scholarly journals A novel Microproteomic Approach Using Laser Capture Microdissection to Study Cellular Protrusions

2019 ◽  
Vol 20 (5) ◽  
pp. 1172 ◽  
Author(s):  
Karine Gousset ◽  
Ana Gordon ◽  
Shravan Kumar Kannan ◽  
Joey Tovar
Keyword(s):  
Laser Capture Microdissection ◽  
Protein Extraction ◽  
Cell Communication ◽  
Protein Composition ◽  
Fluorescent Markers ◽  
Different Types ◽  
Health And Disease ◽  
Multicellular Organisms ◽  
First Time ◽  
Laser Capture

Cell–cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their specific functions have not been elucidated due to the lack of a method for their specific isolation and analysis. In this paper, we described, for the first time, a method to specifically isolate distinct protrusion subtypes, based on their morphological structures or fluorescent markers, using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrate that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Our method confirmed that different types of protrusions have distinct proteomes and it promises to advance the characterization and the understanding of these unique structures to shed light on their possible role in health and disease.

scholarly journals A System of Cytokines Encapsulated in ExtraCellular Vesicles

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Wendy Fitzgerald ◽  
Michael L. Freeman ◽  
Michael M. Lederman ◽  
Elena Vasilieva ◽  
Roberto Romero ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Biological Effects ◽  
Cell Communication ◽  
Health And Disease ◽  
Mediate Cell ◽  
Multicellular Organisms ◽  
Cell Cell

Abstract Cytokines are soluble factors that mediate cell–cell communications in multicellular organisms. Recently, another system of cell–cell communication was discovered, which is mediated by extracellular vesicles (EVs). Here, we demonstrate that these two systems are not strictly separated, as many cytokines in vitro, ex vivo, and in vivo are released in EV-encapsulated forms and are capable of eliciting biological effects upon contact with sensitive cells. Association with EVs is not necessarily a property of a particular cytokine but rather of a biological system and can be changed upon system activation. EV-encapsulated cytokines were not detected by standard cytokine assays. Deciphering the regulatory mechanisms of EV-encapsulation will lead to a better understanding of cell–cell communications in health and disease.

Exploring the extracellular matrix in health and disease using proteomics

2019 ◽  
Vol 63 (3) ◽  
pp. 417-432 ◽  
Author(s):  
Isra N. Taha ◽  
Alexandra Naba
Keyword(s):  
Extracellular Matrix ◽  
Protein Production ◽  
Protein Composition ◽  
Protein Assembly ◽  
Mechanical Support ◽  
Cellular Processes ◽  
Potential Reservoir ◽  
Health And Disease ◽  
Multicellular Organisms ◽  
Biochemical Signals

Abstract The extracellular matrix (ECM) is a complex assembly of hundreds of proteins that constitutes the scaffold of multicellular organisms. In addition to providing architectural and mechanical support to the surrounding cells, it conveys biochemical signals that regulate cellular processes including proliferation and survival, fate determination, and cell migration. Defects in ECM protein assembly, decreased ECM protein production or, on the contrary, excessive ECM accumulation, have been linked to many pathologies including cardiovascular and skeletal diseases, cancers, and fibrosis. The ECM thus represents a potential reservoir of prognostic biomarkers and therapeutic targets. However, our understanding of the global protein composition of the ECM and how it changes during pathological processes has remained limited until recently. In this mini-review, we provide an overview of the latest methodological advances in sample preparation and mass spectrometry-based proteomics that have permitted the profiling of the ECM of now dozens of normal and diseased tissues, including tumors and fibrotic lesions.

scholarly journals A subcellular cookie cutter for spatial genomics in human tissue

2021 ◽  
Author(s):  
Alexander Bury ◽  
Angela Pyle ◽  
Fabio Marcuccio ◽  
Doug Turnbull ◽  
Amy Vincent ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
Human Tissue ◽  
The State ◽  
Precise Location ◽  
Cellular Physiology ◽  
First Time ◽  
Laser Capture ◽  
State Of Art ◽  
Biopsy Technology

Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state of art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of (>20 micron) for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

scholarly journals Dietary Bovine Milk Exosomes Elicit Changes in Microbial Communities in C57BL/6 Mice

2018 ◽  
Author(s):  
Fang Zhou ◽  
Henry A. Paz ◽  
Jiang Shu ◽  
Mahrou Sadri ◽  
Juan Cui ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Gut Microbiome ◽  
Bovine Milk ◽  
Cell Communication ◽  
Intestinal Microbiome ◽  
Operational Taxonomic Units ◽  
Health And Disease ◽  
Cecum Content ◽  
First Time ◽  
Bovine Species

