Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Abstract

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking ("antibody feeding") to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution

“…To determine and quantify the distribution of cell surface versus internalized SK channels, we employed a dual-color fluorescence approach [14]. We first incubated live neurons with apaminbiotin followed by fixation and incubation with Alexa-488 fluorophore conjugated with streptavidin to label SK channels at the cell surface, as described in the methods.…”

The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb

“…At DIV9, neurons were washed with PBS and live labeled with 6E10 diluted in Neurobasal media. For surface staining, neurons were live labeled at 20°C for 60 min, then fixed with 4% PFA with 4% sucrose and stained with Alexa Fluor 488- conjugated secondary antibody (29). For staining of recycled APP, neurons were live labeled with 6E10 for 30 min at 10°C and then incubated in 37°C for 1 hour.…”

Beta-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibition Impairs Synaptic Plasticity via Seizure Protein 6

“…A recent report described an endosomally targeted sterol-linked BACE1 inhibitor that inhibits Aβ production without affecting β-cleavage of neuregulin 1 and neural cell adhesion molecule L1 (58). Both APP and SEZ6 contain NPxY motifs for the endocytosis in their intracellular domains (59,60), indicating that SEZ6 also undergoes endocytosis (19) similar to APP (61). Therefore, this sterol-linked BACE1 inhibitor may also interfere with SEZ6 processing.…”

“…Later, this SEZ6-CTF will be further cleaved by γ-secretase, generating intracellular domain (16). SEZ6 mainly locates in the somatodendritic compartment, specifically in the dendritic plasma membrane, synaptosomes, and postsynaptic density fractions (13,(17)(18)(19), as well as in recycling endosomes (T. Palumaa et al, Nov 2012, Students of Brain Research Symposium, abstract), suggesting that SEZ6 may modulate synaptic function. Similar to BACE1 inhibitor-treated mice (10), Sez6 -/mice have reduced dendritic spine density and spatial memory deficit (17).…”