scholarly journals Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

2018 ◽  
Vol 217 (3) ◽  
pp. 1129-1142 ◽  
Author(s):  
Frederik Johannes Verweij ◽  
Maarten P. Bebelman ◽  
Connie R. Jimenez ◽  
Juan J. Garcia-Vallejo ◽  
Hans Janssen ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Single Cells ◽  
Cell Communication ◽  
Exosome Biogenesis ◽  
Real Time Visualization ◽  
Target Membrane ◽  
Syntaxin 4 ◽  
Optical Reporters ◽  
Druggable Targets ◽  
Insight Into

Exosomes are small endosome-derived extracellular vesicles implicated in cell–cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB–PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB–PM fusion using live total internal reflection fluorescence and dynamic correlative light–electron microscopy. Quantitative analysis demonstrates that MVB–PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB–PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

scholarly journals Multiplexed Profiling of Single-cell Extracellular Vesicles Secretion

2018 ◽  
Author(s):  
Yahui Ji ◽  
Dongyuan Qi ◽  
Linmei Li ◽  
Haoran Su ◽  
Xiaojie Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Single Cells ◽  
Cell Communication ◽  
Deep Understanding ◽  
Surface Markers ◽  
Cell Heterogeneity ◽  
Health And Disease ◽  
Cytokines Secretion

AbstractExtracellular vesicles (EVs) are important intercellular mediators regulating health and disease. Conventional EVs surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EVs secretion. Herein, by using spatially patterned antibodies barcode, we realized multiplexed profiling of single-cell EVs secretion from more than 1000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to deep understanding of previously undifferentiated single cell heterogeneity underlying EVs secretion. Notably, we observed the decrement of certain EV phenotypes (e.g. CD63+EVs) were associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EVs secretion and cytokines secretion simultaneously from the same single cells to investigate multidimensional spectrum of intercellular communications, from which we resolved three functional subgroups with distinct secretion profiles by visualized clustering. In particular, we found EVs secretion and cytokines secretion were generally dominated by different cell subgroups. The technology introduced here enables comprehensive evaluation of EVs secretion heterogeneity at single cell level, which may become an indispensable tool to complement current single cell analysis and EV research.SignificanceExtracellular vesicles (EVs) are cell derived nano-sized particles medicating cell-cell communication and transferring biology information molecules like nucleic acids to regulate human health and disease. Conventional methods for EV surface markers profiling can’t tell the differences in the quantity and phenotypes of EVs secretion between cells. To address this need, we developed a platform for profiling an array of surface markers on EVs from large numbers of single cells, enabling more comprehensive monitoring of cellular communications. Single cell EVs secretion assay led to previously unobserved cell heterogeneity underlying EVs secretion, which might open up new avenues for studying cell communication and cell microenvironment in both basic and clinical research.

scholarly journals Controlled Construction of Stable Network Structure Composed of Honeycomb-Shaped Microhydrogels

Life ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 38 ◽  
Author(s):  
Masayuki Hayakawa ◽  
Satoshi Umeyama ◽  
Ken Nagai ◽  
Hiroaki Onoe ◽  
Masahiro Takinoue
Keyword(s):  
Cell Communication ◽  
Model Systems ◽  
Stable Component ◽  
Photosensitive Polymer ◽  
A Cell ◽  
External Stimuli ◽  
Structural Instabilities ◽  
Insight Into ◽  
Stable Network ◽  
Microfluidic Technique

Recently, the construction of models for multicellular systems such as tissues has been attracting great interest. These model systems are expected to reproduce a cell communication network and provide insight into complicated functions in living systems./Such network structures have mainly been modelled using a droplet and a vesicle. However, in the droplet and vesicle network, there are difficulties attributed to structural instabilities due to external stimuli and perturbations. Thus, the fabrication of a network composed of a stable component such as hydrogel is desired. In this article, the construction of a stable network composed of honeycomb-shaped microhydrogels is described. We produced the microhydrogel network using a centrifugal microfluidic technique and a photosensitive polymer. In the network, densely packed honeycomb-shaped microhydrogels were observed. Additionally, we successfully controlled the degree of packing of microhydrogels in the network by changing the centrifugal force. We believe that our stable network will contribute to the study of cell communication in multicellular systems.

scholarly journals Engineering synthetic morphogen systems that can program multicellular patterning

Science ◽  
2020 ◽  
Vol 370 (6514) ◽  
pp. 327-331 ◽  
Author(s):  
Satoshi Toda ◽  
Wesley L. McKeithan ◽  
Teemu J. Hakkinen ◽  
Pilar Lopez ◽  
Ophir D. Klein ◽  
...  
Keyword(s):  
Communication Systems ◽  
Fluorescent Protein ◽  
Cell Communication ◽  
Positional Information ◽  
Surface Anchoring ◽  
Localized Source ◽  
Anchoring Proteins ◽  
Green Fluorescent ◽  
Insight Into

In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, we asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.

scholarly journals Single-cell transcriptomics allows novel insights into aging and circadian processes

2020 ◽  
Vol 19 (5-6) ◽  
pp. 343-349
Author(s):  
Sara S Fonseca Costa ◽  
Marc Robinson-Rechavi ◽  
Jürgen A Ripperger
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Cellular Level ◽  
Biological Processes ◽  
New Methods ◽  
New Findings ◽  
Single Cell Rna Sequencing ◽  
Aging And Circadian Rhythms ◽  
Insight Into ◽  
Different Time Scales

