Stargazin-related protein γ₇ is associated with signalling endosomes in superior cervical ganglion neurons and modulates neurite outgrowth

Abstract

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca²⁺ channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ₇ subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ₇ is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA–YFP, and with the early endosome marker EEA1, suggesting that γ₇ localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ₇ by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ₇. These findings open the possibility that γ₇ has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling.

Introduction

The Ca²⁺ channel γ-subunit family were so-named because the prototypical member γ₁, was originally identified as a subunit of skeletal muscle calcium channels, because it copurifies with the skeletal muscle Ca²⁺ channel complex (Jay et al., 1990; Powers et al., 1993). In skeletal muscle, γ₁ appears to have a suppressive effect, because γ₁-knockout mice exhibit increased skeletal muscle Ca²⁺ currents (Freise et al., 2000). Subsequent studies have identified seven further putative γ-subunits (Black and Lennon, 1999; Burgess et al., 2001; Klugbauer et al., 2000; Letts et al., 1998; Moss et al., 2002). However, it is unclear whether any of the novel γ-proteins (γ₂–γ₈) are in fact subunits of voltage-gated Ca²⁺ channels. All members of this family are thought to possess four transmembrane domains with cytoplasmic N- and C-termini. The γ₂, γ₃, γ₄ and γ₈ subunits form a subfamily that exclusively localises to the central nervous system (Klugbauer et al., 2000; Letts et al., 1998; Moss et al., 2003; Sharp et al., 2001), whose interaction and functional modulation of Ca_V channels has been investigated in several studies (Kang et al., 2001; Klugbauer et al., 2000; Letts et al., 1998; Moss et al., 2003; Rousset et al., 2001; Sharp et al., 2001). These are now primarily thought to interact, through their PDZ (postsynaptic density-95 protein, Discs large and zona occludens-1 protein) binding motif, with the AMPA subtype of glutamate receptors (Tomita et al., 2003), and have been termed transmembrane AMPA receptor regulatory proteins (TARPs). However, they have also been proposed to provide a bridge between Ca²⁺ channels and AMPA receptors (Kang et al., 2006).

The γ₇ and γ₅ proteins represent a distinct subfamily of stargazin-related proteins (Burgess et al., 2001; Moss et al., 2002), with extremely low sequence identity to γ₁ and approximately 25% identity to γ₂, mainly in the transmembrane domains. We found previously that coexpression of γ₇ with Ca_V2.2 almost abolished the functional expression and markedly suppressed the level of Ca_V2.2 protein. It also had smaller suppressive effects on Ca_V2.1 and Ca_V1.2 currents (Moss et al., 2002). Our conclusion was that γ₇ was not a subunit of these Ca²⁺ channels. In a subsequent study, we showed that γ₇ interacts with the RNA binding protein hnRNP A2 and increases the rate of degradation of certain mRNAs, including that of Ca_V2.2 (Ferron et al., 2008). We concluded that by sequestering hnRNP A2, γ₇ reduces the binding of this RNA binding protein to specific mRNAs, therefore reducing their stability.

The present study was designed to examine the subcellular distribution of γ₇. Here, we show that when γ₇ is expressed in sympathetic superior cervical ganglion (SCG) neurons, it is present in motile particles that show primarily retrograde transport. These subcellular organelles are related to signalling endosomes because γ₇ colocalises with the nerve growth factor receptor (TrkA) and with the early endosome antigen (EEA1).

