Browsing by Subject "Lysosomes"
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Item Doxorubicin Inhibits Cardiomyocyte Autophagic Flux by Suppressing Lysosomal Acidification(2015-04-09) Li, Dan; Sadek, Hesham A.; Hill, Joseph A.; Levine, Beth; Amatruda, James F.The clinical use of doxorubicin is limited by cardiotoxicity. Dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases. However, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of acute doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, I first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, and is reminiscent of the effects seen in patients of chronic doxorubicin cardiomyopathy. Next, via multiple assays I showed that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. Moreover, I went on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, I studied animals with diminished autophagic activity due to haploinsufficiency for Beclin 1. Beclin 1+/- mice exposed to doxorubicin manifested restored cardiac autophagic flux, and were protected in terms of structural and functional changes within the myocardium. Conversely, animals over-expressing Beclin 1 manifested an amplified cardiotoxic response, correlating with their aggravated accumulation of autolysosomes in cardiomyocytes after doxorubicin treatments. In summary, I report here that doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Further, reducing autophagy initiation may protect against doxorubicin cardiotoxicity.Item Endolysosomal Function in Neuronal Maintenance(2018-06-14) Jin, Eugene Jennifer; Bezprozvanny, Ilya; Herz, Joachim; Terman, Jonathan R.; Huber, Kimberly M.; Hiesinger, Peter RobinEndolysosomal degradation of membrane proteins is crucial for the maintenance of synaptic function and neuronal health. Neurons can live for the lifetime of an organism and therefore rely on robust membrane turnover mechanisms to clear old, dysfunctional, excess or possibly also functional membrane proteins. A key regulator of canonical endolysosomal degradation is Rab7, a ubiquitous small GTPase required for endosomal maturation. Based on the observation that Rab7 expression is strongly neuron-enriched during Drosophila development, I first tested specific requirement of Rab7 in neurons. I found that loss of rab7 does not affect development, but causes activity-dependent degeneration that starts at synapses in Drosophila photoreceptors. Four point mutations in Rab7 are associated with the peripheral neuropathy Charcot-Marie-Tooth Type 2B (CMT2B) disease and my data suggest a partial loss of function mechanism. Together, these findings highlight that neurons are particularly sensitive to the dosage of Rab7-dependent endolysosomal degradation. Several other membrane turnover mechanisms, including autophagy and a neuron-specific branch of the endolysosomal system, called 'neuronal-sort-and-degrade' (NSD), are also required for neuronal maintenance. However, it remained unclear what cargoes these different membrane turnover mechanisms degrade, and where cargoes are degraded. Given that NSD is a neuron-specific mechanism whereas Rab7-dependent endolysosomal degradation and autophagy are ubiquitous mechanisms, I hypothesized that NSD may specifically sort and degrade synaptic membrane proteins, whereas the Rab7-dependent canonical endolysosomal degradation and autophagy unbiasedly degrade all membrane proteins. I tested this hypothesis by live imaging of an acidification-sensing degradation probe for a synaptic vesicle (SV)-specific cargo and a general membrane cargo to directly and quantitatively measure the sorting and degradation of these cargoes at Drosophila photoreceptor axon terminals. I found that both cargoes are sorted and degraded locally at axon terminals. Interestingly, the two cargoes are sorted into two distinct 'hub compartments' for degradation. Rab7 and NSD are required for the sorting and degradation of the two cargoes separately: sorting and degradation of general cargo is Rab7-dependent, whereas that of SV cargo is NSD-dependent. In sum, this work highlights neuron-specific mechanisms for cargo-specific membrane protein degradation that keep synapses healthy and functional.Item The Hydrophobic Handoff Between NPC2 and the N-Terminal Domain of NPC1 in the Export of Cholesterol from Lysosomes(2013-05-31) Wang, Michael Leechun; Brown, Michael S.; Goldstein, Joseph L.; Thomas, Philip J.; Roth, Michael G.; Hofmann, Sandra L.; Liang, GuoshengLow density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes where its cholesterol is released. Egress of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, an intrinsic membrane protein of 1278 amino acids and 13 postulated membrane-spanning helices that span the lysosomal membrane. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death in childhood owing to cholesterol accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1(NTD), can be expressed in vitro as a soluble protein of 240 amino acids that retains cholesterol binding activity. This thesis studies NPC2 and NPC1(NTD) in detail as summarized below. Two major differences exist between the cholesterol binding of NPC2 and NPC1(NTD). 