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Cell Reports Methods Feb 2023We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from . These recombinant vesicles compartmentalize proteins within...
We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from . These recombinant vesicles compartmentalize proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.
Topics: Escherichia coli; Recombinant Proteins; Biotechnology; Escherichia coli Proteins
PubMed: 36936078
DOI: 10.1016/j.crmeth.2023.100396 -
Nature Reviews. Molecular Cell Biology Oct 2020The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment... (Review)
Review
The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.
Topics: Animals; Biological Transport; Cell Communication; Extracellular Vesicles; Humans; Mammals; RNA
PubMed: 32457507
DOI: 10.1038/s41580-020-0251-y -
Cell Stem Cell Oct 2021During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human...
During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60 days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.
Topics: Cell Differentiation; Embryonic Development; Humans; Induced Pluripotent Stem Cells; Organogenesis; Organoids; Prosencephalon
PubMed: 34407456
DOI: 10.1016/j.stem.2021.07.010 -
Journal of Extracellular Vesicles Nov 2021The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates...
The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07-1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.
Topics: Cell Line, Tumor; Chromatography, Liquid; Endoplasmic Reticulum; Extracellular Vesicles; Golgi Apparatus; Humans; Membrane Proteins; Mitochondria; Particle Size; Protein Transport; Proteome; Proteomics; Signal Transduction; Tandem Mass Spectrometry
PubMed: 34817906
DOI: 10.1002/jev2.12164 -
International Journal of Molecular... Jun 2023Extensive evidence indicates that the activation of the P2X receptor (P2XR), an ATP-gated ion channel highly expressed in immune and brain cells, is strictly associated... (Review)
Review
Extensive evidence indicates that the activation of the P2X receptor (P2XR), an ATP-gated ion channel highly expressed in immune and brain cells, is strictly associated with the release of extracellular vesicles. Through this process, P2XR-expressing cells regulate non-classical protein secretion and transfer bioactive components to other cells, including misfolded proteins, participating in inflammatory and neurodegenerative diseases. In this review, we summarize and discuss the studies addressing the impact of P2XR activation on extracellular vesicle release and their activities.
Topics: Brain; Extracellular Vesicles; Receptors, Purinergic P2X7; Adenosine Triphosphate
PubMed: 37372953
DOI: 10.3390/ijms24129805 -
Journal of Extracellular Vesicles Aug 2021Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs...
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63, CD81, or CD9 EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.
Topics: Biological Transport; Cell Line; Drug Delivery Systems; Exosomes; Extracellular Vesicles; Genetic Engineering; Green Fluorescent Proteins; Humans; Nanotechnology; Protein Transport; Recombinant Fusion Proteins; Tetraspanins; Workflow
PubMed: 34377376
DOI: 10.1002/jev2.12130 -
Current Opinion in Structural Biology Aug 2022Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components... (Review)
Review
Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.
Topics: Cell Membrane; Clathrin; Clathrin-Coated Vesicles; Coated Vesicles; Cryoelectron Microscopy; Endocytosis; Saccharomyces cerevisiae
PubMed: 35872561
DOI: 10.1016/j.sbi.2022.102427 -
Journal of Controlled Release :... Sep 2023Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in... (Review)
Review
Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in formulation due to their lack of immunogenicity and targeting capabilities. EVs have been shown to have similar regenerative and therapeutic effects to their parental cells and also have potential in disease diagnosis. To improve the therapeutic potential of EVs, researchers have developed various strategies for modifying them, including genetic engineering and chemical modifications which have been examined to confer target specificity and prevent rapid clearance after systematic injection. Formulation efforts have focused on utilising hydrogel and nano-formulation strategies to increase the persistence of EV localisation in a specific tissue or organ. Researchers have also used biomaterials or bioscaffolds to deliver EVs directly to disease sites and prolong EV release and exposure. This review provides an in-depth examination of the material design of EV delivery systems, highlighting the impact of the material properties on the molecular interactions and the maintenance of EV stability and function. The various characteristics of materials designed to regulate the stability, release rate and biodistribution of EVs are described. Other aspects of material design, including modification methods to improve the targeting of EVs, are also discussed. This review aims to offer an understanding of the strategies for designing EV delivery systems, and how they can be formulated to make the transition from laboratory research to clinical use.
Topics: Tissue Distribution; Extracellular Vesicles
PubMed: 37536545
DOI: 10.1016/j.jconrel.2023.07.059 -
Nature Reviews. Materials Jun 2023The extracellular matrix in microenvironments harbors a variety of signals to control cellular functions and the materiality of tissues. Most efforts to synthetically...
The extracellular matrix in microenvironments harbors a variety of signals to control cellular functions and the materiality of tissues. Most efforts to synthetically reconstitute the matrix by biomaterial design have focused on decoupling cell-secreted and polymer-based cues. Cells package molecules into nanoscale lipid membrane-bound extracellular vesicles and secrete them. Thus, extracellular vesicles inherently interact with the meshwork of the extracellular matrix. In this Review, we discuss various aspects of extracellular vesicle-matrix interactions. Cells receive feedback from the extracellular matrix and leverage intracellular processes to control the biogenesis of extracellular vesicles. Once secreted, various biomolecular and biophysical factors determine whether extracellular vesicles are locally incorporated into the matrix or transported out of the matrix to be taken up by other cells or deposited into tissues at a distal location. These insights can be utilized to develop engineered biomaterials where EV release and retention can be precisely controlled in host tissue to elicit various biological and therapeutic outcomes.
PubMed: 38463907
DOI: 10.1038/s41578-023-00551-3 -
Cells Jun 2020Cellular secretion depends on exocytosis of secretory vesicles and discharge of vesicle contents. Actin and myosin are essential for pre-fusion and post-fusion stages of... (Review)
Review
Cellular secretion depends on exocytosis of secretory vesicles and discharge of vesicle contents. Actin and myosin are essential for pre-fusion and post-fusion stages of exocytosis. Secretory vesicles depend on actin for transport to and attachment at the cell cortex during the pre-fusion phase. Actin coats on fused vesicles contribute to stabilization of large vesicles, active vesicle contraction and/or retrieval of excess membrane during the post-fusion phase. Myosin molecular motors complement the role of actin. Myosin V is required for vesicle trafficking and attachment to cortical actin. Myosin I and II members engage in local remodeling of cortical actin to allow vesicles to get access to the plasma membrane for membrane fusion. Myosins stabilize open fusion pores and contribute to anchoring and contraction of actin coats to facilitate vesicle content release. Actin and myosin function in secretion is regulated by a plethora of interacting regulatory lipids and proteins. Some of these processes have been first described in non-neuronal cells and reflect adaptations to exocytosis of large secretory vesicles and/or secretion of bulky vesicle cargoes. Here we collate the current knowledge and highlight the role of actomyosin during distinct phases of exocytosis in an attempt to identify unifying molecular mechanisms in non-neuronal secretory cells.
Topics: Actin Cytoskeleton; Actins; Animals; Exocytosis; Humans; Membrane Fusion; Myosins; Secretory Vesicles
PubMed: 32545391
DOI: 10.3390/cells9061455