Results

Fluorescently tagged γ₇ is associated with organelles in neurites that are subject to transport

When γ₇–YFP or γ₇–CFP was expressed in SCG neurons, it particularly localised to small vesicular organelles within neurites (Fig. 1A–C, neurite edges are indicated by dashed lines), whereas free CFP was uniformly distributed (Fig. 1C). In short-term culture (<48 hours), these neurites have not yet differentiated into axons and dendrites (supplementary material Fig. S1). The γ₇-containing organelles were observed to be motile (Fig. 1B). These organelles moved primarily in a retrograde direction, although anterograde movement was also observed. Fig. 1B depicts several frames from regions of a time-series, pseudo-coloured and merged to emphasise the movement. The image shows that there is a mix of stationary (white) and non-stationary structures (blue, green and red). The average speed of retrograde movement of the γ₇–YFP particles was 1.19±0.08 μm/second (n=7), and the maximum speed was 2.47±0.09 μm/second (n=7). γ₇-containing particles were also densely clustered within the growth-cone bulb of SCG neurites (Fig. 1D), as previously described for the styryl dye FM4-64, following application to growth cones (Bonanomi et al., 2008).

Fig. 1.

γ₇ is present on intracellular organelles subject to transport in neurites. (A) γ₇–YFP was microinjected into an SCG neuron, and images taken after 24 hours. The cell body is located to the left of the frame. Multiple γ₇–YFP-containing organelles are observed in the neurites, some of which are stationary and some of which are motile. The image represents a single frame from a time series movie. Scale bar: 20 μm. (B) Consecutive images, from the field depicted in A, obtained at 3 second intervals, were pseudo-coloured blue (image 1), green (image 2) and red (image 3) and superimposed. Stationary structures appear white, whereas the moving particle appears blue, green and red. Scale bar: 20 μm. (C) Region of neurite from a cell expressing γ₇–YFP (top) and free CFP (middle). The merged image is shown at the bottom. The free CFP is uniformly distributed throughout the neurites. The neurite is outlined with a dashed line in the top panel. Scale bar: 10 μm. (D) Image of a growth cone from a cell expressing γ₇–YFP (left), and CFP (middle). The merged image is shown on the right. Scale bar: 10 μm. γ₇–YFP is present as discrete particles in the bulb of the growth cone.

Colocalisation of γ₇–CFP with organelle markers in neurites

The retrogradely moving γ₇–CFP particles in neurites did not colocalise with the ER marker dsRed2-ER (Fig. 2A) or with a Golgi marker, dsRed2-Golgi (Fig. 2B). Furthermore, γ₇–CFP did not associate with lysosomes labelled with Lysotracker Red (Fig. 2C) or LAMP1 (data not shown), or with mitochondria labelled with Mitotracker Red (Fig. 2D). γ₇–CFP also did not colocalise with CD63-positive organelles in neurites (Fig. 2E). CD63 is a tetraspanin protein that is a marker of late endosomes and multi-vesicular bodies (Pols and Klumperman, 2009). The percentage of particles that colocalised with γ₇-positive particles was determined to be 22.2±3.6% (n=3) for Mitotracker Red, 19.8±4.8% for Lysotracker Red (n=5) and 14.6±10.7% for CD63 antibody (n=4) (see Fig. 6B for summary). This level of colocalisation is likely to represent the baseline that occurs by chance in motile particles.

Fig. 2.

γ₇-containing particles do not colocalise with a number of organelle markers. Cells were micro-injected 6 hours after being placed in culture, and imaged or fixed 18 hours after microinjection. (A) Region of neurite from a cell expressing γ₇–CFP (top) with dsRed2–ER (middle). The merged image is shown at the bottom. (B) Region of neurite from a cell expressing γ₇–CFP (top) and dsRed2-Golgi (middle). The merged image is shown at the bottom. (C) Region of neurite from a cell expressing γ₇–CFP (top) together with Lysotracker Red (middle). The merged image is shown at the bottom. (D) Region of neurite from a cell expressing γ₇–CFP (top) and Mitotracker red (middle). The merged image is shown at the bottom. (E) Region of neurite from a cell expressing γ₇–CFP (top) and stained with anti-CD63 (bottom). The merged image is shown on the right. The neurites in each image are outlined with a dashed line. Scale bars: 10 μm.

Fig. 6.

