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Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient‐limited cells; this translocation was mediated by a rapid post‐transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient‐limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient‐limited cells were treated with 5‐Fluorouracil (5‐FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance. [ABSTRACT FROM AUTHOR]

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal‐lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP‐3 complex mu subunit, the lysosomal‐trafficking regulator LYST, mucopilin‐1, and the Wiskott–Aldrich syndrome‐associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation‐independent mannose‐6‐phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans. [ABSTRACT FROM AUTHOR]

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low β‐glucocerebrosidase (β‐GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant β‐GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based β‐GC activity assay followed by reporter trafficking assay utilizing β‐GC‐mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the β‐GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their β‐GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD. [ABSTRACT FROM AUTHOR]

Previously, we found that age‐dependent accumulation of beta‐amyloid is not sufficient to cause synaptic decline. Late‐endocytic organelles (LEOs) may be driving synaptic decline as lysosomes (Lys) are a target of cellular aging and relevant for synapses. We found that LAMP1‐positive LEOs increased in size and number and accumulated near synapses in aged neurons and brains. LEOs' distal accumulation might relate to the increased anterograde movement in aged neurons. Dissecting the LEOs, we found that late‐endosomes accumulated while there are fewer terminal Lys in aged neurites, but not in the cell body. The most abundant LEOs were degradative Lys or endolysosomes (ELys), especially in neurites. ELys activity was reduced because of acidification defects, supported by the reduction in v‐ATPase subunit V0a1 with aging. Increasing the acidification of aged ELys recovered degradation and reverted synaptic decline, while alkalinization or v‐ATPase inhibition, mimicked age‐dependent Lys and synapse dysfunction. We identify ELys deacidification as a neuronal mechanism of age‐dependent synapse loss. Our findings suggest that future therapeutic strategies to address endolysosomal defects might be able to delay age‐related synaptic decline. [ABSTRACT FROM AUTHOR]

Pseudophosphatases are catalytically inactive but share sequence and structural similarities with classical phosphatases. STYXL1 is a pseudophosphatase that belongs to the family of dual‐specificity phosphatases and is known to regulate stress granule formation, neurite formation and apoptosis in different cell types. However, the role of STYXL1 in regulating cellular trafficking or the lysosome function has not been elucidated. Here, we show that the knockdown of STYXL1 enhances the trafficking of β‐glucocerebrosidase (β‐GC) and its lysosomal activity in HeLa cells. Importantly, the STYXL1‐depleted cells display enhanced distribution of endoplasmic reticulum (ER), late endosome and lysosome compartments. Further, knockdown of STYXL1 causes the nuclear translocation of unfolded protein response (UPR) and lysosomal biogenesis transcription factors. However, the upregulated β‐GC activity in the lysosomes is independent of TFEB/TFE3 nuclear localization in STYXL1 knockdown cells. The treatment of STYXL1 knockdown cells with 4‐PBA (ER stress attenuator) significantly reduces the β‐GC activity equivalent to control cells but not additive with thapsigargin, an ER stress activator. Additionally, STYXL1‐depleted cells show the enhanced contact of lysosomes with ER, possibly via increased UPR. The depletion of STYXL1 in human primary fibroblasts derived from Gaucher patients showed moderately enhanced lysosomal enzyme activity. Overall, these studies illustrated the unique role of pseudophosphatase STYXL1 in modulating the lysosome function both in normal and lysosome‐storage disorder cell types. Thus, designing small molecules against STYXL1 possibly can restore the lysosome activity by enhancing ER stress in Gaucher disease. [ABSTRACT FROM AUTHOR]

Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue‐containing small immature secretory granules, which then fuse with each other and reach their normal 3–3.5 μm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome‐to‐TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans. [ABSTRACT FROM AUTHOR]

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease. A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here, we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathepsin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age‐dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains. Synopsis Lack of glial LRRK2/dLRRK in the immortalized microglial cells or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, and exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathespin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, dopaminergic neurodegeneration, and locomotor deficits in an age‐dependent manner. [ABSTRACT FROM AUTHOR]

AP‐3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP‐3 cause Hermansky‐Pudlak syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP‐3 carries cargo directly from the trans‐Golgi to the lysosomal vacuole. Despite the pathway's importance and its discovery two decades ago, rapid screens and selections for AP‐3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate‐based assessment of AP‐3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP‐3 carrier vesicles and is adaptable to high‐throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention. [ABSTRACT FROM AUTHOR]

