Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Zaborowski, Mikołaj; Cheah, Pike See; Zhang, Xuan; Bushko, Isabella; Lee, Kyungheon; Sammarco, Alessandro; Zappulli, Valentina; Maas, Sybren L. N.; Allen, Ryan M.; Rumde, Purva; György, Bence; Aufiero, Massimo; Schweiger, Markus; Lai, Charles P.; Weissleder, Ralph; Lee, Hakho; Vickers, Kasey C.; Tannous, Bakhos A.; Breakefield, Xandra O.

doi:10.1038/s41598-019-53554-y

Cited by 20 publications

(25 citation statements)

References 41 publications

(64 reference statements)

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“…The palmitoylation of both cysteines in this sequence at the endoplasmic reticulum-Golgi intermediate compartment drives GAP43 protein to the inner surface of the plasma membrane of growing axons (GAP43 is reviewed in [ 34 ]). Another option for the anchoring of a reporter in the EVs membrane could be the use of a heterologous transmembrane domain, as in the Gaussia luciferase construct reported by [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles

et al. 2020

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Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

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Section: Discussionmentioning

confidence: 99%

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles

et al. 2020

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“…Subsequent studies showed that membranebound Gluc may undergo proteolytic cleavage, leading to the release of protein fragments into the extracellular space in an active form. [147] Following this observation, we developed a novel membrane-bound Gluc based assay to quantitatively track the shedding of membrane proteins from EVs in vitro and in vivo. [147] Furthermore, we utilized this assay to demonstrate that ectodomain shedding in EVs is continuous and mediated by proteases.…”

Section: Indirect Tracking Methodsmentioning

confidence: 99%

“…[147] Following this observation, we developed a novel membrane-bound Gluc based assay to quantitatively track the shedding of membrane proteins from EVs in vitro and in vivo. [147] Furthermore, we utilized this assay to demonstrate that ectodomain shedding in EVs is continuous and mediated by proteases. [147] Takahashi et al took another approach and used lacadherin, a membrane-associated protein often found in EVs, and designed a fusion protein between Gluc and a truncated form of this protein, which labeled cancer cells and their secreted EVs, allowing their in vivo visualization.…”

Section: Indirect Tracking Methodsmentioning

confidence: 99%

“…[147] Furthermore, we utilized this assay to demonstrate that ectodomain shedding in EVs is continuous and mediated by proteases. [147] Takahashi et al took another approach and used lacadherin, a membrane-associated protein often found in EVs, and designed a fusion protein between Gluc and a truncated form of this protein, which labeled cancer cells and their secreted EVs, allowing their in vivo visualization. [148] To monitor EV uptake at the cellular level, fluorescent-based techniques were developed by several groups.…”

Section: Indirect Tracking Methodsmentioning

confidence: 99%

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Small but Fierce: Tracking the Role of Extracellular Vesicles in Glioblastoma Progression and Therapeutic Resistance

Schweiger

Tannous

2020

Adv. Biosys.

