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

Abstract

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.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission

“…There is little doubt that clefts of ribbon synapses acidify during activity, as this has been revealed through direct imaging of SE-pHluorin fused to an extracellular Ca 2ϩ -channel subunit (␣ 2 ␦ 4 ), which placed SE-pHluorin within the cleft adjacent to AZs (Wang et al, 2014;Beckwith-Cohen et al, 2019). Their approach was approximated in this study where a "kisser" probe (Stawarski et al, 2018) placed SE-pHluorin and mCherry in the cleft adjacent to presynaptic VGCCs (Fig. 7 A, B).…”

“…FAP probes were conjugated with a pH-sensitive fluorogen as follows: PM-impermeable se-Red-pH dye (Spectra Genetics) was added to the bath of a fillet-dissected female larvae at a final concentration of 100 nM for 15 min. The full sequences of PMCA-kisser-FAP and Cac-kisser-pHerry-TM is given in the supplementary materials of Stawarski et al (2018).…”

“…The intracellular domain of PMCA-kisser-FAP duplicates select elements of the Drosophila PMCA C terminus and localizes PMCA-kisser-FAP to the microenvironment of the PMCA (Fig. 3B) (Stawarski et al, 2018). Fluorogens are effectively nonfluorescent before binding a FAP tag.…”

“…7) where the cleft is only ϳ20 nm wide in Drosophila (Pawlu et al, 2004) similar to synaptic clefts within the mouse brain (Peters et al, 1991). As described previously (Stawarski et al, 2018), Cac-kisser probes were designed to localize to the Drosophila presynaptic VGCC (Cacophony). In this case, Cac-kisser-pHerry-TM is comprised of an intracellular carboxy terminus based on Cac, a TM of hPDGFR, and SE-pHluorin fused to mCherry exposed to the extracellular space (Fig.…”

A reductionist approach to understanding the nervous system: the Harold Atwood legacy

“…Jeffrey Dason et al characterize the role of a Type II phosphatidylinositol 4-kinase in synaptic function (Cantarutti, Burgess, Brill, & Dason, 2018). Gregory Macleod et al describe novel 'kisser' probes for resolving the distribution of membrane proteins (Stawarski, Hernandez, Justs, & Macleod, 2018). Ken Dawson-Scully et al report a role for BK channels in tolerance of synaptic transmission to acute oxidative stress (Bollinger, Sial, & Dawson-Scully, 2018).…”