1) Competitive binding studies and crystal structures indicate that the two proteins bind cholesterol in opposite orientations. NPC2 binds the iso-octyl side chain, leaving the 3ß hydroxyl exposed, whereas NPC1 binds the 3ß-hydroxyl, leaving the side chain partially exposed. 2) Kinetic studies of cholesterol binding reveal that NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1(NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC). Its rapid cholesterol binding allows NPC2 to transfer cholesterol to and from liposomes. Unlike NPC2, NPC1(NTD) cannot rapidly transfer its bound cholesterol to liposomes. However, NPC1(NTD) can accomplish this delivery when NPC2 is present. Furthermore, cholesterol binding to NPC1(NTD) is accelerated by >15-fold when the sterol is first bound to NPC2 and then transferred to NPC1(NTD). These data led us to advance a model in which NPC2 can mediate bi-directional transfer of cholesterol to or from NPC1(NTD). In cells, we envision that NPC2 accepts cholesterol in the lysosomal lumen and transports it to membrane-bound NPC1, thus accounting for the requirement for both proteins for lysosomal cholesterol export. Amino acid residues important or binding or transfer of cholesterol on NPC2 and NPC1(NTD) were identified through alanine scan mutagenesis. For both NPC2 and NPC1(NTD), residues that decreased binding mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the opening of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most severe mutations disrupting binding were P120S for NPC2 and P202A/F203A for NPC1(NTD); and those that disrupted transfer were V81D for NPC2 and L175Q/L176Q for NPC1(NTD). Furthermore, the functional significance of both the binding and transfer of cholesterol by NPC2 and NPC1(NTD) in the egress of cholesterol from lysosomes was confirmed. The above binding- or transfer-defective mutants of NPC2 and NPC1 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2 or NPC1-deficient cells, respectively, in contrast to wild-type NPC2 and NPC1. With these data, we envision that NPC2 binds cholesterol the instant that it is released from LDL, either as the free sterol or after cleavage of lipoprotein-derived cholesteryl esters by lysosomal acid lipase. This binding would prevent cholesterol from crystallizing in the lysosomal lumen. According to the model, NPC2 can transfer its bound cholesterol to NPC1(NTD) directly, thus avoiding the necessity for the insoluble cholesterol to transit the water phase. This transfer of cholesterol from NPC2 to NPC1(NTD) has a special functional relevance in light of the near-absolute insolubility of cholesterol in water, and we have named this process a "hydrophobic handoff."Item Illuminating Endocytic Organelles with pH-Resposive [sic] Nanomaterials(2017-02-20) Wang, Chensu; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming; Danuser, Gaudenz; Yoo, Hyuntae; Zhong, QingEndosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of key cell physiological processes that include protein/lipid metabolism, nutrient sensing and cell survival. To support quantitative investigation of these processes in living cells, we have developed a library of ultra-pH sensitive (UPS) fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatial-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution. Deployment in cells enabled quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. These biological applications benchmarked the robustness and adaptability of this nanotechnology-enabled 'detect and perturb' strategy. As a translational application, we leveraged the technology in high-throughput screening assays that successfully identified chemical agents in the promotion of autophagolysosomal activity through TFEB activation. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles conferred hepatoprotection against diet-induced steatosis in murine models and prolonged survival in Caenorhabditis elegans. These results highlight the therapeutic potential of small-molecule TFEB activators to ameliorate metabolic syndrome and extend lifespan.Item Methods for Identifying Subcellular Targeting Ligands and Selected Applications(2015-09-09) Umlauf, Benjamin J.; Albanesi, Joseph P.; Brown, Kathlynn C.; Schmid, Sandra; Kohler, Jennifer J.Subcellular localization plays an essential role in targeting drug therapies as generally the pro-drug or linker relies on physical conditions of a particular subcellular compartment to function. We developed two methods that allow for selecting targeting ligands that both internalize specifically in cancer cells as well as accumulate in a defined subcellular location. The first method utilizes endocytic selection pressure to identify targeting peptides from a phage displayed peptide library that internalize specifically in cancer cells via a defined mechanism of endocytosis while the second couples together a traditional biopanning selection protocol with a secondary immunofluorescent screen to directly identify functional targeting peptides. In addition we also present a method for reducing