γ₇–YFP-containing particles show partial colocalisation with CFP–Ca_V2.2 in SCG neurites. (A) An SCG neuron expressing γ₇–YFP (top), and CFP–Ca_V2.2 (middle). The merged image is shown at the bottom, with areas of colocalisation in white, indicated by arrows. Scale bar: 10 μm. (B) Bar chart summarising colocalisation data for γ₇–YFP or γ₇–CFP together with the markers shown. Data are taken from the number of cells (n) given, in at least two different experiments for Lysotracker Red (black bar, n=5), Mitotracker Red (white bar, n=3), CD63 antibody (grey bar, n=4), EEA1 in neurites (red bar, n=6), EEA1 in somata (red hatched bar, n=8), TrkA–YFP (yellow bar, n=5) and CFP–Ca_V2.2 (cyan bar, n=4). Statistical significance of the percentage of particles showing colocalisation in the last four conditions was compared with the low level of colocalisation seen for Mitotracker Red, where colocalisation of the motile particles was considered to be a chance occurrence. **P<0.01; ***P<0.001 (One-way ANOVA and Bonferroni's post test).

Colocalisation of γ₇–CFP with endosome markers

When TrkA–YFP was coexpressed with γ₇–CFP, they were substantially colocalised in retrogradely moving particles (Fig. 3A). These generally originated from neurites that terminated in a growth cone (data not shown). γ₇–CFP is shown in Fig. 3Ai and TrkA–YFP in Fig. 3Aii. The merged images (Fig. 3Aiii) clearly show colocalisation in a large proportion of particles, calculated to be 46.5±7.9% of γ₇–CFP clusters (n=5, see Fig. 6B for summary). Furthermore, the colocalisation was continuous over time and during movement, as shown by the kymograph (Fig. 3B). These results suggest that γ₇–CFP is associated with signalling endosomes, which are a form of early endosome containing activated TrkA (Cui et al., 2007).

Fig. 3.

γ₇–CFP-containing particles colocalise with TrkA–YFP in SCG neurons. (A) A region of neurite expressing γ₇–CFP and TrkA–YFP was imaged over time. Three examples are given at different time points, 59, 165 and 266 seconds after the start of time series collection. For each example the top panel (i) is γ₇–CFP, the middle panel (ii) is TrkA–YFP and the bottom panel (iii) shows the merged image. Regions of colocalisation are white. Scale bar: 44 μm. (B) Kymograph of γ₇–CFP (i) and TrkA–YFP (ii) movement, showing colocalisation in white (iii). Vertical lines represent non-motile particles, whereas diagonal lines represent motile particles, most of which show retrograde movement. Scale bars: 60 seconds (vertical) and 44 μm (horizontal).

In addition, γ₇–CFP particles also partially colocalised with particles expressing the early endosome marker EEA1. This was present in particles both within SCG cell bodies (Fig. 4A), where there was 61.3±4.4% (n=8) colocalisation with γ₇–CFP, and within the SCG neurites (Fig. 4B), where there was 73.9±5.6% (n=6) colocalisation (see Fig. 6B for summary). We also found that both the number (Fig. 4C) and average size (Fig. 4D) of the EEA1-containing particles was increased when γ₇–CFP was coexpressed.

Fig. 4.

γ₇–CFP-containing particles colocalise with EEA1 in SCG neurons. (A) Cell body from a cell expressing γ₇–CFP (top), and stained with anti-EEA1 (middle). The merged image is shown at the bottom, with areas of colocalisation in white. Scale bar: 10 μm. (B) Region of neurite from a cell expressing γ₇–CFP (top), and stained with anti-EEA1 (middle). The merged image is shown at the bottom, with areas of colocalisation in white. Neurites are outlined with a dashed line. Scale bar: 10 μm. (C) Bar chart showing numbers of EEA1-containing puncta in γ₇–CFP-expressing (black bar, n=9 cells), compared with CFP-expressing (white bar, n=8) somata, *P<0.05 (Student's t-test). The value is the average number of particles in all the optical sections of each cell. (D) Bar chart showing average area of EEA1-containing puncta in γ₇–CFP-expressing (black bar, n=9), compared with CFP-expressing (white bar, n=8) somata, measured throughout the z-stack of optical sections. ***P<0.001 (Student's t-test).

