Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Genetic dissection of intercellular interactions in vivo by membrane-permeable protein.

ABSTRACT: Unraveling cell-cell interaction is fundamental to understanding many biological processes. To date, genetic tools for labeling neighboring cells in mammals are not available. Here, we developed a labeling strategy based on the Cre-induced intercellular labeling protein (CILP). Cre-expressing donor cells release a lipid-soluble and membrane-permeable fluorescent protein that is then taken up by recipient cells, enabling fluorescent labeling of neighboring cells. Using CILP, we specifically labeled endothelial cells surrounding a special population of hepatocytes in adult mice and revealed their distinct gene signatures. Our results highlight the potential of CILP as a platform to reveal cell-cell interactions and communications in vivo.

SUBMITTER: Zhang S

PROVIDER: S-EPMC9910597 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Genetic dissection of intercellular interactions in vivo by membrane-permeable protein.

Zhang Shaohua S Zhang Qianyu Q Liu Zixin Z Liu Kuo K He Lingjuan L Lui Kathy O KO Wang Lixin L Zhou Bin B

Proceedings of the National Academy of Sciences of the United States of America 20221227 1

Unraveling cell-cell interaction is fundamental to understanding many biological processes. To date, genetic tools for labeling neighboring cells in mammals are not available. Here, we developed a labeling strategy based on the Cre-induced intercellular labeling protein (CILP). Cre-expressing donor cells release a lipid-soluble and membrane-permeable fluorescent protein that is then taken up by recipient cells, enabling fluorescent labeling of neighboring cells. Using CILP, we specifically label ...[more]

PMID: 36574652

Similar Datasets

Massively Parallel Dissection of RNA in RNA-protein Interactions in vivo

Project description:Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to molecular biology. Here, we present MPRNA-immunoprecipitation (MPRNA-IP), an adaptation of the previously developed massively parallel RNA assay (MPRNA) for in vivo high-throughput dissection of RNA-protein interactions, and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, we are able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest including NORAD, MS2, and human telomerase RNA, and we use it to interrogate the RNA-binding preferences of the DNA-looping factor CTCF. By blending modern and classical approaches, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions.

2024-04-15 | GSE221537 | GEO

Fully quantified spectral imaging reveals in vivo membrane protein interactions.

Project description:Here we introduce the fully quantified spectral imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling.

| S-EPMC4755892 | biostudies-literature

Generation of neighbor-labeling cells to study intercellular interactions in vivo.

Project description:Understanding cell-cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell-cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2-3 weeks to generate the labeling cells and 1-2 weeks to test their labeling ability.

| S-EPMC7615221 | biostudies-literature

Genetic dissection of plexin signaling in vivo.

Project description:Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during development.

| S-EPMC3926053 | biostudies-literature

Peptoid-based reprogrammable template for cell-permeable inhibitors of protein-protein interactions.

Project description:The development of inhibitors of intracellular protein-protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors.

| S-EPMC8528041 | biostudies-literature

Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling.

Project description:Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of 'kiss-and-run' interactions between immune cells 'Labelling Immune Partnerships by SorTagging Intercellular Contacts' (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4+ T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

| S-EPMC5853129 | biostudies-literature

Novel pyrrolopyrimidine-based α-helix mimetics: cell-permeable inhibitors of protein−protein interactions.

Project description:There is considerable interest in developing non-peptidic, small-molecule α-helix mimetics to disrupt α-helix-mediated protein−protein interactions. Herein, we report the design of a novel pyrrolopyrimidine-based scaffold for such α-helix mimetics with increased conformational rigidity. We also developed a facile solid-phase synthetic route that is amenable to divergent synthesis of a large library. Using a fluorescence polarization-based assay, we identified cell-permeable, dual MDMX/MDM2 inhibitors, demonstrating that the designed molecules can act as α-helix mimetics.

| S-EPMC3079198 | biostudies-literature

Membrane-mediated protein interactions drive membrane protein organization.

Project description:The plasma membrane's main constituents, i.e., phospholipids and membrane proteins, are known to be organized in lipid-protein functional domains and supercomplexes. No active membrane-intrinsic process is known to establish membrane organization. Thus, the interplay of thermal fluctuations and the biophysical determinants of membrane-mediated protein interactions must be considered to understand membrane protein organization. Here, we used high-speed atomic force microscopy and kinetic and membrane elastic theory to investigate the behavior of a model membrane protein in oligomerization and assembly in controlled lipid environments. We find that membrane hydrophobic mismatch modulates oligomerization and assembly energetics, and 2D organization. Our experimental and theoretical frameworks reveal how membrane organization can emerge from Brownian diffusion and a minimal set of physical properties of the membrane constituents.

| S-EPMC9712761 | biostudies-literature

Nanopodia--thin, fragile membrane projections with roles in cell movement and intercellular interactions.

Project description:Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 ?m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per ?m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 ?m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

| S-EPMC4161280 | biostudies-literature

An expanded genetic code in Candida albicans to study protein-protein interactions in vivo.

Project description:For novel insights into the pathogenicity of Candida albicans, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p-azido-L-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair (EcTyrtRNA(CUA)) derived from Escherichia coli, which was previously reported to be orthogonal in Saccharomyces cerevisiae. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and EcTyrtRNA(CUA) for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

| S-EPMC3675983 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data