Phagocytosis is a complex cellular uptake process involving multiple distinct steps of cargo recognition, uptake, phagosome maturation and eventual phagolysosome resolution. Emerging literature shows that heterogeneity of phagocytosis at multiple steps at a single cell level influences the population outcome. However, the determinants of phagocytic heterogeneity are not clear. Here we show that the variance in the endocytic capacity of individual cells in a macrophage population determines subsequent phagocytic uptake and trafficking. Our results document the extensive heterogeneity in the endocytic uptake of individual macrophages, and show that cells with higher endocytic capacity preferentially phagocytose diverse cargo, including pathogenic Mycobacterium tuberculosis. Interestingly, M. tuberculosis infected cells sustain the higher endocytic capacity following infection. Modulating endocytic capacity by inhibiting endocytosis reduces phagocytic uptake. Differential uptake of M. tuberculosis into cells with different endocytic capacities correlates with the efficiency of phagocytic delivery to lysosomes, thus contributing further to phagocytic as well as mycobacterial heterogeneity. Thus, variance in endocytic capacity is a determinant of generating heterogeneity in phagocytosis at multiple steps. [ABSTRACT FROM AUTHOR]

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease. [ABSTRACT FROM AUTHOR]

The late endosomes/endo‐lysosomes of vertebrates contain an atypical phospholipid, lysobisphosphatidic acid (LBPA) (also termed bis[monoacylglycero]phosphate [BMP]), which is not detected elsewhere in the cell. LBPA is abundant in the membrane system present in the lumen of this compartment, including intralumenal vesicles (ILVs). In this review, the current knowledge on LBPA and LBPA‐containing membranes will be summarized, and their role in the control of endosomal cholesterol will be outlined. Some speculations will also be made on how this system may be overwhelmed in the cholesterol storage disorder Niemann‐Pick C. Then, the roles of intralumenal membranes in endo‐lysosomal dynamics and functions will be discussed in broader terms. Likewise, the mechanisms that drive the biogenesis of intralumenal membranes, including ESCRTs, will also be discussed, as well as their diverse composition and fate, including degradation in lysosomes and secretion as exosomes. This review will also discuss how intralumenal membranes are hijacked by pathogenic agents during intoxication and infection, and what is the biochemical composition and function of the intra‐endosomal lumenal milieu. Finally, this review will allude to the size limitations imposed on intralumenal vesicle functions and speculate on the possible role of LBPA as calcium chelator in the acidic calcium stores of endo‐lysosomes. [ABSTRACT FROM AUTHOR]

Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra‐ and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions. [ABSTRACT FROM AUTHOR]

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER‐to‐phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles. [ABSTRACT FROM AUTHOR]

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP‐activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell. [ABSTRACT FROM AUTHOR]

β‐Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor‐targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol‐derived activity‐based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre‐labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme. [ABSTRACT FROM AUTHOR]

For the past five decades, the lysosome has been characterized as an unglamorous cellular recycling center. This notion has undergone a radical shift in the last 10 years, with new research revealing that this organelle serves as a major hub for metabolic signaling pathways. The discovery that master growth regulators, including the protein kinase mTOR (mechanistic target of rapamycin), make their home at the lysosomal surface has generated intense interest in the lysosome's key role in nutrient sensing and cellular homeostasis. The transcriptional networks required for lysosomal maintenance and function are just being unraveled and their connection to lysosome‐based signaling pathways revealed. The catabolic and anabolic pathways that converge on the lysosome connect this organelle with multiple facets of cellular function; when these pathways are deregulated they underlie multiple human diseases, and promote cellular and organismal aging. Thus, understanding how lysosome‐based signaling pathways function will not only illuminate the fascinating biology of this organelle but will also be critical in unlocking its therapeutic potentials. After decades of being known as just the cellular recycling center, the lysosome has recently emerged as a central hub for metabolic signaling. Signaling pathways that call the lysosome home connect this organelle to key anabolic and catabolic processes that control many facets of cellular metabolism. Metabolic signals emanating from the lysosome are contributors to multiple human diseases, emphasizing the key role of the lysosome as a cellular site for the regulation of human health. [ABSTRACT FROM AUTHOR]