Further evidence that γ₇ is associated with early endosomes comes from our finding that γ₇–CFP was associated with EGF receptors in Cos-7 cells visualised after incubation of live cells with EGF–TR (Fig. 5A). In these experiments, 39.2±3.2% of γ₇–CFP clusters colocalised with EGF–TR (n=5). These particles were also colocalised in SCG neuronal somata (Fig. 5B), where 55.3±5.6% of γ₇–CFP clusters colocalised with EGF–TR (n=5). However, in neurites, very few EGF–TR-containing particles were observed (Fig. 5C), and only 14.0±7.1% of γ₇–CFP clusters colocalised with EGF–TR (n=5). This is to be expected because although EGF receptors have been reported in the soma of sympathetic neurons, they have not been observed in neurites (Jia et al., 2007).

Fig. 5.

γ₇–CFP-containing particles show partial colocalisation with EGFR in Cos-7 cells and SCG neurons. (A) A Cos-7 cell expressing γ₇–CFP (left), and incubated with Texas-Red-conjugated EGF at 37°C for 1 hour (middle). The merged image is shown on the right, with areas of colocalisation in white. Scale bar: 20 μm. (B) An SCG neuron expressing γ₇–CFP (left), and incubated with Texas-Red-conjugated EGF at 37°C for 1 hour (middle). The merged image is shown on the right, with areas of colocalisation in white. Scale bar: 10 μm. (C) Region of neurite from a cell expressing γ₇–CFP (top panel), and incubated with Texas-Red-conjugated EGF at 37°C for 1 hour (middle). The merged image is shown at the bottom, with no areas of colocalisation. Neurites are outlined with dashed lines. Scale bar: 20 μm.

N-type Ca²⁺ channels are widely expressed in SCG neurons, and are the main Ca²⁺ channel subtype responsible for transmitter release at presynaptic terminals in these neurons (Brock and Cunnane, 1999). We therefore wished to examine whether Ca_V2.2 would colocalise with γ₇. Interestingly, we observed that coexpression of γ₇–YFP with CFP–Ca_V2.2 resulted in its colocalisation with 58.4±8.0% (n=4) of γ₇–YFP clusters (Fig. 6A,B).

Endogenous γ₇ is partially colocalised with EEA1

To determine whether endogenous γ₇, which we had previously shown to have a punctuate distribution (Ferron et al., 2008), was also colocalised with endosomal markers, we examined its colocalisation with EEA1 in SCG neurons (Fig. 7). Colocalisation was observed in both somata and neurites (Fig. 7A), such that about 45% of γ₇-containing particles colocalised with an EEA1 particle (Fig. 7B). This suggests that the colocalisation of γ₇–CFP with endosomal markers was not an artifact of overexpression.

Fig. 7.

Partial colocalisation of endogenous γ₇ with EEA1 in SCG neurons. (A) Left, Cell body from an SCG neuron stained for γ₇ (top), and with anti-EEA1 (middle). The merged image is shown in the bottom panel, with areas of colocalisation in yellow. Scale bar: 10 μm. Right, Region of neurite from a neuron stained for γ₇ (top), and with anti-EEA1 (middle panel). The merged image is shown at the bottom, with areas of colocalisation in yellow, and indicated by arrows. Neurites are outlined with dashed lines. Scale bar: 10 μm. (B) Bar chart showing percentage colocalisation of γ₇-containing and EEA1-containing puncta in SCG somata (black bar, n=11 cells), and neurites (grey bar, n=17 cells).

