The application of 'kisser' probes for resolving the distribution and microenvironment of membrane proteins in situ

摘要

Membrane proteins play a lead role in the formation and function of synapses, but, despite revolutions in immunology and molecular genetics, limitations persist in our ability to investigate membrane proteins in the context of an intact synapse. Here, we introduce a simple but novel approach to resolving the distribution of endogenous membrane proteins in either live or fixed tissues. The technique involves transgenic expression of a protein with an extracellular tag, a generic transmembrane domain, and an intracellular terminus that mimics the intracellular anchoring motifs of the endogenous protein of interest. We provide three examples where these kisser probes can be used to answer questions regarding the synaptic distribution of endogenous proteins and their microenvironment that would be difficult to resolve by other contemporary means: (i) the live distribution of untagged proteins at the neuromuscular junction (Cacophony and Shaker), (ii) the relative distribution of an untagged protein (PMCA) in pre- versus post-synaptic membranes separated by only 20 nm across the cleft of a fixed synapse, and (iii) the live targeting of functional probes (chemical and protein fluorescent pH reporters) to membrane protein-defined subcellular domains.