Lysosomes are membrane‐enclosed organelles that mediate the intracellular degradation of macromolecules. They play an essential role in calcium regulation and have emerged as key signaling hubs in controlling the nutrient response. Maintaining lysosomal integrity and function is therefore crucial for cellular homeostasis. Different forms of stress can induce lysosomal membrane permeabilization (LMP), resulting in the translocation to the cytoplasm of intralysosomal components, such as cathepsins, inducing lysosomal‐dependent cell death (LDCD). Here, we review recent advances that have furthered our understanding of the molecular mechanisms of LMP and the methods used to detect it. We discuss several endolysosomal damage‐response mechanisms that mediate the repair or elimination of compromised lysosomes and summarize the role of LMP and cathepsins in LDCD and other cell death pathways. Finally, with the emergence of lysosomes as promising therapeutic targets for several human diseases, we review a variety of therapeutic strategies that seek to either destabilize lysosomes or to maintain, enhance or restore lysosomal function. Lysosomes mediate intracellular degradation through the action of lysosomal hydrolases. Lysosomal membrane permeabilization (LMP) results in translocation to the cytoplasm of the intraluminal contents and consequent lysosome‐dependent cell death. [ABSTRACT FROM AUTHOR]

Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule‐organizing center in a “cloud,” and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biological functions, such as cargo degradation, antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biological events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid‐protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the molecular machineries that control this mobility. [ABSTRACT FROM AUTHOR]

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V‐ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V‐ATPase proton pumps and led to the discovery of protein splicing of the V‐ATPase catalytic subunit. The Vma− phenotype, characteristic of loss‐of‐V‐ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V‐ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V‐ATPase assembly factors and helped unravel the complex pathways for V‐ATPase assembly. In later years, exploration of the evolutionary history of several V‐ATPase subunits provided new information about the enzyme's structure and function. This review highlights V‐ATPase work in the Stevens’ lab between 1987 and 2017. [ABSTRACT FROM AUTHOR]

Despite the rapidly expanding use of antibody‐based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody‐opsonized tumor cells is limited. Here we report the formation of a phagosome‐associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2‐positive cancer cells in the presence of the HER2‐specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome‐associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody‐opsonized cancer cells by macrophages. [ABSTRACT FROM AUTHOR]

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB‐lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell‐free MVB‐lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS ( homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa‐SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab‐cascade mechanism harmonizes MVB maturation with lysosome fusion. [ABSTRACT FROM AUTHOR]

The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 'anti-ligases' that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme ( DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the 'ubiquitome' of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase- DUb fusions. [ABSTRACT FROM AUTHOR]

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith-Magenis chromosome region 8 ( SMCR8) and WD repeat-containing protein 41 ( WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 ( mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72-containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology. [ABSTRACT FROM AUTHOR]

Mammalian cells acquire most exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co-ordinated actions of Niemann-Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post-endosomal cholesterol trafficking. Numerous sterol-transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome-peroxisome membrane contact (LPMC) established by a non-STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5-bisphosphate also serves as a novel and important path for LDL-cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells. [ABSTRACT FROM AUTHOR]

The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions. [ABSTRACT FROM AUTHOR]

Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome. [ABSTRACT FROM AUTHOR]

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase ( NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor ( FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G ( IgG)-stimulated reactive oxygen species ( ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C ( PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR-mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG-opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR-triggered, PKC/Syk-dependent local assembly and activation of NOX2. [ABSTRACT FROM AUTHOR]

LYST‐1 is a Caenorhabditis elegansBEACH domain containing protein (BDCP) homologous to LYST and NBEAL2, BDCPs controlling organelle biogenesis that are implicated in human disease. Unlike the three other BDCPs encoded by C. elegans, mutations in lyst‐1 lead to smaller lysosome‐related organelles (LROs), smaller lysosomes, increased numbers of LROs and decreased numbers of early endosomes. lyst‐1(−) mutations do not obviously disrupt protein trafficking to lysosomes or LROs, however, the formation of gut granules is diminished. While the trafficking pathways that mediate the delivery of proteins to lysosome‐related organelles (LROs) are becoming increasingly clear, how LRO size and numbers are controlled is relatively unstudied. BEACH domain containing proteins (BDCPs) have emerged as potential regulators of these processes. Caenorhabditis elegans lyst‐1 encodes a BDCP that is homologous to both human LYST, which is mutated in Chediak‐Higashi syndrome, and NBEAL2, which is mutated in gray platelet syndrome. We show that mutations in lyst‐1 lead to fragmentation of intestinal specific LROs called gut granules. Protein trafficking to gut granules appears to be unaffected in lyst‐1(−) mutants; however, gut granule formation and function is disrupted. We find that lysosomes are smaller in both intestinal cells and coelomocytes of lyst‐1(−) mutants. We show that mutations in the three other BDCPs encoded by C. elegans do not similarly alter gut granule morphology. While recycling and late endosomes appear to be unaltered, mutations in lyst‐1 cause a significant reduction in the number of RAB‐5::GFP containing early endosomes, an effect that has not been reported for any other BDCP mutant. [ABSTRACT FROM AUTHOR]

