Stoichiometric analysis of oligomerization of membrane proteins on living cells using coiled-coil labeling and spectral imaging.

Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for ß2-adorenergic receptor (ß2AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag-probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that ß2ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation.

Case Report: A Domestic Sponge Brush Used to Clean a Milk Feeding Bottle: The Source of Neonatal Meningitis Caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a relatively rare cause of neonatal meningitis, and most patients have serious underlying diseases, prematurity, immunodeficiency, or anatomical abnormalities. We report the case of a 7-day-old girl with meningitis caused by P. aeruginosa. She was born full-term and had no immunodeficiency or anatomical abnormalities as far as our investigation ascertained. Through the use of anti-Pseudomonas antibiotics, she recovered without any complications other than a slight hearing disability revealed by audiology testing. P. aeruginosa was also isolated from a domestic sponge brush used to clean her milk bottle. Physicians should consider P. aeruginosa as a possible pathogen of neonatal meningitis even in full-term infants with no immunodeficiency or anatomical abnormalities. Physicians should give advice concerning appropriate hygiene practices to be applied to the neonate's environment.

[A visualization tool for oligomerization and internalization of membrane proteins in living cells: coiled-coil labeling method].

Genetic fusion of fluorescent/luminescent proteins to a target protein for specific labeling in living cells has been widely used to investigate the intracellular trafficking and oligomerization of the proteins. However, several limitations of fluorescent/luminescent proteins, such as considerable size, difficulty in controlling labeling ratio in multicolor labeling, can obscure true behaviors of the target proteins. To overcome these difficulties, post-translational labeling methods using pairs of small genetically-encodable 'tags' and synthetic 'probes' targeting the tags have been widely studied in recent years. We have developed a quick tag-probe labeling method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target protein and the K4 probe (KIAALKE)4 labeled with a fluorophore. The labeling is cell-surface-specific and completed within 1 min, therefore suitable for monitoring oligomerization/internalization of membrane proteins on living cell surface. Taking advantage of easiness in multicolor labeling, we show that the oligomeric state of membrane proteins can be precisely analyzed based on fluorescence resonance energy transfer. By using this method, we found that ß2 adrenergic receptors do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function. Furthermore, the degree of internalization of the ß2 receptors following agonist stimulation was evaluated by ratiometric detection of pH decrease in endosomes.