Effective expression of the green fluorescent fusion proteins in cultured Aplysia neurons

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Abstract

The green fluorescent fusion protein and its isoforms are extensively used to monitor gene expression, protein localisation and their dynamics in relations to fundamental cellular processes. However, it has not yet been effectively applied to Aplysia neurons that serve as a powerful model to study the mechanisms underlying neuroplasticity. We report here the development of a procedure combining in vitro transcription of mRNA encoding fluorescent-tagged proteins and its subsequent injection into the cytoplasm to image, in real-time, protein dynamics in cultured Aplysia neurones. To illustrate the efficiency of the procedure we report here the visualisation of actin, microtubules and vesicle trafficking. The results presented here introduce a reliable and effective method to express green fluorescent protein (GFP) fusion proteins in cultured Aplysia neurons.

Introduction

The green fluorescent protein (GFP) emits green light when excited by blue light without the use of a substrate and may thus be viewed in living or fixed tissue (Lippincott and Smith, 1997, Tsien, 1998, Baumann and Reyes, 1999, Chamberlain and Hahn, 2000). Since its discovery and isolation from the jellyfish Aequorea victoria (Chalfie et al., 1994), it has been efficiently used to image gene expression, to monitor cellular protein localisation and its dynamics (for review Stearns, 1995, Gerdes and Kaether, 1996, Day et al., 2001, Van Roessel and Brand, 2002). The growing family of fluorescent protein variants enables more sophisticated studies of protein function within living cells (Ellenberg et al., 1999, Periasamy and Day, 1999, Day et al., 2001).

GFP fusion proteins have been used in neurobiological studies as a tracer for individual neurons during development of neuronal networks and as markers for proteins distribution within single neurons in relation to various physiological functions (Spergel et al., 2001).

Aplysia neurons have been extensively used to study the biophysical, cellular and molecular mechanisms that underlie simple forms of behaviour, and in particular the mechanisms underlying short and long-term neuronal plasticity (for review see the Nobel lecture of Kandel, 2001a, Kandel, 2001b). In addition, cultured Aplysia neurons are extensively used to study the mechanisms of growth cones motility, navigation, regeneration and synaptogenesis (for review see Spira et al., 1996, Spira et al., 2001, Spira et al., 2003, Suter and Forscher, 2000). The relationship between these processes and protein dynamics is mainly based on retrospective immunolabelling of various proteins, microinjections of fluorescent probes and electron microscopy (for example see Bailey and Kandel, 1993, Gitler and Spira, 1998, Gitler and Spira, 2002, Spira et al., 2001, Scheafer et al., 2002).

The studies performed using Aplysia to elucidate fundamental mechanisms underlying neuroplasticity could be significantly facilitated by the use of GFP fusion proteins. Nevertheless, attempts to express GFP fusion proteins in Aplysia neurons were only partially successful. In 1996, Kaang introduced the gene encoding GFP using a pNEX derivative vector (Kaang, 1996a, Kaang, 1996b). The pNEX3 vector was reported as a plasmid fostering the most efficient expression of β-galactosidase from the lacZ gene in Aplysia neurons. However, the rate of successful expression of GFP following DNA injection of pNEX3-GFP plasmid was low, approximately 20% (Kim and Kaang, 1998). Furthermore, no significant fluorescent signal was detected in the axon or in the neurites.

Here we report the development of an effective procedure for the expression of EGFP-tagged protein in Aplysia neurones. The method consists of in vitro transcription of mRNA encoding EGFP-fusion proteins and its subsequent intracellular microinjection into the cytoplasm. On-line confocal imaging of EGFP tagged actin, tubulin and EGFP-labelled vesicle used to demonstrate the efficiency of the method.

Section snippets

L-15 supplemented for marine species (msL-15)

Leibovitz's L-15 Medium (GibcoBRL, Paisley, Scotland) was supplemented for marine species according to Schacher and Proshansky (1983) by the addition of 12.5 g/l NaCl, 6.86 g/l d(+) Glucose·H2O, 3.15 g/l anhydrous MgSO4, 344 mg/l KCl, 192 mg/l NaHCO3, 5.7 g/l MgCl2·6H2O and 1.49 g/l CaCl2·2H2O. Penicillin, streptomycin and amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel) were added up to final concentrations of 100 U/ml, 0.1 mg/ml and 0.25 μg/ml, respectively.

Culture medium

Consists of

EGFP-actin construct

EGFP cDNA was amplified from pEGFP-N1 vector (Clontech) by polymerase chain reaction (PCR) with two specific primers: 5′ GGCCATGGTGAGCAAGG3′ and 5′ CTTGTACAGCTCGTCCATG3′ containing HindIII and SmaI restriction sites, respectively, in their 5′ ends. The PCR product was digested with HindIII and SmaI and subcloned into corresponding sites of pBluscript II SK (Stratagen). Aplysia actin, (provided by Dr DesGrosiller, Montreal University) was amplified by PCR from pBluscript containing the actin

Discussion

In the present study we report the development of an efficient method for expression of GFP fusion proteins in cultured Aplysia neurons.

Functional genes have been expressed in Aplysia neurons following the injection of DNA plasmids (Kaang, 1996a, Kaang, 1996b, Manseau et al., 2001, Lee et al., 2001), nevertheless, these proteins were expressed at low levels and visualised only within the domain of the cell body.

Attempts to express mRNA encoding the lacz, gene in Aplysia neurons were not very

Acknowledgements

This study was supported by grants from the Israel Science Foundation (No. 620/98), the US-IsraelBi-National Research Foundation (No. 97-00297-1) and the Charles E. Smith Family Laboratory for Collaborative Research in Psychobiology, the Hebrew University of Jerusalem. M.E. Spira is the Levi DeViali Professor in Neurobiology.

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    Present address: Institute of Biology, College de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 5, France.

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