amplification bias during phage display experiments. Finally, we demonstrate the utility of a novel peptide, termed H1299.3 selected via the endocytic pressure method, to serve as targeting ligand for a novel immunotherapy. H1299.3 targeted liposomes facilitate delivery of antigenic peptide specifically in MHC class 1 molecules of cancer cells resulting in activation of secondary immune response against the cancer cells. Thus, we present two novel methods for identify targeting ligands with selective internalization that accumulate in defined subcellular localizations as well as a novel application for a newly identified targeting ligand.Item Molecular Basis of Cooperativity in pH-Triggered Supramolecular Self-Assembly(2017-06-14) Li, Yang; Siegwart, Daniel J.; Gao, Jinming; Sumer, Baran; Zhang, XuewuResponsive nanomaterials have become an attractive biosensing platform because of their versatility in varying the size, composition, shape and other physicochemical properties to address the deficiency of conventional sensors such as low sensitivity and specificity. Compared to small molecular sensors, nanoparticle sensors often deploy a multitude of non-covalent interactions (hydrogen bonding, hydrophobic and electrostatic interactions) and the resulting system frequently displays cooperative behaviors. pH is an important physiological parameter that plays a critical role in cellular and tissue homeostasis. Dysregulated pH has been recognized as a universal hallmark of cancer. pH-sensitive nanoparticles have been widely used for tumor imaging, study of endosome/lysosome biology and cancer-targeted drug delivery. Recently, we have established a library of ultra-pH sensitive (UPS) nanoprobes with sharp pH transitions that are finely tunable in a broad range of physiological pH (4-8). The UPS nanoprobes showed significantly improved sensitivity and biological precision over commonly used small molecular and polymeric pH sensors. Here, we performed the mechanistic study of sharp pH response and binary on/off switch, which are absent in common small molecular and polymeric pH sensors or buffers, in pH-triggered supramolecular self-assembly process. Hydrophobic nanophase separation drove cooperative deprotonation of protonated unimers into neutral copolymers inside micelles. This divergent proton distribution characteristic of a representative PDPA copolymers was not observed in commonly used small molecular and polymeric bases (e.g., PEI). The cooperative deprotonation dynamics can explain the significantly decreased pKa and sharpened pH response. Combination of theoretical modeling and experimental validation allowed identification of key structural parameters on impacting pKa and sharpness in pH transition. Inspired by the impact of counter-ions on the self-assembly of UPS block copolymers, we reported a novel specific anion-induced micellization process. In vitro and in vivo experiments suggested an "capture and integration" mechanism underlying the binary tumor margin delineation performance of UPS nanoparticles. Results from this study offer molecular insights to help establish the general principles in nanophase transition and supramolecular self-assembly for the development of new nanomaterials-based sensors with binary on/off switch in chemical and biological sensing.Item On Cholesterol Transport Between Membranes(August 2021) Trinh, Michael Nguyen; Mendell, Joshua T.; Chen, Zhijian J.; Abrams, John M.; Brown, Michael S.; Goldstein, Joseph L.The studies described in this dissertation focus on investigation of the pathways for transport of cholesterol from one organelle to another in animal cells. Cells have evolved elaborate transport mechanisms to assure an optimum cholesterol content within their membranes. Dysregulation of cholesterol transport causes common diseases, including atherosclerosis. The major source of cellular cholesterol comes from Low Density Lipoprotein (LDL). When plasma membranes are low in cholesterol, cells produce LDL receptors which bind LDL and mediate its uptake by endocytosis and its delivery to lysosomes. Within lysosomes the cholesteryl esters of LDL are hydrolyzed. The free cholesterol binds to a soluble lysosomal protein called Niemann Pick C2 (NPC2) which delivers it to a membrane-embedded protein called NPC1 which inserts the cholesterol into the lysosome membrane. From there the cholesterol moves to the plasma membrane (PM) through a pathway that is unknown. When the PM becomes saturated with cholesterol, any excess is transported to the endoplasmic reticulum (ER) to repress production of LDL receptors and to be stored in lipid droplets. The work described here 1) showed that triazole antifungal drugs inhibit lysosomal cholesterol export by binding to the membrane domain of NPC1 2) used itraconazole to solve the crystal structure of NPC1 at 3.3Å, 3) revealed that cholesterol is transported out of lysosomes through interactions between two or more NPC1 molecules, and 4) utilized CRISPR-Cas9 whole-genome knockout screens to identify all the genes involved in the transport and uptake of LDL cholesterol. From