergo a striking reorganization, changing from dozens of globular structures to a tubular network, in a process that requires the phosphatidylinositol-3-kinase-Akt-mTOR signalling pathway. Ultimately, lysosome tubulation is thought to promote pinocytic retention and antigen presentation. We show that lysosome tubulation is accompanied by a rapid boost in lysosome volume and holding capacity during phagocyte activation with lipopolysaccharides. Unexpectedly, lysosome expansion was paralleled with the induction of lysosomal proteins, which was independent of TFEB and TFE3, transcription factors known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of lysosome expansion via mTOR-dependent increase in translation of mRNAs encoding key lysosomal proteins including LAMP1 and V-ATPase subunits. Collectively, we identified a mechanism of rapid organelle expansion and remodelling driven by selective enhancement of protein synthesis.
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Oliver, C., R. Dromy und T. K. Hart. „Density gradient separation of two populations of lysosomes from rat parotid acinar cells.“ Journal of Histochemistry & Cytochemistry 37, Nr. 11 (November 1989): 1645–52. http://dx.doi.org/10.1177/37.11.2553801.
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Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.
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Werneburg, Nathan W., M. Eugenia Guicciardi, Steven F. Bronk und Gregory J. Gores. „Tumor necrosis factor-α-associated lysosomal permeabilization is cathepsin B dependent“. American Journal of Physiology-Gastrointestinal and Liver Physiology 283, Nr. 4 (01.10.2002): G947—G956. http://dx.doi.org/10.1152/ajpgi.00151.2002.
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Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-α (TNF-α) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-α. Thus the aims of this study were to examine the mechanisms involved in TNF-α-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-α/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-α or sphingosine in Cat B(+/+) but not Cat B(−/−) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(−/−) liver. C6ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-α cytotoxic signaling.
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Christensen, Kenneth A., Jesse T. Myers und Joel A. Swanson. „pH-dependent regulation of lysosomal calcium in macrophages“. Journal of Cell Science 115, Nr. 3 (01.02.2002): 599–607. http://dx.doi.org/10.1242/jcs.115.3.599.
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Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6×10-4 M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A1 or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm,since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.
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Nguyen, Van-Nghia, und Haidong Li. „Recent Development of Lysosome-Targeted Organic Fluorescent Probes for Reactive Oxygen Species“. Molecules 28, Nr. 18 (15.09.2023): 6650. http://dx.doi.org/10.3390/molecules28186650.
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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.
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Jerome, W. Gray, und Patricia G. Yancey. „The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism“. Microscopy and Microanalysis 9, Nr. 1 (31.01.2003): 54–67. http://dx.doi.org/10.1017/s1431927603030010.
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Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.
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Li, Chenghao, Zhuo Zheng und Meishan Jin. „The Significance of Lysosome in the Diagnosis and Subclassification of Alzheimer’s Disease“. Science of Advanced Materials 15, Nr. 2 (01.02.2023): 233–42. http://dx.doi.org/10.1166/sam.2023.4441.
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Lysosomes are the main degradation organelles in eukaryotic cells, and their dysfunction is closely related to Alzheimer’s disease (AD). Our goal is to identify the lysosomal molecular subtype of AD and explore the mechanisms. By differential analysis, 50 differentially expressed lysosomal genes in AD were identified. R-package “ROCR” was used to calculate ROC curves and AUC values for lysosomal genes. “ConsensusClusterPlus” was used for consistent clustering of the AD data set. The contents of 28 kinds of immune cells in AD samples were calculated using the R-package “GSVA”. The R package “limma” was used to analyze the differences of autophagy genes. R package “WGCNA” carried out weighted co-expression network analysis. Two lysosomes subtypes were identified in AD cohort by lysosome diagnostics molecular, known as cluster 1/2. The results showed that 13 immune cells were significantly different between cluster 1 and cluster 2. A total of 76 differentially autopaghy genes were identified. The enrichment analysis showed that the key module genes were mainly concentrated in the synapses. We identified two subtypes based on lysosome gene expressions, preliminarily revealing that the heterogeneity of AD may be mainly caused by lysosomes. The role of lysosomes may be related to autophagy and synapses.