ABSTRACTExosomes and exosome-like vesicles participate in cell-to-cell communication in animals, plant and bacteria. Dietary exosomes in bovine milk are bioavailable in non-bovine species, but a fraction of milk exosomes reaches the large intestine. We hypothesized that milk exosomes alter the composition of the gut microbiome in mice. C57BL/6 mice were fed AIN-93G diets, defined by their content of bovine milk exosomes and RNA cargos: exosome/RNA depleted (ERD) versus exosome/RNA-sufficient (ERS) diets. Feeding was initiated at age three weeks and cecum content was collected at ages 7, 15 and 47 weeks. Microbial communities were identified by 16S rRNA gene sequencing. The dietary intake of exosomes and age had significant effects on the microbial communities in the cecum. At the phylum level, the abundance of Verrucomicrobia was greater in mice fed ERD compared to ERS, and the abundance of both Firmicutes and Tenericutes was smaller in mice fed ERD compared to ERS at age 47 weeks. At the family level, the abundance of Anaeroplasmataceae was greater in mice fed ERD compared to ERS, and the abundance of Bifidobacteriaceae, Lachnospiraceae, and Dehalobacteriaceae was significantly greater in mice fed ERS than mice fed ERD at age 15 weeks. Exosome feeding significantly altered the abundance of 52 operational taxonomic units; diet effects were particularly strong in the Lachnospiraceae, Ruminococcaceae and the Verrucomicrobiaceae families. We conclude that exosomes in bovine milk alter microbial communities in non-bovine species, suggesting that exosomes and their cargos participate in the crosstalk between bacterial and animal kingdoms.IMPORTANCEVirtually all living cells, including bacteria communicate through exosomes, which can be found in all body fluids. Exosomes and the RNA cargos have been implicated in all aspects of health and disease, e.g., metastasis of cancer, neuronal signaling and embryonic development. Previously, we reported that exosomes and their microRNA cargos are not solely derived from endogenous synthesis, but may also be obtained from dietary sources such as bovine milk in non-bovine mammals. Here, we report for the first time that bovine milk exosomes communicate with the intestinal microbiome and alters microbial communities in mice. This is the first report suggesting that the gut microbiome facilitates the signaling by dietary exosomes across kingdoms: animal (cow) → bacteria → animal (mouse).

scholarly journals Mapping Genomic Scaffolds to Chromosomes Using Laser Capture Microdissection in Application to Hawaiian Picture-Winged Drosophila

2017 ◽  
Vol 152 (4) ◽  
pp. 204-212 ◽  
Author(s):  
Lin Kang ◽  
Phillip George ◽  
Donald K. Price ◽  
Igor Sharakhov ◽  
Pawel Michalak
Keyword(s):  
Laser Capture Microdissection ◽  
De Novo ◽  
Chromosome Mapping ◽  
Drosophila Species ◽  
Sequencing Technologies ◽  
Physical Maps ◽  
New Information ◽  
Genome Assemblies ◽  
First Time ◽  
Laser Capture

Next-generation sequencing technologies have led to a decreased cost and an increased throughput in genome sequencing. Yet, many genome assemblies based on short sequencing reads have been assembled only to the scaffold level due to the lack of sufficient chromosome mapping information. Traditional ways of mapping scaffolds to chromosomes require a large amount of laboratory work and time to generate genetic and/or physical maps. To address this problem, we conducted a rapid technique which uses laser capture microdissection and enables mapping scaffolds of de novo genome assemblies directly to chromosomes in Hawaiian picture-winged Drosophila. We isolated and sequenced intact chromosome arms from larvae of D. differens. By mapping the reads of each chromosome to the recently assembled scaffolds from 3 Hawaiian picture-winged Drosophila species, at least 67% of the scaffolds were successfully assigned to chromosome arms. Even though the scaffolds are not ordered within a chromosome, the fast-generated chromosome information allows for chromosome-related analyses after genome assembling. We utilize this new information to test the faster-X evolution effect for the first time in these Hawaiian picture-winged Drosophila species.

scholarly journals Connexins: Key Mediators of Endocrine Function

2011 ◽  
Vol 91 (4) ◽  
pp. 1393-1445 ◽  
Author(s):  
Domenico Bosco ◽  
Jacques-Antoine Haefliger ◽  
Paolo Meda
Keyword(s):  
Cell Communication ◽  
Endocrine Function ◽  
Kidney Cortex ◽  
Cell Contact ◽  
Signal Molecules ◽  
Cell Coupling ◽  
Β Cells ◽  
Extracellular Release ◽  
Different Types ◽  
Multicellular Organisms

The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane “hemi-channels,” for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing β-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

scholarly journals Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

2019 ◽  
Author(s):  
Sualyneth Galarza ◽  
Alfred J. Crosby ◽  
ChangHui Pak ◽  
Shelly R. Peyton
Keyword(s):  
Physiological Role ◽  
Protein Composition ◽  
Culture Conditions ◽  
Quiescent State ◽  
Dependent Manner ◽  
Synthetic Hydrogel ◽  
Health And Disease ◽  
And Control ◽  
First Time

Bioengineers designed numerous instructive brain extracellular matrix (ECM) environments that have tailored and tunable protein composition and biomechanics in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under “normal” culture conditions. Here we introduce a synthetic hydrogel, that for the first time demonstrates maintenance of astrocyte quiescence, and control over activation on demand. With this synthetic brain hydrogel, we show the brain-specific integrin-binding and matrix metalloprotease (MMP)-degradable domains of proteins control astrocyte star-shaped morphologies, and we can achieve an ECM condition that maintains astrocyte quiescence with minimal activation. In addition, we can induce activation in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. We envision this synthetic brain hydrogel as a new tool to study the physiological role of astrocytes in health and disease.

Plant peptide hormone signalling

2015 ◽  
Vol 58 ◽  
pp. 115-131 ◽  
Author(s):  
Ayane Motomitsu ◽  
Shinichiro Sawa ◽  
Takashi Ishida
Keyword(s):  
Communication Systems ◽  
Cell Communication ◽  
Peptide Hormone ◽  
Peptide Hormones ◽  
Target Cells ◽  
Signalling Molecules ◽  
Receptor Complexes ◽  
Environmental Responses ◽  
Plant Life Cycle ◽  
Multicellular Organisms

The ligand–receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone–receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

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