Calnuc is a ubiquitous Ca2+-binding protein present on the trans-Golgi network ( TGN) and endosomes. However, the precise role of Calnuc in these organelles is poorly characterized. We previously highlighted the role of Calnuc in the transport of LRP9, a new member of a low-density lipoprotein ( LDL) receptor subfamily that cycles between the TGN and endosomes. The objective of this study was to explore the role of Calnuc in the endocytic sorting of mannose-6-phosphate receptor ( MPR) and Sortilin, two well-characterized lysosomal receptors that transit between the TGN and endosomes. Using biochemical and microscopy assays, we showed that Calnuc depletion [by small interfering RNA ( siRNA)] causes the misdelivery to and degradation in lysosomes of cationic-independent mannose-6-phosphate receptor ( CI-MPR) and Sortilin due to a defect in the endosomal recruitment of retromers, which are key components of the endosome-to-Golgi retrieval machinery. Indeed, we demonstrated that Calnuc depletion impairs the activation and membrane association of Rab7, a small G protein required for the endosomal recruitment of retromers. Overall, our data indicate a novel role for Calnuc in the endosome-to- TGN retrograde transport of lysosomal receptors through the regulation of Rab7 activity and the recruitment of retromers to endosomes. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3,5-bisphosphate ( PtdIns(3,5) P2) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5) P2 using freeze-fracture electron microscopy. GST-ATG18-4× FLAG was used to label PtdIns(3,5) P2 and its binding to phosphatidylinositol 3-phosphate ( PtdIns(3)P) was blocked by an excess of the p40phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5) P2 was concentrated in intramembrane particle ( IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5) P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5) P2 metabolism is defective. In HeLa cells, PtdIns(3,5) P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5) P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5) P2-rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. [ABSTRACT FROM AUTHOR]

Endosomes are transportation nodes, mediating selective transport of soluble and transmembrane cargos to and from the Golgi apparatus, plasma membrane and lysosomes. As endosomes mature to become multivesicular bodies (MVBs), Endosomal Sorting Complexes Required for Transport (ESCRTs) selectively incorporate transmembrane cargos into vesicles that bud into the endosome lumen. Luminal vesicles and their cargoes are targeted for destruction when MVBs fuse with lysosomes Common assays of endosomal luminal targeting, including fluorescence microscopy and monitoring of proteolytic cargo maturation, possess significant limitations. We present a quantitative assay system called LUCID (LUCiferase reporter of Intraluminal Deposition) that monitors exposure of chimeric luciferase-cargo reporters to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal from the chimera depends upon luminal delivery of the reporter rather than proteolytic degradation. The system presents several advantages, including rapidity, microscale operation and a high degree of reproducibility that enables detection of subtle phenotypic differences. Luciferase reporters provide linear signal over an extremely broad dynamic range, allowing analysis of reporter traffic even at anemic levels of expression. Furthermore, LUCID reports transport kinetics when applied to inducible trafficking reporters. [ABSTRACT FROM AUTHOR]

The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) proteins in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans-Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. Here we show that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, we show that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation-independent mannose 6-phosphate receptor. Finally, we find that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells. [ABSTRACT FROM AUTHOR]

Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process. [ABSTRACT FROM AUTHOR]

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs 2D particle tracking, trajectory analysis and fitting of diffusion models to the calculated mean square displacement. It allows for spatial analysis of detected vesicle patterns including calculation of the radial distribution function and particle-based colocalization. Importantly, all analysis tools are supported by Monte Carlo simulations of synthetic images. This allows the user to assess the reliability of the analysis and to study alternative scenarios. We demonstrate the functionality of SpatTrack by performing a detailed imaging study of internalized fluorescence-tagged Niemann Pick C2 ( NPC2) protein in human disease fibroblasts. Using SpatTrack, we show that NPC2 rescued the cholesterol-storage phenotype from a subpopulation of late endosomes/lysosomes ( LE/ LYSs). This was paralleled by repositioning and active transport of NPC2-containing vesicles to the cell surface. The potential of SpatTrack for other applications in intracellular transport studies will be discussed. [ABSTRACT FROM AUTHOR]

Mutations in the large BEACH domain-containing protein LYST causes Chediak-Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome-related organelles in various cell types. Dysfunctional secretion of enlarged lysosome-related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA-depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor ( EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein-3 ( AP-3) and cation-independent mannose-6-phosphate receptor ( CI-MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events. [ABSTRACT FROM AUTHOR]

The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid ( HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1. [ABSTRACT FROM AUTHOR]