these screens in the latter study, we discovered that a specific phospholipid, phosphatidylserine (PS), is required for PM-to-ER cholesterol transport. These studies provide supporting evidence towards a vision of one-way directional transport of LDL-derived cholesterol from lysosomes to the PM to the ER.Item Regulation of Traffic into and out of the Yeast Endosome by the VPS9P Cue Domain and the VPS5P Domain(2004-05-04) Davies, Brian Andrew; Roth, Michael G.The presence of membrane bound compartments in eukaryotic cells enables the generation of discrete environments in which distinct and sometimes competing chemical reactions occur. The mammalian lysosome represents an example of this principle. The lysosome is an acidic, hydrolase-rich compartment that functions in macromolecular degradation. The delivery of material to the lysosome both from biosynthetic and endocytic pathways is a highly regulated process, and defects in the lysosomal trafficking system have been linked to congenital diseases including mucolipidosis type II (I-cell disease). An analogous trafficking system functions in the fungi Saccharomyces cerevisiae to deliver biosynthetic and endocytic cargo to the yeast vacuole. Genetic and biochemical analyses of the yeast vacuolar protein sorting pathway have defined the steps in this process and identified more than 40 gene products involved. Soluble vacuolar hydrolases are diverted from the secretory pathway through interaction with a vacuolar protein sorting receptor in the trans-Golgi compartment. This receptor then facilitates transport to the endosome where the biosynthetic and endocytic pathways coincide. The receptor is recycled back to the Golgi while the vacuolar hydrolases and endocytic material are conveyed to their ultimate destination. I have been interested in two aspects of this pathway in yeast, focusing on traffic into and out of the endosome. The first question addressed is the identification of the cytosolic components that mediate receptor recycling (Chapters 2 and 3). The Sorting Nexin-1 homolog Vps5p is demonstrated to form a complex with Vps17p to mediate this process. Vps5p and Vps17p also interact with Vps26p, Vps29p and Vps35p and the lipid phosphatidylinositol-3-phosphate to recycle the receptor. The significance of these protein-protein interactions and the lipid-protein interaction in Vps5p function is examined. The second question addressed is the regulation of traffic into the endosome by modulators of the guanine nucleotide exchange factor Vps9p (Chapters 4 and 5). Ubiquitin is identified as one such regulator, and the Vps9p CUE domain is demonstrated to be a new ubiquitin binding motif. The mechanism by which the CUE domain binds ubiquitin is addressed, and the functional relevance of Vps9p ubiquitin binding and ubiquitylation are examined in vivo.Item The Role of a Neuron-Specific V-ATPase in Synapse Specification, Function, and Maintenance(2012-08-13) Williamson, Wallace Ryan; Hiesinger, Peter RobinThe Vacuolar-type (V-) adenosine triphosphatase (ATPase) is a proton-pumping nanomachine consisting of two multi-subunit, reversibly associating protein sectors, the cytosolic V1 sector and the membrane-bound Vo sector. The V1/Vo holoenzyme hydrolyses ATP to translocate protons across biological membranes thereby modulating lumenal and extracellular pH. Additionally, accumulating evidence suggests that the Vo sector has a role in membrane fusion when dissociated from the V1 sector, its proton-pumping partner. Published evidence for this includes a null allele for the neuron-specific a subunit in the Drosophila Vo sector, v100, which leads to defects in synaptic function that are unrelated to pH regulation. My project emerged from the need to explain why v100 has two additional phenotypes that are absent in other synaptic function mutants: functional and structural degeneration in photoreceptor cells and patterning defects in the visual system neuropil. I proposed that v100 has a previously undocumented role on a neuron-specific endo/lysosomal pathway in addition to its documented role in neurotransmitter secretion. To test my hypothesis in the context of only one of the two purported v100 functions, I generated transgenic animals with v100 mutations designed to specifically disrupt either acidification or membrane fusion. Using these genetic tools, I discovered that v100 has an essential role in sorting cargo into an endo-lysosomal pathway that concomitantly requires v100 for the acidification-dependent maturation of degradation-competent organelles. This 'sort-and-degrade' mechanism for v100 defines a neuron-specific degradation pathway that is required for synaptic specification, function, and maintenance. In developmental stages, v100 is required to 'sort-and-degrade' guidance receptors as part of the synapse specification program. In the adult, the 'sort-and-degrade' mechanism provides additional degradative capacity to neurons, a cell type that must often maintain homeostasis for unusually long periods of time. Finally, I provide evidence that the role for V100 in membrane fusion requires