Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Summary

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface. The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Abstract

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

Introduction

During their navigation, axons integrate multiple environmental cues that guide them towards their target. These cues activate guidance receptors at the surface of axon terminals, the growth cones, which in turn initiate an appropriate signaling pathway. Thus, the temporal and spatial regulation of the cell surface distribution of the receptors is critical to set the sensitivity of the growth cone¹. In this context, midline crossing by commissural axons is an excellent model to investigate the regulation of receptor cell surface levels. In the developing spinal cord, commissural axons are initially attracted towards the ventral floor plate where they cross ....

Protocol

1. Cloning Strategy to Tag PlexinA1 Receptor with pHluorin

1. Choose an appropriate expression vector as a backbone (e.g. the mouse receptor plexinA1 expressing vector, a kind gift of Dr. Andreas Puschel¹¹).
   Note: This plexinA1 vector was engineered to achieve efficient HA- or VSV-tagged receptor insertion in the plasma membrane.
2. Amplify by PCR the ecliptic pHluorin coding sequence using the adequate plasmid as a template (e.g. pHluorin-tagged GABA A recept.......

Discussion

This protocol provides a step-by-step procedure to follow the dynamics of an axon guidance receptor both in cell culture and in the developmental context of the chick embryo spinal cord.

To design a de novo pHluorin tagged protein, two points need to be considered regarding the cloning strategy. First, the pHluorin tag should be exposed to the lumen of the acidic endosomes, and consequently, to the extracellular compartment in order to visualize the plasma membrane receptor pool. Thus.......

Materials

Name	Company	Catalog Number	Comments
COS7 cells	ATCC	CRL-1651
DMEM GlutaMAX	GIBCO	61965-026
Sodium pyruvate	GIBCO	11360-039
Amphotericin B	Sigma	A2942
Fetal bovine serum	GIBCO	10270-106
Penicillin/Streptomycin	GIBCO	15140-122
Exgen500 reagent	Euromedex Fermentas	ET0250
PBS -Ca2+ -Mg2+	GIBCO	14190-094
Fast green dye	Sigma	F7252
32% Paraformaldehyde aqueous solution	Electron Microscopy	15714-S	Dilute extemporaneously in PBS to achieve a 4% solution
Gelatin from cold water fish skin	Sigma	G7041
Sucrose	Sigma	S0389
Cryomount	Histolab	00890
Hoechst 34580	Invitrogen	H21486
Mowiol 4-88	Fluka	81381
Consumables
Bottom-glass 35 mm dish	MatTek	P35G-1.5-14-C
5 ml Syringe	Terumo	SS-05S
Needles 0.9 mm x 25 mm	Terumo	NN-2025R
Capillaries	CML	PP230PO	capillaries are stretched manually in the flame
Superfrost Plus Slides	Thermo Scientific	4951PLUS
Material
Curved scissors	FST	129-10
Microscalpel	FST	10316-14
Forceps	FST	Dumont #5 REF#11254
Equipment/software
Time lapse microscope	Zeiss	Observer 1
Temp module S	PECON for Zeiss
CO2 module S	PECON for Zeiss
Metamorph software	Metamorph
Eggs incubator	Sanyo	MIR154
Electroporator apparatus	Nepa Gene CO., LTD	CUY21
Electrodes	Nepa Gene CO., LTD	CUY611P7-4	4 mm platinum electrodes
Fluorescence stereomicroscope	LEICA	MZ10F
Cryostat	MICROM	HM550
Confocal microscope	Olympus	FV1000, X81
Fluoview software	Olympus
CLC Main Workbench software	CLC Bio