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Flannery, Andrew R., Cecilia Czibener und Norma W. Andrews. „Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes“. Journal of Cell Biology 191, Nr. 3 (01.11.2010): 599–613. http://dx.doi.org/10.1083/jcb.201003021.
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Syt VII is a Ca2+ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca2+-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes.
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Ebner, Michael, Philipp Alexander Koch und Volker Haucke. „Phosphoinositides in the control of lysosome function and homeostasis“. Biochemical Society Transactions 47, Nr. 4 (05.08.2019): 1173–85. http://dx.doi.org/10.1042/bst20190158.
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Abstract Lysosomes are the main degradative compartments of mammalian cells and serve as platforms for cellular nutrient signaling and sterol transport. The diverse functions of lysosomes and their adaptation to extracellular and intracellular cues are tightly linked to the spatiotemporally controlled synthesis, turnover and interconversion of lysosomal phosphoinositides, minor phospholipids that define membrane identity and couple membrane dynamics to cell signaling. How precisely lysosomal phosphoinositides act and which effector proteins within the lysosome membrane or at the lysosomal surface recognize them is only now beginning to emerge. Importantly, mutations in phosphoinositide metabolizing enzyme cause lysosomal dysfunction and are associated with numerous diseases ranging from neurodegeneration to cancer. Here, we discuss the phosphoinositides and phosphoinositide metabolizing enzymes implicated in lysosome function and homeostasis and outline perspectives for future research.
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Williams, M. A., und M. Fukuda. „Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail.“ Journal of Cell Biology 111, Nr. 3 (01.09.1990): 955–66. http://dx.doi.org/10.1083/jcb.111.3.955.
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Human lysosome membrane glycoprotein h-lamp-1 is a highly N-glycosylated protein found predominantly in lysosomes, with low levels present at the cell surface. The signal required for delivery of h-lamp-1 to lysosomes was investigated by analyzing the intracellular distribution of h-lamp-1 with altered amino acid sequences expressed from mutated cDNA clones. A cytoplasmic tail tyrosine residue found conserved in chicken, rodent, and human deduced amino acid sequences was discovered to be necessary for efficient lysosomal transport of h-lamp-1 in COS-1 cells. In addition, the position of the tyrosine residue relative to the membrane and carboxyl terminus also determined lysosomal expression. Supplanting the wild-type h-lamp-1 cytoplasmic tail onto a cell surface reporter glycoprotein was sufficient to cause redistribution of the chimera to lysosomes. A similar chimeric protein replacing the cytoplasmic tyrosine residue with an alanine was not expressed in lysosomes. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface, and unlike wild-type lysosomal membrane glycoproteins, were unable to undergo endocytosis. These data indicate that lysosomal membrane glycoproteins are sorted to lysosomes by a cytoplasmic signal containing tyrosine in a specific position, and the sorting signal may be recognized both in the trans-Golgi network and at the cell surface.