The multivesicular body ( MVB) is a specialized Rab7+ late endosome ( LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MVB, but contain orthologs of the ESCRT ( Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin-dependent lysosomal degradation of invariant surface glycoproteins ( ISG65) (Leung et al., Traffic 2008;9:1698-1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes. [ABSTRACT FROM AUTHOR]

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non-mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Fertilization triggers cell remodeling from each gamete to a totipotent zygote. Using Caenorhabditis elegans as a model system, it has been revealed that lysosomal degradation pathways play important roles in cellular remodeling during this developmental transition. Endocytosis and autophagy, two pathways leading to the lysosomes, are highly upregulated during this period. A subset of maternal membrane proteins is selectively endocytosed and degraded in the lysosomes before the first mitotic cell division. Autophagy is also induced shortly after fertilization and executes the degradation of paternally inherited embryonic organelles, e.g. mitochondria and membranous organelles. This mechanism underlies the maternal inheritance of the mitochondrial genome. Autophagy is also required for the removal of extra P-granule (germ granules in C. elegans) components in somatic cells of early embryos and thereby for the specific distribution of P-granules to germ cells. This review focuses on recent advances in the study of the physiological roles and mechanisms of lysosomal pathways during early development in C. elegans. [ABSTRACT FROM AUTHOR]

Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phago-some maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes (TLs), which have been implicated in retention of pinocytic uptake and phagosome matura-tion. Moreover, dendritic cells exposed to lipopolysac-charides (LPSs) convert their punctate class II major histocompatibility complex compartment, a lysosome-related organelle, into a tubular network that is thought to be involved in antigen presentation. Other than a requirement for microtubules and kinesin, little is known about the molecular mechanisms that drive lysosome tubulation. Here, we show that macrophage cell lines readily form TLs after LPS exposure, with a require-ment for the Rab7 GTPase and its effectors RILP (Rab7-/'nteracting /ysosomal protein) and FYC01 (coiled-coil domain-containing protein 7), which respectively modu-late the dynein and kinesin microtubule motor proteins. We also show that Arl8B, a recently identified lysoso-mal GTPase, and its effector SKIP, are also important for TL biogenesis. Finally, we reveal that TLs are sig-nificantly more motile than punctate lysosomes within the same LPS-treated cells. Therefore, we identify the first molecular regulators of lysosome tubulation and we show that TLs represent a more dynamic lysosome population. [ABSTRACT FROM AUTHOR]

Traffic through endosomes and lysosomes is controlled by small G-proteins of the Rab5 and Rab7 families. Like humans, Saccharomyces cerevisiae has three Rab5s (Vps21, Ypt52 and Ypt53) and one Rab7 (Ypt7). Here, we elucidate the functional roles and regulation of the yeast Rab5s. Using GFP-tagged cargoes, a novel quantitative multivesicular body ( MVB) sorting assay, and electron microscopy, we show that MVB biogenesis and thus MVB cargo sorting is severely impaired in vps21Δ ypt52Δ double mutants. Ypt53, the third Rab5 paralog, is hardly expressed during normal growth but its transcription is strongly induced by cellular stress through the calcineurin-Crz1 pathway. The requirement for Rab5 activity in stress tolerance facilitated identification of Msb3/Gyp3 as the principal Rab5 GAP ( GTPase accelerating protein). In vitro GAP assays verified that Vps21 is a preferred Gyp3 target. Moreover, we demonstrate that Gyp3 spatially restricts active Vps21 to intermediate endosomal compartments by preventing Vps21 accumulation on lysosomal vacuoles. Gyp3, therefore, operates as a compartmental insulator that helps to define the spatial domain of Vps21 signaling in the endolysosomal pathway. [ABSTRACT FROM AUTHOR]

The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively little is known about the parasite interactions with the host hepatocyte. Using immunofluorescence, live cell imaging and electron microscopy, we show that Plasmodium berghei parasites are surrounded by vesicles from the host late endocytic pathway. We found that these vesicles are acidic and contain the membrane markers Rab7a, CD63 and LAMP1. When host cell vesicle acidification was disrupted using ammonium chloride or Concanamycin A during the late liver stage of infection, parasite survival was not affected, but schizont size was significantly decreased. Furthermore, when the host cell endocytic pathway was loaded with BSA-gold, gold particles were found within the parasite cytoplasm, showing the transport of material from the host endocytic pathway toward the parasite interior. These observations reveal a novel Plasmodium-host interaction and suggest that vesicles from the host endolysosomal pathway could represent an important source of nutrients exploited by the fast-growing late liver stage parasites. [ABSTRACT FROM AUTHOR]