Endogenous γ₇ influences neurite outgrowth

We had previously observed an indication of altered neurite morphology following γ₇ overexpression in peripheral neurons (Moss et al., 2002). Furthermore, in our previous study we showed that it was possible to knock down endogenous γ₇ in cultured neurons with shRNA (Ferron et al., 2008). Therefore, to examine whether endogenous γ₇ has a role in neurite outgrowth and morphology in sympathetic neurons, we transfected SCGs with a mixture of three γ₇ shRNAs (Ferron et al., 2008), and found that this resulted in a reduction in levels of endogenous γ₇ (Fig. 8A,B). It also caused a reduction in neurite outgrowth compared with control shRNA (Fig. 9A,B). Cells were transfected with the shRNAs and GFP, and cultured for 4–6 days to allow γ₇ knockdown to occur, then re-plated, and neurite outgrowth was measured 24 hours later (Fig. 9A). The total neurite length per cell was markedly reduced 4, 5 and 6 days after transfection with γ₇ shRNA, compared with control shRNA, and the pooled data are shown in Fig. 9B.

Fig. 8.

Quantification of knockdown of γ₇ with shRNA in SCG neurons. (A) Examples of SCG neurons transfected with GFP and either the control gnu shRNA (top) or the γ₇ shRNA mixture (bottom). Left, GFP; centre, endogenous γ₇; right, merged image. Scale bar: 20 μm. (B) Bar chart showing level of γ₇ in GFP-positive SCG somata in control shRNA-transfected cells (black bar, n=17 cells), compared with cells transfected with γ₇ shRNA (white bar, n=17 cells). Data are expressed as a percentage of control level. **P<0.003 (Student's t-test).

Fig. 9.

Knockdown of γ₇ with shRNA results in a reduction of neurite outgrowth in SCG neurons. (A) Examples of SCG neurons transfected with GFP and either the control gnu shRNA (top) or the γ₇ shRNA mixture (bottom). Images represent composites. Scale bar: 100 μm. (B) Neurite length for γ₇ shRNA mixture (white bar, n=28). expressed as a percentage of control shRNA (black bar, n=34). ***P<0.00001 (Student's t-test). (C) Examples of PC12 cells transfected with GFP and either the control gnu shRNA (top) or rat γ₇ shRNA mix (bottom). Scale bar: 100 μm. (D) Recovery of neurite outgrowth in PC12 cells by transfection with human γ₇. Neurite outgrowth was measured following transfection with the constructs stated, and expressed as a percentage of the neurite length obtained with the negative control shRNA (Ctrl, black bar, n=43), for the mixture of shRNAs against rat γ₇ (white bar, n=79), rat γ₇ shRNAs plus human γ₇ (grey bar, n=36) and human γ₇ alone as a control (hatched bar, n=74). ***P<0.001 (One-way ANOVA and post-hoc Bonferroni test).

We then examined in more detail the effect of γ₇ knockdown on neurite outgrowth, using the PC12 neuronal cell line, which represents a model for these peripheral neurons. As before, we used a mixture of three shRNAs specifically complementary to mRNA encoding rat γ₇, and used Drosophila gnu shRNA as a control (Ferron et al., 2008). We found that transfection of rat γ₇ shRNAs markedly decreased the length of PC12 neurites, compared with the control shRNA, when measured 9 days after transfection and 3 days after the start of differentiation with NGF (Fig. 9C,D). Importantly, the effect was reversed by cotransfection of full-length human γ₇ with γ₇ shRNA (Fig. 9D). No increase in apoptosis was observed following transfection with γ₇ shRNA, using the nuclear dye Hoechst 33258 (data not shown).

