Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain.

Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT™ laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.

Chemical crosslinkers enhance detection of receptor interactomes.

Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor-protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor-protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.

Are nicotinic acetylcholine receptors coupled to G proteins?

It was, until recently, accepted that the two classes of acetylcholine (ACh) receptors are distinct in an important sense: muscarinic ACh receptors signal via heterotrimeric GTP binding proteins (G proteins), whereas nicotinic ACh receptors (nAChRs) open to allow flux of Na+, Ca2+, and K+ ions into the cell after activation. Here we present evidence of direct coupling between G proteins and nAChRs in neurons. Based on proteomic, biophysical, and functional evidence, we hypothesize that binding to G proteins modulates the activity and signaling of nAChRs in cells. It is important to note that while this hypothesis is new for the nAChR, it is consistent with known interactions between G proteins and structurally related ligand-gated ion channels. Therefore, it underscores an evolutionarily conserved metabotropic mechanism of G protein signaling via nAChR channels.