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Nguyen, Thuy T. P., Grace A. Hernandez, Gilles Rademaker, Matthew Luy, Alexander Li, Longhui Qiu, Joao A. Paulo, Rushika M. Perera und Joseph D. Mancias. „Abstract A082: Defining the lysosome proteome during pancreatic cancer tumor evolution and metastasis“. Cancer Research 84, Nr. 17_Supplement_2 (15.09.2024): A082. http://dx.doi.org/10.1158/1538-7445.pancreatic24-a082.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a five-year survival rate of only 13%, characterized by rapid proliferation, high metastatic potential, and resistance to conventional therapies. PDAC tumors are heavily reliant on nutrient-scavenging pathways such as autophagy and the lysosome – a degradative organelle that plays an essential role in the digestion and recycling of diverse cellular material. Despite their importance, the specific function of lysosomes over the course of tumor evolution and metastasis in PDAC tumors remains poorly understood. To understand the function of the lysosome in PDAC, we utilize a biochemical method known as LysoIP-based proteomics, which isolates intact lysosomes for subsequent analysis using mass spectrometry-based proteomics. This approach allows for a comprehensive analysis of lysosomal composition that can uncover unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early-stage, late-stage, and metastatic disease. Employing this strategy, we successfully captured lysosomal proteins from primary tumors (pancreas), as well as lung and liver metastases. Our preliminary analysis revealed an enrichment of resident lysosomal membrane and luminal proteins in the datasets, confirming the successful isolation of intact lysosomes from these complex tissues. Additionally, we identified non-resident lysosomal proteins (termed cargo) that are enriched and co-evolved with advancing tumor stages. Notably, our results reveal distinct differences in the lysosomal proteome of tumor cells across different tissues. These findings emphasize the critical role of lysosomal function in tumor progression and suggest that targeting lysosomal components could be a promising therapeutic strategy for treating metastatic PDAC. Citation Format: Thuy T.P Nguyen, Grace A. Hernandez, Gilles Rademaker, Matthew Luy, Alexander Li, Longhui Qiu, Joao A. Paulo, Rushika M. Perera, Joseph D. Mancias. Defining the lysosome proteome during pancreatic cancer tumor evolution and metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A082.
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Boonen, Marielle, Isabelle Hamer, Muriel Boussac, Anne-Françoise Delsaute, Bruno Flamion, Jérôme Garin und Michel Jadot. „Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes“. Biochemical Journal 395, Nr. 1 (15.03.2006): 39–47. http://dx.doi.org/10.1042/bj20051647.
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Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.
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Andrade, Luciana O., und Norma W. Andrews. „Lysosomal Fusion Is Essential for the Retention of Trypanosoma cruzi Inside Host Cells“. Journal of Experimental Medicine 200, Nr. 9 (01.11.2004): 1135–43. http://dx.doi.org/10.1084/jem.20041408.
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Trypomastigotes, the highly motile infective forms of Trypanosoma cruzi, are capable of infecting several cell types. Invasion occurs either by direct recruitment and fusion of lysosomes at the plasma membrane, or through invagination of the plasma membrane followed by intracellular fusion with lysosomes. The lysosome-like parasitophorous vacuole is subsequently disrupted, releasing the parasites for replication in the cytosol. The role of this early residence within lysosomes in the intracellular cycle of T. cruzi has remained unclear. For several other cytosolic pathogens, survival inside host cells depends on an early escape from phagosomes before lysosomal fusion. Here, we show that when lysosome-mediated T. cruzi invasion is blocked through phosophoinositide 3-kinase inhibition, a significant fraction of the internalized parasites are not subsequently retained inside host cells for a productive infection. A direct correlation was observed between the lysosomal fusion rates after invasion and the intracellular retention of trypomastigotes. Thus, formation of a parasitophorous vacuole with lysosomal properties is essential for preventing these highly motile parasites from exiting host cells and for allowing completion of the intracellular life cycle.
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Domagala, Antoni, Klaudyna Fidyt, Malgorzata Bobrowicz, Joanna Stachura, Kacper Szczygiel und Malgorzata Firczuk. „Typical and Atypical Inducers of Lysosomal Cell Death: A Promising Anticancer Strategy“. International Journal of Molecular Sciences 19, Nr. 8 (01.08.2018): 2256. http://dx.doi.org/10.3390/ijms19082256.
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Lysosomes are conservative organelles with an indispensable role in cellular degradation and the recycling of macromolecules. However, in light of recent findings, it has emerged that the role of lysosomes in cancer cells extends far beyond cellular catabolism and includes a variety of cellular pathways, such as proliferation, metastatic potential, and drug resistance. It has been well described that malignant transformation leads to alterations in lysosomal structure and function, which, paradoxically, renders cancer cells more sensitive to lysosomal destabilization. Furthermore, lysosomes are implicated in the regulation and execution of cell death in response to diverse stimuli and it has been shown that lysosome-dependent cell death can be utilized to overcome apoptosis and drug resistance. Thus, the purpose of this review is to characterize the role of lysosome in cancer therapy and to describe how these organelles impact treatment resistance. We summarized the characteristics of typical inducers of lysosomal cell death, which exert its function primarily via alterations in the lysosomal compartment. The review also presents other anticancer agents with the predominant mechanism of action different from lysosomal destabilization, the activity of which is influenced by lysosomal signaling, including classical chemotherapeutics, kinase inhibitors, monoclonal antibodies, as well as photodynamic therapy.