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(30%), mouse double minute 2 (MDM2) amplifications (<10%) and/ or overexpression (50%), p16INK4a deletions (30-40%), and loss of heterozygosity on chromosome 10 in 50-80% of all cases. [3] Around 5-10% of all GBMs harbor isocitrate dehydrogenase (IDH) mutations with a better prognosis compared to tumors carrying the wildtype form known to exhibit a much more aggressive clinical behavior, particularly in adults. [4,5] It is now appreciated that IDH-wildtype and IDH-mutant gliomas represent distinct clinical and genetic entities, and efforts have been made to restrict the term "glioblastoma" to tumors without IDH mutations. [6,7] The standard-of-care treatment consists of maximal surgical resection of the tumor in combination with radiation and temozolomide (TMZ) chemotherapy. However, GBM is insidious and treatments have not been effective in preventing eventual disease progression as recurrence remains inevitable. [8-11] Transcriptomic, genomic, and epigenomic characterization have unveiled the highly heterogeneous nature of GBM with complex interactions among different cells within as well as cells surrounding the tumor. [3,12-17] Comprehensive longitudinal analyses of the tumor transcriptome have uncovered intricate intertumoral heterogeneity and classified GBM into three distinct subtypes: classical (CL), proneural (PN), and mesenchymal (MES). [18] In addition to this intertumoral heterogeneity, single-cell RNA sequencing demonstrated the presence of numerous transcriptome subtypes within the same tumor. [14] Further, single-cell lineage tracking and expression analysis of specimens from The Cancer Genome Atlas (TCGA) revealed that malignant cells in GBM exist in four main cellular states: neural-progenitor-like, oligodendrocyte-progenitor-like, astrocyte-like, and mesenchymal-like. [19] The number and frequency of cells in each defined state varies between patients and is influenced by mutations in the neurofibromin 1 (NF1) locus and copy number amplifications of EGFR, cyclin dependent kinase 4 (CDK4), and platelet-derived growth factor receptor alpha (PDGFRA) loci. The intricate combination of inter-as well as intratumoral heterogeneity plays a key role in rendering conventional and experimental targeted therapy approaches ultimately ineffective. [20-22] The majority of glioma cells lack the capacity to recapitulate a phenocopy of the original tumor and only a small subpopulation of neural stem-like cells within the tumor, called glioma stem cells (GSCs) or tumor initiating cells, have that ability upon xenotransplantation in immunocompromised mice. [23-25] Evidence suggests that these GSCs act as a driving Glioblastoma is the most common and aggressive brain tumor in adults. Most patients die within a year and long-term survival remains rare, owing to a combination of rapid progression/degeneration, lack of successful treatments, and high recurrence rates. Extracellular vesicles are cell-derived membranous structures involved in numerous physiological and pathological processes. In the context of cancer, th...

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“…Many methods have been developed for EV preparation, including ultracentrifugation (UC) method [21], filter centrifugation (FC), size exclusion chromatography (SEC) [22,23], affinity purification (AP), immunoprecipitation (IP), sucrose cushion ultracentrifugation (SCU) [24], density gradient centrifugation (DGC), and polymer-based precipitation (PBP) method aka pellet-down method [25]. The SEC method is based on the differential elution profiles of particles of different sizes running through a porous polymer (gel filtration matrix).…”

Section: Introductionmentioning

confidence: 99%

Macrophage-Derived Small Exosomes: Efficient Transmission and Cytotoxicity to Cancer Cells

Lu¹,

Eguchi²,

Sogawa³

et al. 2021

Preprint

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Tumor-associated macrophages are a key component in the tumor microenvironment, secreting extracellular vesicles (EVs) such as exosomes and other various factors for intercellular communication. However, macrophage-derived EVs heterogeneity and their cytotoxicity to cancer cells has not been well understood. Here, we aimed to separately isolate various types of macro-phage-EVs by size exclusion chromatography (SEC) method and investigate EV transmission and cytotoxicity to oral cancer cells. For fluorescence-labeling of cellular and EV membranes, palmitoylation signal-fused GFP and tdTomato were expressed in THP-1 monocytic cells and HSC-3 oral cancer cells, respectively. We found that fluorescence-labeled EVs secreted by macrophages were highly transmissive to oral cancer cells than those from parental monocytic cells. In a co-culture system and conditioned medium (CM), a macrophage-secreted unidentified factor was cytotoxic to oral cancer cells. We fractionated macrophage-derived EVs by the SEC method and performed western blotting to characterize various EV types. Three fractions were characterized: small exosomes (EXO-S: < 50 nm) fraction containing HSP90α, HSP90β, CD63 (EV marker) and β-actin; large exosomes (EXO-L: 50-200 nm) fraction containing CD9 (EV marker) and HSP90β; large EVs (100-500 nm) fraction. Notably, the macrophage-derived small exosomes fraction was cytotoxic to oral cancer cells, while large exosomes and large EVs were not. There-fore, it was implicated that macrophage-derived small exosomes are cytotoxic with high trans-mission potential to cancer cells.

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Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Cited by 20 publications

References 41 publications

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles

Small but Fierce: Tracking the Role of Extracellular Vesicles in Glioblastoma Progression and Therapeutic Resistance

Macrophage-Derived Small Exosomes: Efficient Transmission and Cytotoxicity to Cancer Cells

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