Discussion

We have previously described the identification of two genes that encode γ₅ and γ₇, by their homology with the mouse stargazin gene (Cacng2), and have cloned and expressed the cDNA for both human and mouse γ₇ (Moss et al., 2002). Together with the other γ-like proteins, γ₇ belongs to the claudin superfamily, members of which have diverse roles in cellular physiology (Sanders et al., 2001). Some members of the family of γ-subunits or TARPs have been shown to have a role in AMPA receptor function (Tomita et al., 2003). Despite not having a classical C-terminal PDZ motif, γ₇ has also recently been shown to have effects on AMPA receptor trafficking (Kato et al., 2007), and γ₅ has been found to affect Ca²⁺-permeable AMPA receptors in Bergmann glia (Soto et al., 2009). Nevertheless, in a recent proteomic study, only a small proportion of AMPA receptors were found to be associated with γ subunits, with γ₅ and γ₇ being particularly rare (Schwenk et al., 2009), despite the fact that γ₇ is widely expressed in brain (Moss et al., 2002). This suggests that these γ-subunits have other roles. Knockout of γ₇ has recently been found to have little effect on cerebellar function (Yamazaki et al., 2010), unlike knockout of γ₂ in Stargazer mice (Letts et al., 1998); however, the double-knockout (γ₂ and γ₇) mouse is more severely affected than γ₂-knockout mice (Yamazaki et al., 2010), suggesting that there is redundancy in γ-subunit function.

Presence of γ₇ in organelles within neurites that are retrogradely transported

In this study, we have found that when γ₇–YFP or γ₇–CFP are expressed in SCG neurons, they are localised in endosome-like organelles that are transported primarily in a retrograde direction, away from growth cones. Many studies have shown that growth cones contain vesicles (Cheng and Reese, 1988), and both constitutive and stimulated endocytosis have been demonstrated (Bonanomi et al., 2008; Diefenbach et al., 1999). Constitutively formed endocytotic vesicles have also been shown to undergo retrograde transport (Diefenbach et al., 1999). We have examined the properties of γ₇-containing vesicles in neurites, and showed that they do not colocalise with mitochondria, lysosomes, ER or late endosomes. However, we found that the γ₇-containing organelles do share some characteristics with signalling endosomes (Zweifel et al., 2005), because the transport is primarily retrograde and the organelles colocalise with TrkA and EEA1. We also found that endogenous γ₇-containing particles were partially colocalised with EEA1 in somata and neurites.

The speed of γ₇–CFP-containing vesicles in SCG neurites is in close agreement with the rate of movement of quantum-dot-labelled NGF in DRG neurites, which showed an average rate of 1.3 μm/second, with a speed during active movement of 2.1 μm/second (Cui et al., 2007). Signalling endosomes are thought to transport activated growth factor receptors to the soma, and are essential for maintaining neurite outgrowth (Cosker et al., 2008). Furthermore, we know that SCG neurons contain endogenous γ₇ (Ferron et al., 2008), and we have observed here that knockdown of endogenous γ₇ with shRNA reduced neurite outgrowth both in SCGs and in PC12 cells, where we showed that neurite outgrowth is restored by expression of human γ₇.

YFP–Ca_V2.2 was also observed to colocalise with γ₇–CFP in retrogradely transported vesicles, and it is possible that this represents Ca_V2.2 that was inserted into the growth cone membrane and then endocytosed during the extensive remodelling of the growth cone cell surface that occurs in the early phase of axon outgrowth (Bonanomi et al., 2008).

In our previous study (Ferron et al., 2008), we identified by coimmunoprecipitation from a PC12 cell line stably transfected with γ₇–HA, that the RNA binding protein hnRNP A2 coimmunoprecipitates with γ₇. This protein has been shown to be involved in the stability, trafficking and localisation of mRNAs containing a specific binding motif termed A2RE (Ainger et al., 1997; Shan et al., 2003). We showed that mRNA encoding Ca_V2.2 contained an A2RE motif and was immunoprecipitated with hnRNP A2. However, other mRNAs containing A2RE motifs are also likely to be similarly affected, and with Ca_V2.2, might be involved in the observed consequences of knockdown of γ₇ on neurite outgrowth.

