细胞功能测定

Tissue-specific gene expression is a major determinant in the elaboration of cells with distinctive phenotypes and functions, which is crucial for the development and homeostasis of multicellular organisms. Fluorescent in situ hybridization (FISH) is a powerful method for asses ...

Here we describe how to use fluorescence in situ hybridization and immunofluorescence staining to determine the in situ distributions of specific mRNAs and proteins in Chlamydomonas reinhardtii. This unicellular eukaryotic green alga is a major model organism in cell biological r ...

The localization of specific mRNAs into dendrites and/or axons is an important mechanism to enrich �proteins at their sites of function and influence neuronal development, plasticity, and repair. The fluorescence in situ hybridization (FISH) methods described here have provided h ...

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demons ...

Localization of mRNAs plays pivotal roles in different cell types, including neurons and the cells in the developing stages. To visualize the dynamic movements of mRNAs in living cells, many methods have been emerged in the past decade. However, it has not been realized to visualize endogenous m ...

Over the past decade, a variety of oligonucleotide-based probes have been developed that allow for direct visualization of RNA molecules in living cells. Of these, molecular beacons have garnered a particularly high degree of interest due to their simple yet exquisite unimolecular stem ...

The molecular beacon technology is an established approach for visualizing native mRNAs in living cells. These probes need to efficiently hybridize to accessible RNA regions in order to spatially and temporally resolve the dynamic steps of the RNA life cycle. A refined method using two comp ...

Visualization of RNA in live cells is a challenging task due to the transient character of most RNA molecules and the lack of adequate methods to label RNA noninvasively. Here, we describe a system for regulated RNA synthesis and visualization of RNA in live Escherichia coli cells based on protein co ...

It is now well understood that G protein-coupled receptor (GPCR)-mediated cell signalling is subject to extensive spatial–temporal control, and that a meaningful understanding of this complexity requires techniques to study signalling at the molecular and sub-cellular level. T ...

Protein kinase C (PKC) signaling drives many important cellular processes and its dysregulation results in pathophysiologies such as cancer (Gokmen-Polar et al., Cancer Res 61:1375–1381, 2001). Because PKC is activated acutely and allosterically, it is difficult to monitor the cellu ...

The ubiquitous Protein Kinase A (PKA) signaling pathway is responsible for the regulation of numerous processes including gene expression, metabolism, cell growth, and cell proliferation. This method details how to monitor real-time PKA activity dynamics in mammalian cells using f ...

G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling receptors. Their actions are highly regulated, both under physiological conditions and in response to clinically relevant drugs. A key element in this regulation is control of the number ...

G-protein-coupled 7 transmembrane domain receptors (GPCR-7TMR) represent the largest class of membrane protein drug targets. They respond to a plethora of ligands ranging from small molecules to polypeptide hormones. Upon activation, almost all GPCR-7TMRs undergo desensitiz ...

The heptahelical G protein-coupled receptors (GPCRs) receive and transmit a wide range of extracellular stimuli and induce a wide array of cellular responses by activating signaling kinases. It has become increasingly evident that the agonist-stimulated GPCR complexed with the ad ...

Many transmembrane receptors are regulated by associations with scaffold proteins containing PDZ domains, which interact with receptor carboxyl-termini to control receptor signaling, trafficking, and localization. We describe here approaches for detecting and charac ...

Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biol ...

β-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of β-arrestin/receptor interactions and how these complexes, and complexes with ot ...

Protein–protein interaction is a widely existing phenomenon and is essential for almost all biological processes, extending from the formation of cellular macromolecular structures and enzymatic complexes to the regulation of signal transduction pathways. Proteins inte ...

G-protein coupled, seven-transmembrane (7-TM) receptors (GPCRs) comprise a diverse class of signaling molecules involved in cellular physiology and pathology. In recent years, intracellular biosensors have been developed to monitor changes in intracellular cAMP in real time, ...

Advances in imaging assays based on resonance energy transfer (RET) have made it possible to study protein/protein interactions in living cells under physiological conditions. It is now possible to measure the kinetics of changes in these interactions in response to ligand stimulati ...