Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes (original) (raw)
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The delivery of endocytosed cargo to lysosomes
Biochemical Society Transactions, 2009
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 VAMP...
Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum
Journal of Cellular and Molecular Medicine, 2009
Protein molecules destined for the extracellular space are cotranslationally isolated from cytosol by recruitment at the endoplasmic reticulum (ER). In contrast with the reducing environment of the cytoplasm, the ER lumen has an oxidizing milieu which is similar with what the secreted molecule encounters outside the cell [1, 2]. Once translation and quality control processes occurring within the ER lumen are completed, proteins are exported by the secretory pathway which links the ER to the plasma membrane [3, 4]. Although this secretory function of the ER is well documented, little is known about reverse pathways which transport extracellular molecules from the plasma membrane to the ER. These pathways maybe particularly important in the ER stress induced apoptosis, or in the homeostasis of cholesterol, for which receptor molecules are located within the ER [5-7]. ER-mediated phagocytosis is another example of ER targeting from outside the cell that has been recently proposed as an adaptation of intracellular pathogens to avoid destruction in host cells [8]. Living cells require a continuous supply of molecules that are internalized from the environment by multiple endocytic pathways. These routes may lead to lysosomes where the cargo is degraded. However, many toxins, viruses and lipid vesicles such as melanosomes and exosomes are able to escape the cell degradative pathways [9-12]. Lysosomes are bypassed by some endocytosed viruses that escape from endosomes in the cytoplasm through fusion pores generated at low endosomal pH. Remarkable strides have been made recently in tracing some bacterial toxins, but also viruses within the ER of the host cell. Indeed, some toxins, including cholera toxin, find their way to the ER after endocytosis, by taking the retrograde pathway starting from endosomes and passing through the Golgi to the ER [13]. Although some retrograde endocytic pathways are well characterized, the direct routes that transport molecules from the plasma membrane to the ER have only recently started to be deciphered. At least two viruses, simian virus 40 (SV40) and polyomavirus are targeted to the ER of the infected cell for processing and retrotranslocation in the cytoplasm [14, 15]. Interestingly, the internalized viruses are
Nature Cell Biology, 2019
lthough the presence of vesicles around cells in mammalian tissues or fluids was first described in the late 1960s 1,2 , it was not until 2011 that the generic term 'extracellular vesicles' (EVs) was proposed to define all lipid bilayer-enclosed extracellular structures 3 (Fig. 1). EVs can form by outward budding of the plasma membrane or by an intracellular endocytic trafficking pathway involving fusion of multivesicular late endocytic compartments (multivesicular bodies (MVBs)) with the plasma membrane, as described in the 1980s 4,5 (Fig. 2). Such fusion events result in the extracellular release of the intraluminal vesicles (ILVs) of these compartments, generating a subtype of EVs termed 'exosomes' 6 that have the same size as ILVs (< 200 nm in diameter). Exosome secretion was initially proposed as a mechanism through which cells eliminate unnecessary proteins 4,5. However, work in the late 1990s indicated that it could serve an intercellular communication purpose, especially in immune responses and cancer 7,8. Strong support for this concept came in 2007, when exosomes were shown to contain mRNAs and microRNAs that, when transferred to recipient cells, remained functional and changed cellular behaviour 9. Concomitantly, EVs called microvesicles, microparticles or ectosomes, presumably released from the plasma membrane, were also shown to transfer functional proteins and RNA between cells 10,11. EVs of sizes similar to exosomes 12-14 or larger 15-17 have been observed to bud from the plasma membrane of the main cell body or membrane extensions (for example, microvilli, filopodia, cilia and flagella) (Fig. 2) of various cell types. EVs in the size range of exosomes share the same biophysical characteristics in terms of size, density and membrane orientation (Fig. 1) and, therefore, current methods cannot separate them efficiently. Depending on the cell type, environmental conditions and other factors (for example, infection or artificial expression of molecules), the relative proportion of released MVB-derived exosomes versus other small EVs is highly variable and preparations obtained by most current protocols may contain a mixture of small EVs of endosomal (namely, exosomes) and nonendosomal origin 18 , as well as other lipid-based but non-vesicular structures, such as lipoproteins of various densities (intermediate-, high-, low-or very-low-density lipoprotein) 19 (Fig. 1), and the newly identified exomeres 20. Recently 18 , exosomes enriched in late endosome components were defined as a small EV subtype bearing the tetraspanins CD63 (a protein accumulating in MVBs) and CD9/ CD81 (which are mainly at the plasma membrane), although this definition awaits validation in other cells and conditions. Numerous functions have been ascribed to EVs in many pathophysiological situations, including cancer, immune responses, ESCRT. The endosomal sorting complex required for transport (ESCRT) is a family of proteins that associate in successive complexes (ESCRT-0,-I,-II and-III) at the membrane of MVBs to regulate cargo targeting into and the formation of ILVs 23. The presence of tumour susceptibility gene 101 protein (TSG101) and other ESCRT proteins or ESCRT-accessory molecules, for example, ALIX (encoded by PDCD6IP) and vacuolar protein sorting-associated protein 4 (VPS4), in the preparations of small EVs has often been offered as proof of their MVB origin. However, ESCRT factors are involved in various budding and membrane scission events at the plasma membrane, all of which require ESCRT-I/II/III and accessory molecules 23 , leading to EV release (see examples in refs. 24,25). ESCRT-0 components are generally not described in plasma membrane budding and release models. Thus, ESCRT-0 dependence of EV secretion could be a means to demonstrate their MVB origin. Accordingly, depletion of the ESCRT-0 components HRS (encoded by HGS) or signal transducing adapter molecule 1 (STAM1) in HeLa cells 26 decreases the secretion of small EVs bearing CD63 and major histocompatibility complex-II/CD81, which correspond to exosomes according to ref. 18. However, such approaches may induce
Defining endocytic pathways tocharacterise the cellular uptake ofextracellular vesicles
2017
There is a need for vectors that, with high efficiency, can deliver small and macromolecular therapeutics into cells and defined intracellular locations. Extracellular vesicles (EVs), including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they Firstly I would like to thank my principle supervisor Prof. Arwyn Jones for all of his support, guidance and understanding throughout my time in the lab and throughout the project. Special thanks for the time and patience always found when in need. Many thanks also to my co-supervisor Dr. Pete Watson and other members of the Watson group whose expertise and rigor helped to constantly develop the project from start to finish.
The Journal of Cell Biology, 2016
Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell inte...
The Delivery of Endocytosed Material from Late Endosomes to Lysosomes
2000
Lysosomes are membrane-bound organelles containing many hydrolytic enzymes, which are optimally active at an acid pH. They are distinguished from endosomes by the absence of the two mannose-6-phosphate receptors (MPRs) and recycling cell surface receptors. They are characteristically observed by electron microscopy to be organelles of ~0.5 μm diameter and often have electron-dense cores (Holtzmann, 1989). Lysosomes are usually regarded as the terminal degradation compartment of the endocytic pathway (Kornfeld and Mellman, 1989), and play important roles in the degradation of phagocytosed material (Funato et al., 1997), in autophagy (Lawrence and Brown, 1992), in crinophagy (Noda and Farquhar, 1992) and in the proteolysis of cytosolic proteins transported across the lysosomal membrane by a carriermediated mechanism (Cuervo and Dice, 1996). However, the view that lysosomes are simply a ‘garbage-disposal unit’ has been challenged, both by a better understanding of how endocytosed mater...
Cell, 1983
We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.