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Dielschneider, Rebecca, Hannah Eisenstat, James B. Johnston und Spencer B. Gibson. „Lysosome Membrane Permeabilization Causes Cell Death in Primary Chronic Lymphocytic Leukemia Cells“. Blood 124, Nr. 21 (06.12.2014): 930. http://dx.doi.org/10.1182/blood.v124.21.930.930.
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Abstract Introduction: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in North America. Despite many therapeutic advances over the past decade, drug resistance and disease recurrence are common. Novel therapeutic approaches are therefore required to treat CLL. One novel target identified in a variety of cancers, including acute myeloid leukemia, is the lysosome. In transformed cancerous cells, lysosomes were found to be sensitive to permeabilization by lysotropic agents in a process called lysosome membrane permeabilization. Permeabilization of lysosomes releases their acidic and proteolytic contents into the cytoplasm causing lysosome-mediated cell death. The therapeutic strategy of targeting lysosomes has yet to be determined in CLL. Methods: Primary CLL cells were purified from patient peripheral blood using negative selection and separated on a ficoll gradient. Three different lysosome-targeting drugs used in the clinic for other purposes were investigated: a quinolone, a fluoroquinolone antibiotic, and a cationic drug (CAD). To determine the mechanism of action, various dyes were used to stain lysosomes, mitochondria, and reactive oxygen species. Fluorescence was visualized under the confocal microscope and quantified using flow cytometry. To determine the role of reactive oxygen species (ROS) the antioxidants α-tocopherol, lycopene, N-acetyl cysteine, and glutathione were added to cells. To determine the role of proteases the inhibitors zVADfmk, Ca-074-Me, Chymostatin, and E64 were added to cells. Results: All the lysotropic agents except the antibiotic effectively killed CLL cells isolated from patients. The CAD had the greatest activity and was significantly more cytotoxic to the CLL cells as compared to T cells from the same patients and peripheral blood mononuclear cells from non-CLL donors. Treatment was equally effective in p53-proficient and p53-deficient CLL cells, demonstrating that the most aggressive and drug-resistant CLL cells were sensitive to this CAD. Mechanistic studies revealed that lysosome membrane permeabilization occurred within minutes and led to an increase in ROS and loss of mitochondrial membrane potential. The permeabilization of lysosomes was further confirmed by the translocation of transcription factor EB (TFEB) into the nucleus indicating promotion of lysosomal biogenesis. Lipid ROS were necessary to induce cell death, as only lipophilic antioxidants prevented cell death. Lipophilic antioxidants did not prevent lysosomal permeabilization, but did prevent downstream mitochondrial dysfunction. Inhibitors of caspases and lysosomal cathepsins failed to prevent cell death in CLL cells. Conclusions: Results show that the lysosome-targeting quinolone and CAD effectively permeabilize lysosomes and induce lysosome-mediated cell death in primary human CLL cells. This unique mechanism of cell death in CLL is dependent on the generation of lipid ROS, but not on the action of caspases or cathepsins. Overall, targeting lysosomes may be an effective strategy to selectively kill CLL cells regardless of p53 expression. Future studies are focused on the lysosomal differences in B cells and CLL cells. Disclosures No relevant conflicts of interest to declare.
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Kuk, Myeong Uk, Yun Haeng Lee, Jae Won Kim, Su Young Hwang, Joon Tae Park und Sang Chul Park. „Potential Treatment of Lysosomal Storage Disease through Modulation of the Mitochondrial—Lysosomal Axis“. Cells 10, Nr. 2 (17.02.2021): 420. http://dx.doi.org/10.3390/cells10020420.
