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

Summary

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

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.

Introduction

In establishing the function of newly identified proteins, investigation of the subcellular localization and trafficking of the protein in question can provide important clues about the likely role/s of the protein^1,2. Bioinformatic analysis of the transcriptome of the developing neocortex³ provided us with a list of genes exhibiting altered expression during mouse brain corticogenesis. We then adopted a gene knockout approach to ascertain that the protein encoded by one of these genes, sez6, has a key role in neuron development. We observed that the Seizure-related gene 6, or Sez6, protein is located in developing dendrites and is also ....

Protocol

1. Dissociated Hippocampal Neuron Culture

1. Prepare coverslips (borosilicate glass):
   1. Wash in 100% ethanol.
   2. Air dry under UV irradiation.
   3. Coat with Poly-D-lysine (0.5 mg/ml in 0.15 M borate buffer, overnight at 4 °C).
   4. On the next day (the day of culture) wash 3x in PBS then coat with laminin (2.5 μg/ml, natural mouse laminin) + 5% v/v heat-inactivated fetal calf serum (FCS) diluted in PBS for 2 hr at 37 °C.
2. Dissect embryonic day 18 (E18) rat hippocampi and collect into PBS containing calcium and magnesium, chilled on ice. NOTE: all experimental protocols involving animals were....

Results

The dual-color fluorescent immunostaining technique presented here is useful for labeling extracellular domains of transmembrane proteins in living cells (shown schematically in Figure 1). During the incubation period, the immunoglobulins bind accessible epitopes and a proportion of the population of protein molecules, together with bound antibody, is endocytosed. In addition, newly synthesized protein may reach the cell surface via forward trafficking and recycled protein molecules may be returned to th.......

Discussion

The technique described here is complementary to that of cell-surface biotinylation (reviewed by Arancibia-Càrcamo et al.)¹² and it is the method of choice for preserving information about the subcellular localization of the internalized protein, provided a suitable primary antibody to an extracellular epitope is available. In addition, quantitation of protein trafficking/internalization over time can be performed (by fixing coverslips at different times throughout the live cell incubation with p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS with Ca and Mg	Invitrogen	14040182
Neurobasal medium	Invitrogen	21103-049
B27 supplement	Invitrogen	17504-044
L-Glutamine	Invitrogen	25030-081
Papain Dissociation System	Worthington Biochemical Corporation	PDS
Bovine Serum Albumin	Sigma Aldrich Australia	A9418
Hank's balanced salt solution without calcium, magnesium, phenol red	Invitrogen (Gibco)	14175-079
Poly-D-Lysine	Sigma Aldrich Australia	P0899
Natural mouse laminin	Invitrogen	23017-015	Thaw on ice prior to making aliquots
Fetal bovine serum HyClone	Thermo Fisher
Fluorodeoxyuridine	Sigma Aldrich Australia	F0503
Uridine	Sigma Aldrich Australia	U3003
18 mm Round glass coverslips	Menzel Gläser	CB00180RA1
VECTASHIELD aqueous mounting medium	Vector Laboratories	H1400
Donkey anti-rabbit Dylight 649	Jackson ImmunoResearch Laboratories	711-495-152
AffiniPure Fab fragment Goat anti-Rabbit 1gG (H+L)	Jackson ImmunoResearch Laboratories	111-007-003
Paraformaldehyde	Sigma Aldrich Australia	P6148	TOXIC - handle in fume hood
Triton-X-100	Sigma Aldrich Australia	T8787
Alexa Fluor 488-conjugated donkey anti-rabbit 2° antibody	Invitrogen - Molecular Probes	A21206