a direct, physical interaction with Syntaxin-1, an interaction that can be specifically disrupted in vitro and in vivo. In brief, my results provide mechanistic insight into the acidification-independent role of v100 and reveal the existence of a neuron-specific endo-lysosomal pathway on which v100 functions to 'sort-and-degrade' cargo in order to meet the special needs of a neuron in development, function, and maintenance.Item Using Advanced Fluorescence Microscopy to Analyze Intracellular Trafficking of FcRn In Live Cells(2013-01-17) Gan, Zhuo; Ward, E. Sally; Ober, Raimund J.The MHC class I-related Fc receptor (FcRn) regulates the in vivo half-life of immunoglobulin G (IgG) and transports IgG across cell barriers. The intracellular trafficking of FcRn is central to its diverse functions. FcRn, like all receptors, is transferred to lysosomes for constitutive degradation to maintain a balance between synthesis and breakdown. Using live cell imaging, a novel lysosomal delivery pathway for FcRn has been observed. Unlike signaling receptors that enter the intraluminal vesicles in late endosomes, FcRn remains on the limiting membrane of late endosomes and is delivered to lysosomes through a selective, primarily tubule-mediated process. Following transfer, FcRn is rapidly internalized into the lysosomal lumen. By contrast, LAMP1 remains on the limiting membrane of lysosomes. Rab5 can persist on late endosomes, which can not only fuse with lysosomes, but can also give rise to tubulovesicular carriers that enter the recycling pathway. Thus, late endosomes are functionally plastic. These observations have relevance to understanding lysosomal delivery pathways. A combination of MUltifocal plane Microscopy (MUM) and localized photoactivation ('LP-MUM') has been developed to investigate the intracellular recycling pathway of FcRn. LP-MUM has been used to activate photoactivatable GFP (PAGFP) tagged proteins in individual sorting endosomes within cells, followed by imaging in two focal planes simultaneously. This approach has enabled the tracking of small, motile and dense transport carriers (TCs) that deliver FcRn to different destinations within the cell. The Rab GTPases, SNX4 and APPL1 play important roles in various steps of receptor trafficking pathways, and their associations with TCs has also been investigated. Four distinct itineraries taken by TCs at various stages of FcRn recycling have been characterized. In addition, the effectors associated with TCs on different pathways have been identified. APPL1+ TCs can transfer FcRn from the plasma membrane to pre-existing sorting endosomes. Interendosomal TCs migrate between sorting endosomes and are Rab4+/SNX4+ but Rab11-/APPL1-. Post-endosomal TCs that deliver FcRn to the plasma membrane are Rab11+ but Rab4-/SNX4-/APPL1-. Unexpectedly, a novel class of 'looping' TCs that leave a sorting endosome and return to the same endosome after several minutes has also been observed. The 'looping' TCs are Rab11+/Rab4+/SNX4+. The analyses of these TCs should have general relevance to other receptors and cargo on the recycling pathway.Item Vibrio Effector Protein, VopQ Targets the Host Lysosome to Manipulate Autophagy(2014-07-23) Sreelatha, Anju; Tu, Benjamin; Orth, Kim; Goodman, Joel M.; Shiloh, MichaelVibrio parahaemolyticus is a gram-negative marine bacterium that is the major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood. Vibrio parahaemolyticus harbors two Type III secretion systems. The first, T3SS1, orchestrates a temporally regulated cell death mediated by autophagy, membrane blebbing, followed by cell rounding and eventual lysis of the host cell. One T3SS1 effector protein, VopQ is both necessary and sufficient to induce rapid autophagy during the first hour of infection. Herein, I characterize the biochemical activity of the virulence factor VopQ, a novel Vibrio parahaemolyticus protein with no homology to any proteins outside of the Vibrio species. VopQ binds to the conserved Vo domain of the V-ATPase that is enriched on the lysosomal membrane and causes deacidification of the lysosomes within minutes of entry into the host cell. VopQ forms an ~18 angstrom gated channel that facilitates outward-rectified flux of ions across lipid bilayers. These studies show how a bacterial pathogen uses a novel, targeted pore forming effector to alter autophagic flux by manipulating the partitioning of small molecules and ions. Additionally, we demonstrate that VopQ is also a potent inhibitor of vesicular membrane fusion using in vitro membrane fusion. The inhibition of membrane fusion appears to be independent of VopQ’s pore-forming activity. VopQ inhibits the final step of membrane fusion by inhibiting trans-SNARE complex formation. In order to delineate the two inhibitory functions of VopQ, deacidification and membrane fusion, I use mutational, biochemical and crystallographic studies. Elucidating the molecular mechanism of VopQ not only provides a better understanding of Vibrio parahaemolyticus pathology but also offers new insight into the host cell mechanisms of autophagy and vesicle fusion.