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Lysosomal storage disease (LSD) is an inherited metabolic disorder caused by enzyme deficiency in lysosomes. Some treatments for LSD can slow progression, but there are no effective treatments to restore the pathological phenotype to normal levels. Lysosomes and mitochondria interact with each other, and this crosstalk plays a role in the maintenance of cellular homeostasis. Deficiency of lysosome enzymes in LSD impairs the turnover of mitochondrial defects, leading to deterioration of the mitochondrial respiratory chain (MRC). Cells with MRC impairment are associated with reduced lysosomal calcium homeostasis, resulting in impaired autophagic and endolysosomal function. This malicious feedback loop between lysosomes and mitochondria exacerbates LSD. In this review, we assess the interactions between mitochondria and lysosomes and propose the mitochondrial–lysosomal axis as a research target to treat LSD. The importance of the mitochondrial–lysosomal axis has been systematically characterized in several studies, suggesting that proper regulation of this axis represents an important investigative guide for the development of therapeutics for LSD. Therefore, studying the mitochondrial–lysosomal axis will not only add knowledge of the essential physiological processes of LSD, but also provide new strategies for treatment of LSD.
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Schulze, Ryan J., Eugene W. Krueger, Shaun G. Weller, Katherine M. Johnson, Carol A. Casey, Micah B. Schott und Mark A. McNiven. „Direct lysosome-based autophagy of lipid droplets in hepatocytes“. Proceedings of the National Academy of Sciences 117, Nr. 51 (07.12.2020): 32443–52. http://dx.doi.org/10.1073/pnas.2011442117.
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Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.
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Rodríguez, Ana, Paul Webster, Javier Ortego und Norma W. Andrews. „Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells“. Journal of Cell Biology 137, Nr. 1 (07.04.1997): 93–104. http://dx.doi.org/10.1083/jcb.137.1.93.
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Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+containing buffers to streptolysin O–permeabilized cells induced exocytosis of ∼10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.
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Burlando, Bruno, Barbara Marchi, Isabella Panfoli und Aldo Viarengo. „Essential role of Ca2+-dependent phospholipase A2in estradiol-induced lysosome activation“. American Journal of Physiology-Cell Physiology 283, Nr. 5 (01.11.2002): C1461—C1468. http://dx.doi.org/10.1152/ajpcell.00429.2001.
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The mechanism of lysosome activation by 17β-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+that was completely prevented by preincubating the cells with the Ca2+chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+-dependent PLA2(cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+-independent PLA2, ( E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2 H-pyran-2-one (BEL). The Ca2+ionophore A-23187 yielded similar effects on [Ca2+]iand lysosomes. Exposure to estradiol also resulted in cPLA2translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+-dependent mechanism involving activation of Ca2+-dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.
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Jerome, W. Gray, Brian E. Cox, Evelyn E. Griffin und Jody C. Ullery. „Lysosomal Cholesterol Accumulation Inhibits Subsequent Hydrolysis of Lipoprotein Cholesteryl Ester“. Microscopy and Microanalysis 14, Nr. 2 (03.03.2008): 138–49. http://dx.doi.org/10.1017/s1431927608080069.
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Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.
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Wang, Tuanlao, Ka Khuen Wong und Wanjin Hong. „A Unique Region of RILP Distinguishes It from Its Related Proteins in Its Regulation of Lysosomal Morphology and Interaction with Rab7 and Rab34“. Molecular Biology of the Cell 15, Nr. 2 (Februar 2004): 815–26. http://dx.doi.org/10.1091/mbc.e03-06-0413.