These results provoke the hypothesis that γ₇ is a protein that is involved more generally in intracellular transport, which possibly has a role in transport of mRNAs encoding transmembrane proteins, or perhaps a more global role. There are indications that Ca_V2.2 mRNA is subject to transport, because it has been identified in dendritic growth cones (Crino and Eberwine, 1996). Furthermore, N-type Ca²⁺ channels are localised to the presynaptic terminals of peripheral neurons, such as DRG neurons, and local synthesis of transmembrane proteins has recently been demonstrated in axons and axonal growth cones (Brittis et al., 2002; Lin and Holt, 2007). Our finding that shRNA knockdown of endogenous γ₇ markedly decreases neurite outgrowth might indicate that the presence of γ₇ in growth cones maintains a particular level of expression of the products of certain mRNAs.

Materials and Methods

cDNA constructs

The following xFP-tagged cDNAs were used: γ₇–eYFP and γ₇–eCFP (Ferron et al., 2008), TrkA–YFP (Valdez et al., 2007) and eCFP–Ca_V2.2. When CFP–Ca_V2.2 was expressed, Ca_Vβ1b and α₂δ-1 were also included in a ratio 3:2:2. Constructs were cloned into the pcDNA3.1 expression vector, for expression in neurons, unless otherwise stated. Other constructs included dsRed2-ER (Clontech) and dsRed2-Golgi (Clontech), and human γ₇ (GenBank accession number NM031896).

Cell culture

COS-7 cells were cultured and transfected as previously described (Campbell et al., 1995). Primary culture of SCG neurons was performed essentially as previously described (Ferron et al., 2008). Rats (postnatal day 17) were killed by either CO₂ inhalation or cervical dislocation, according to UK Home Office Schedule 1 Guidelines. SCGs were dissected and ganglia were desheathed and lightly gashed before successive collagenase (Sigma) and trypsin (Sigma) treatment, both at 3 mg/ml. To produce a single-cell suspension, ganglia were dissociated by trituration and centrifugation. Dissociated cells were plated onto glass-bottomed plates (MatTeK, Ashland, OR) pre-coated with laminin (Sigma), using 1 ganglion per five plates. Cells were maintained with Liebovitz L-15 medium (Sigma), supplemented with 24 mM NaHCO₃, 10% FBS (Gibco), 33 mM glucose (Sigma), 20 mM L-glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin (Gibco) and 50 ng/ml NGF.

Microinjection

γ₇–YFP or γ₇–CFP in pcDNA3.1, together with other plasmids, were injected into SCG neurons usually in a 1:1 ratio, 18–24 hours after they were placed in culture. Microinjection was performed using an Eppendorf microinjection system on a Zeiss Axiovert 200M microscope using the following settings: 100–150 hPa injection pressure, an injection time of 0.2 seconds and constant pressure of between 40 and 50 hPa. γ₇–XFP cDNA was injected at 25–50 ng/μl diluted in 200 mM KCl.

Transfection of PC12 cells

PC12 cells were grown in Dulbecco's modified Eagle's medium, containing 7.5% foetal bovine serum and 7.5% horse serum. Differentiation was with serum-free medium containing nerve growth factor (NGF; 100 ng/ml murine 7S; Invitrogen, Paisley, UK), which was replenished every 48 hours. Cells were used after 5–7 days of differentiation. Six days before differentiation, PC12 cells were transfected using an Amaxa Nucleofector, according to manufacturer protocol (Lonza), DNA mixes contain GFP (0.5 μg) and either control gnu shRNA or γ₇ shRNA (1.5 μg), as previously described (Ferron et al., 2008).

Transfection of SCG neurons

SCG neurons were transfected using a biolistic PSD-1000/He unit, after 1 day in culture, according to the manufacturer's protocol (Bio-Rad). DNA mixes contained GFP (1 μg) and either control gnu shRNA or γ₇ shRNA (3 μg).