Abstract Aging and circadian rhythms are two biological processes that affect an organism, although at different time scales. Nevertheless, due to the overlap of their actions, it was speculated that both interfere or interact with each other. However, to address this question, a much deeper insight into these processes is necessary, especially at the cellular level. New methods such as single-cell RNA-sequencing (scRNA-Seq) have the potential to close this gap in our knowledge. In this review, we analyze applications of scRNA-Seq from the aging and circadian rhythm fields and highlight new findings emerging from the analysis of single cells, especially in humans or rodents. Furthermore, we judge the potential of scRNA-Seq to identify common traits of both processes. Overall, this method offers several advantages over more traditional methods analyzing gene expression and will become an important tool to unravel the link between these biological processes.

scholarly journals Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 624 ◽  
Author(s):  
Smita Mohanty ◽  
Bharat P Chaudhary ◽  
David Zoetewey
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Modification ◽  
Cell Communication ◽  
Reticulum Cell ◽  
Enzyme Complex ◽  
Single Polypeptide Chain ◽  
Domains Of Life ◽  
And Function ◽  
Insight Into

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

scholarly journals Polyamine catabolism and disease

2009 ◽  
Vol 421 (3) ◽  
pp. 323-338 ◽  
Author(s):  
Robert A. Casero ◽  
Anthony E. Pegg
Keyword(s):  
Dominant Role ◽  
Therapeutic Benefit ◽  
Spermine Oxidase ◽  
Polyamine Catabolism ◽  
Disease States ◽  
Disease Aetiology ◽  
Multiple Cell ◽  
Druggable Targets ◽  
Acetylpolyamine Oxidase ◽  
Insight Into

In addition to polyamine homoeostasis, it has become increasingly clear that polyamine catabolism can play a dominant role in drug response, apoptosis and the response to stressful stimuli, and contribute to the aetiology of several pathological states, including cancer. The highly inducible enzymes SSAT (spermidine/spermine N1-acetyltransferase) and SMO (spermine oxidase) and the generally constitutively expressed APAO (N1-acetylpolyamine oxidase) appear to play critical roles in many normal and disease processes. The dysregulation of polyamine catabolism frequently accompanies several disease states and suggests that such dysregulation may both provide useful insight into disease mechanism and provide unique druggable targets that can be exploited for therapeutic benefit. Each of these enzymes has the potential to alter polyamine homoeostasis in response to multiple cell signals and the two oxidases produce the reactive oxygen species H2O2 and aldehydes, each with the potential to produce pathological states. The activity of SSAT provides substrates for APAO or substrates for the polyamine exporter, thus reducing the intracellular polyamine concentration, the net effect of which depends on the magnitude and rate of any increase in SSAT. SSAT may also influence cellular metabolism via interaction with other proteins and by perturbing the content of acetyl-CoA and ATP. The goal of the present review is to cover those aspects of polyamine catabolism that have an impact on disease aetiology or treatment and to provide a solid background in this ever more exciting aspect of polyamine biology.

scholarly journals Osteoclast Activity and Subtypes as a Function of Physiology and Pathology—Implications for Future Treatments of Osteoporosis

2011 ◽  
Vol 32 (1) ◽  
pp. 31-63 ◽  
Author(s):  
K. Henriksen ◽  
J. Bollerslev ◽  
V. Everts ◽  
M. A. Karsdal
Keyword(s):  
Bone Resorption ◽  
Bone Quality ◽  
Cell Communication ◽  
Bone Microenvironment ◽  
Cell Type ◽  
Drug Induced ◽  
Current Review ◽  
Heterogeneous Cell Population ◽  
Future Treatments ◽  
Insight Into

Abstract Osteoclasts have traditionally been associated exclusively with catabolic functions that are a prerequisite for bone resorption. However, emerging data suggest that osteoclasts also carry out functions that are important for optimal bone formation and bone quality. Moreover, recent findings indicate that osteoclasts have different subtypes depending on their location, genotype, and possibly in response to drug intervention. The aim of the current review is to describe the subtypes of osteoclasts in four different settings: 1) physiological, in relation to turnover of different bone types; 2) pathological, as exemplified by monogenomic disorders; 3) pathological, as identified by different disorders; and 4) in drug-induced situations. The profiles of these subtypes strongly suggest that these osteoclasts belong to a heterogeneous cell population, namely, a diverse macrophage-associated cell type with bone catabolic and anabolic functions that are dependent on both local and systemic parameters. Further insight into these osteoclast subtypes may be important for understanding cell–cell communication in the bone microenvironment, treatment effects, and ultimately bone quality.

scholarly journals Caught in the Act: Mechanistic Insight into Supramolecular Polymerization-Driven Self-Replication from Real-Time Visualization

2020 ◽  
Vol 142 (32) ◽  
pp. 13709-13717 ◽  
Author(s):  
Sourav Maity ◽  
Jim Ottelé ◽  
Guillermo Monreal Santiago ◽  
Pim W. J. M. Frederix ◽  
Peter Kroon ◽  
...  
Keyword(s):  
Real Time ◽  
Mechanistic Insight ◽  
Real Time Visualization ◽  
Self Replication ◽  
Supramolecular Polymerization ◽  
Insight Into
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