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Rab7 and Rab34 are implicated in regulation of lysosomal morphology and they share a common effector referred to as the RILP (Rab-interacting lysosomal protein). Two novel proteins related to RILP were identified and are tentatively referred to as RLP1 and RLP2 (for RILP-like protein 1 and 2, respectively). Overexpression of RILP caused enlarged lysosomes that are positioned more centrally in the cell. However, the morphology and distribution of lysosomes were not affected by overexpression of either RLP1 or RLP2. The molecular basis for the effect of RILP on lysosomes was investigated, leading to the demonstration that a 62-residue region (amino acids 272-333) of RILP is necessary for RILP's role in regulating lysosomal morphology. Remarkably, transferring this 62-residue region unique to RILP into corresponding sites in RLP1 rendered the chimeric protein capable of regulating lysosome morphology. A correlation between the interaction with GTP-bound form of both Rab proteins and the capability of regulating lysosomes was established. These results define a unique region in RILP responsible for its specific role in regulating lysosomal morphology as well as in its interaction with Rab7 and Rab34.
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Colbaugh, P. A., M. Stookey und R. K. Draper. „Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.“ Journal of Cell Biology 108, Nr. 6 (01.06.1989): 2211–19. http://dx.doi.org/10.1083/jcb.108.6.2211.
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We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.
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Eurell, Thomas E., Jo Ann C. Eurell, David J. Schaeffer, David R. Mattie und Carl L. Alden. „Lysosomal Changes in Renal Proximal Tubular Epithelial Cells of Male Sprague Dawley Rats following Decalin Exposure“. Toxicologic Pathology 18, Nr. 4 (November 1990): 637–42. http://dx.doi.org/10.1177/01926233900184p201.
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A histochemical stain for acid phosphatase served as a marker for lysosomal alterations in renal tubular cells associated with male rat hyaline droplet nephropathy. Morphometric analysis and quantitative histochemistry were used to compare the size and acid phosphatase stain reaction of lysosomes in tubular epithelial cells of treated and control animals. Decalin exposure increased the size and significantly ( p < 0.01) reduced the acid phosphatase stain intensity of individual lysosomes. However, there was no significant different ( p > 0.05) between the acid phosphatase stain intensity of treated and control animals when analyzed on a whole cell basis. The increase in size of the lysosomes without a proportional increase in whole cell acid phosphatase stain intensity indicates a dilution or a failure to accommodate in the acid phosphatase concentration (stain intensity/μm2) per lysosome. All acid phosphatase stain reaction product was contained within intact lysosomes, mitigating against the hypothesis of lysosomal enzyme leakage as the cause of cell death in decalin-induced alpha 2U globulin nephropathy.
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Selmi, S., und B. Rousset. „Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway“. Biochemical Journal 253, Nr. 2 (15.07.1988): 523–32. http://dx.doi.org/10.1042/bj2530523.
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Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n
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Saric, Amra, Victoria E. B. Hipolito, Jason G. Kay, Johnathan Canton, Costin N. Antonescu und Roberto J. Botelho. „mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells“. Molecular Biology of the Cell 27, Nr. 2 (15.01.2016): 321–33. http://dx.doi.org/10.1091/mbc.e15-05-0272.
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Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.
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Jing, Yongwei, Masahiko Kobayashi und Atsushi Hirao. „Abstract LB258: Therapeutic advantage of targeting lysosome as signaling hub for metabolic conditions in malignant gliomas“. Cancer Research 83, Nr. 8_Supplement (14.04.2023): LB258. http://dx.doi.org/10.1158/1538-7445.am2023-lb258.