Measurement of neurite outgrowth

Neurons and PC12 cells were replated, 4–6 days after transfection, and cultured in the presence of NGF for a further 24 hours, before measurement of neurite length. Cells were observed using a Zeiss 200M fluorescent microscope. The neurite length of transfected cells was determined using NeuronJ software (Meijering et al., 2004).

Immunocytochemistry and staining

Cells were fixed and permeabilised for immunocytochemistry essentially as previously described (Brice et al., 1997). The primary antibodies used were CD63 (Abcam), EEA1 (BD Biosciences), tau (Millipore), microtubule-associated protein 2 (MAP2, EnCor Biotechnology) and γ₇ (Moss et al., 2002). Endogenous γ₇ was detected using a cyanine 5-tyramide system kit (Perkin Elmer). Secondary goat anti-mouse (Molecular Probes, Eugene, Oregon) or goat anti-rabbit (Sigma) antibodies conjugated to Texas Red or biotin were applied at 10 μg/ml and 5 μg/ml, respectively. When used, Texas-Red-conjugated streptavidin was applied at 3.33 μg/ml. For Epidermal Growth Factor (EGF) receptor localisation, live cells were incubated with EGF conjugated to Texas Red (EGF–TR, Invitrogen) for 1 hour at 37°C. Mitotracker Red (Invitrogen) and Lysotracker Red (Invitrogen) were used to visualise mitochondria and lysosomes in the neurites using the manufacturer's recommended staining protocol. In some experiments, apoptosis was assessed using the dye Hoechst 33258. Cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) to reduce photobleaching, and examined on a confocal laser-scanning microscope (Ziess LSM 510 Meta), using a 40× (1.3 NA) or 63× (1.4 NA) oil-immersion objective.

Live-cell confocal imaging

Time-series imaging of live cells was performed 24 hours after cDNA injection. Cells were maintained at 37°C, and each plate was imaged for no more than 30 minutes. Cells were selected if they exhibited xFP fluorescence in the processes and the soma. The confocal microscope was used at 512×512 pixel resolution. The 458 and 514 nm lines of an argon laser were used to excite the specimen, together with 543 HeNe diode laser for CFP, YFP or dsRED excitation, respectively. Z-stacks were generated by taking slices with a thickness of 1 μm.

Kymograph generation

Neurons were imaged in L15 medium containing 10 mM HEPES and incubated at 37°C throughout. Imaging was performed using the laser lines: 405 nm (4.5%) and 488 nm (2.0%). No bleed-through was present. Images were obtained using 40× objective and 1.5× zoom. A region of interest (500×50 pixels in size) was drawn on a section of neurite between 50 and 200 μm from the soma. Imaging was performed every 385 ms and images were obtained from the CFP and YFP channel with a pixel dwell time of 3.15 μs. The time-series was performed over 10 minutes.

Image analysis

Image analysis for colocalisation was performed using ImageJ. Images were split into channels and adjusted to 0.15 μm pixel resolution The different colour channels were then balanced for intensity and range and the non-specific noise reduced using a Gaussian filter. The ‘Find maxima’ function was then applied to images to count the local maxima points. The resultant maxima map was then enlarged by two pixels by using the ‘dilate’ function twice. The numbers of independent particles were then counted in the separate channels and in the colocalised channel. Percentage colocalisation was calculated by taking the number of colocalised particles divided by the total number of particles in the γ₇ channel.

Analysis of the image time-series was performed using ImageJ. Kymographs were produced using the Multiple Kymograph plug-in. A line was drawn across the neurite length and a kymograph was generated using a bin of 3 pixels. A median (2.0) filter was applied to the kymographs generated in each channel and the image intensity and range matched for each image channel. Kymographs were overlaid using the ‘Projection’ function set to ‘maximum’.

Particle sizes were calculated from image stacks of sympathetic neurons stained for EEA1. A threshold was applied to EEA1 staining and the number and size of the particles counted in each image slice using the ‘Analyze Particles’ function of ImageJ. The mean size and frequency was calculated for each stack.