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Abstract Lysosome, as the digestive system of the cell, participated in numerous cell biological processes, such as macromolecular degradation, cell adhesion/migration, and apoptosis by regulating metabolic status and pro-growth signaling. Therefore, lysosome could be a promising therapeutic target for cancer therapy, but it remains unclear how lysosome is involved in cancer malignancy. In this study, to investigate the roles of function lysosomes in the regulation of malignant status, we evaluated the proteolytic activity of lysosomes using BODIPY-dye conjugated BSA (DQ-BSA) in malignant gliomas characterized by high aggressivity, destructivity, and invasiveness, glioblastoma multiforme (GBM). While most GBM patient-derived cells showed higher lysosomal proteolytic activity than an immortalized human neural progenitor cell line derived from the embryonic brain, levels of lysosome activity positively co-related with sphere formation. Multi-omics analysis demonstrated that the level of lysosomal proteolytic activity was co-related with metabolic status, including activated mitochondria biogenesis and amino acid transport. The orthotopic xenograft model revealed that lysosome activity in vitro is a critical biomarker representing malignant phenotypes of GBM. We found that the MiT/TFE family, a master regulator of lysosomal biogenesis, controlled the malignant progression of GBM, including therapy resistance, indicating that quality control of lysosomes is a critical determinant for the malignant properties of gliomas. To develop a novel therapeutic approach for targeting lysosomal function, we performed screening of FDA-approved compounds and found that lysosomotropic agents, which induced galectin-3 punctation as an indicator of lysosomal membrane damage, efficiently suppressed tumor growth. Ifenprodil, a clinically available drug that acts as a lysosomotropic agent, increased intracellular Ca2+ levels, resulting in mitochondrial reactive oxygen species-mediated cytotoxicity. Furthermore, we found that the efficacy of ifenprodil was remarkably enhanced by metabolic modifications, such as amino acid restriction, in vitro and in vivo. Collectively, these data indicate that lysosomes as signaling hubs for metabolic conditions play critical roles in the regulation of the malignant status of GBM patients. Our findings propose that lysosomal biogenesis is a promising target to develop a novel therapeutic approach for GBM. Citation Format: Yongwei Jing, Masahiko Kobayashi, Atsushi Hirao. Therapeutic advantage of targeting lysosome as signaling hub for metabolic conditions in malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB258.
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Swanson, J., E. Burke und S. C. Silverstein. „Tubular lysosomes accompany stimulated pinocytosis in macrophages.“ Journal of Cell Biology 104, Nr. 5 (01.05.1987): 1217–22. http://dx.doi.org/10.1083/jcb.104.5.1217.
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A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.
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Bright, N. A., B. J. Reaves, B. M. Mullock und J. P. Luzio. „Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles“. Journal of Cell Science 110, Nr. 17 (01.09.1997): 2027–40. http://dx.doi.org/10.1242/jcs.110.17.2027.
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Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates by fluid phase endocytosis using a pulse-chase protocol. The gold particles present in dense core lysosomes and late endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-loaded dense core lysosomes at 37 degrees C, but accumulation was reversibly blocked by incubation at 20 degrees C. Time course experiments indicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptor-positive/lysosomal glycoprotein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the two BSA-gold conjugates were mixed in swollen late endosomal structures. Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late endosomes and hybrid endosome-lysosome structures. Subsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-formed from the resultant hybrid organelles prior to subsequent cycles of fusion and re-formation.
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Beauvarlet, Jennifer, Rabindra Nath Das, Karla Alvarez-Valadez, Isabelle Martins, Alexandra Muller, Elodie Darbo, Elodie Richard et al. „Triarylpyridine Compounds and Chloroquine Act in Concert to Trigger Lysosomal Membrane Permeabilization and Cell Death in Cancer Cells“. Cancers 12, Nr. 6 (18.06.2020): 1621. http://dx.doi.org/10.3390/cancers12061621.
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Lysosomes play a key role in regulating cell death in response to cancer therapies, yet little is known on the possible role of lysosomes in the therapeutic efficacy of G-quadruplex DNA ligands (G4L) in cancer cells. Here, we investigate the relationship between the modulation of lysosomal membrane damage and the degree to which cancer cells respond to the cytotoxic effects of G-quadruplex ligands belonging to the triarylpyridine family. Our results reveal that the lead compound of this family, 20A promotes the enlargement of the lysosome compartment as well as the induction of lysosome-relevant mRNAs. Interestingly, the combination of 20A and chloroquine (an inhibitor of lysosomal functions) led to a significant induction of lysosomal membrane permeabilization coupled to massive cell death. Similar effects were observed when chloroquine was added to three new triarylpyridine derivatives. Our findings thus uncover the lysosomal effects of triarylpyridines compounds and delineate a rationale for combining these compounds with chloroquine to increase their anticancer effects.
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Perou, C. M., und J. Kaplan. „Chediak-Higashi syndrome is not due to a defect in microtubule-based lysosomal mobility“. Journal of Cell Science 106, Nr. 1 (01.09.1993): 99–107. http://dx.doi.org/10.1242/jcs.106.1.99.
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Chediak-Higashi Syndrome is an autosomal recessive disorder that affects intracellular vesicle formation. The diagnostic feature of Chediak-Higashi Syndrome is the presence of ‘giant’ lysosomes clustered near the nucleus. Lysosome morphology in macrophages is maintained by microtubules and microtubule-based motors, such as kinesin. Dramatic changes in lysosome morphology can be induced by lowering cytoplasmic pH or by adding phorbol esters. When macrophages from beige mice (a murine homolog of Chediak-Higashi Syndrome) were subjected to these protocols they were able to alter their lysosomal distribution and morphology to the same degree as macrophages from control mice. These results indicate that lysosomes in Chediak cells are capable of interacting with the microtubule-based motor system, suggesting that the defective gene product is not an altered microtubular element involved in lysosomal movement.
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Folick, Andrew, Holly D. Oakley, Yong Yu, Eric H. Armstrong, Manju Kumari, Lucas Sanor, David D. Moore, Eric A. Ortlund, Rudolf Zechner und Meng C. Wang. „Lysosomal signaling molecules regulate longevity in Caenorhabditis elegans“. Science 347, Nr. 6217 (01.01.2015): 83–86. http://dx.doi.org/10.1126/science.1258857.
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Lysosomes are crucial cellular organelles for human health that function in digestion and recycling of extracellular and intracellular macromolecules. We describe a signaling role for lysosomes that affects aging. In the worm Caenorhabditis elegans, the lysosomal acid lipase LIPL-4 triggered nuclear translocalization of a lysosomal lipid chaperone LBP-8, which promoted longevity by activating the nuclear hormone receptors NHR-49 and NHR-80. We used high-throughput metabolomic analysis to identify several lipids in which abundance was increased in worms constitutively overexpressing LIPL-4. Among them, oleoylethanolamide directly bound to LBP-8 and NHR-80 proteins, activated transcription of target genes of NHR-49 and NHR-80, and promoted longevity in C. elegans. These findings reveal a lysosome-to-nucleus signaling pathway that promotes longevity and suggest a function of lysosomes as signaling organelles in metazoans.
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Ju, Xiangwu, Yiwu Yan, Qiang Liu, Ning Li, Miaomiao Sheng, Lifang Zhang, Xiao Li et al. „Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture“. Journal of Virology 89, Nr. 20 (05.08.2015): 10347–58. http://dx.doi.org/10.1128/jvi.01411-15.
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ABSTRACTAs a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death.IMPORTANCEThe integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.
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Gowrishankar, Swetha, und Shawn M. Ferguson. „Lysosomes relax in the cellular suburbs“. Journal of Cell Biology 212, Nr. 6 (14.03.2016): 617–19. http://dx.doi.org/10.1083/jcb.201602082.
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Lysosomes support cellular homeostasis by degrading macromolecules and recycling nutrients. In this issue, Johnson et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507112) reveal a heterogeneity in lysosomal pH and degradative ability that correlates with lysosome subcellular localization, raising questions about the functional implications and mechanisms underlying these observations.
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Luzio, J. Paul, Michael D. J. Parkinson, Sally R. Gray und Nicholas A. Bright. „The delivery of endocytosed cargo to lysosomes“. Biochemical Society Transactions 37, Nr. 5 (21.09.2009): 1019–21. http://dx.doi.org/10.1042/bst0371019.
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In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.
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Ke, Po-Yuan. „Molecular Mechanism of Autophagosome–Lysosome Fusion in Mammalian Cells“. Cells 13, Nr. 6 (13.03.2024): 500. http://dx.doi.org/10.3390/cells13060500.
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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.