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J. Biol. Chem., Vol. 277, Issue 16, 14227-14237, April 19, 2002

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Differential Use of Myristoyl Groups on Neuronal Calcium Sensor Proteins as a Determinant of Spatio-temporal Aspects of Ca²⁺ Signal Transduction^*

Dermott W. O'Callaghan, Lenka Ivings, Jamie L. Weiss, Michael C. Ashby, Alexei V. Tepikin, and Robert D. Burgoyne^§

From the Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom

Received for publication, December 10, 2001, and in revised form, February 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca²⁺, hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin , also translocated to this region after a rise in Ca²⁺ concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin  in the absence of calcium, but in the presence of 10 µM Ca²⁺, both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca²⁺. A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca²⁺ concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca²⁺ concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca²⁺/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of ~12 s after a short lag period when Ca²⁺ was elevated. These results demonstrate that closely related Ca²⁺ sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca²⁺ signal transduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcium is a widely used intracellular signal that regulates many different cellular processes in a very specific manner. An aspect that has received considerable attention in recent years is the varied use of Ca²⁺ signal mechanisms that determine spatio-temporal aspects of the changes in Ca²⁺ concentration (1). The existence of highly localized changes in Ca²⁺ concentration in addition to global Ca²⁺ changes has been increasingly highlighted, and these local Ca²⁺ signals are likely to contribute to the specificity of Ca²⁺ actions (2). The specificity of Ca²⁺ signaling is also the result, in part, of the existence of many different Ca²⁺-binding proteins, which act as Ca²⁺ sensors in the transduction of Ca²⁺ signals. Localization of Ca²⁺ sensors could be a significant factor, and the effect of localization of Ca²⁺ sensors on signal transduction has been examined for two such proteins. Calmodulin responds to global Ca²⁺ changes by binding Ca²⁺ and then subsequently to target proteins; it can also translocate into the nucleus (3-5). Protein kinase C isoforms also show specific patterns of translocation to the plasma membrane (6). Analysis of the factors that determine the localization of other Ca²⁺ sensors is likely to be crucial for further understanding of Ca²⁺ signal transduction, but has so far been little studied.

The neuronal calcium sensor (NCS)¹ proteins are a family of high affinity Ca²⁺-binding proteins that can sense Ca²⁺ elevations above resting Ca²⁺ concentration in the range of 0.1-1.0 µM (7). The NCS family includes proteins expressed only in retinal photoreceptors (recoverin (Ref. 8) and the GCAPs (Ref. 9)) or in neurons, in some cases most highly in particular cell types (hippocalcin (Ref. 10), neurocalcins/VILIPs (Ref. 11)) or with more widespread expression (NCS-1/frequenin (Refs. 12 and 13)). All the NCS family members share four characteristic EF hands and a myristoylated N terminus. In the NCS proteins, three (or only two in some family members) of the four EF hands are capable of binding calcium and the EF hand nearest the N terminus (EF1) is altered so that it cannot bind Ca²⁺ (7). The myristoyl tail is a fatty acid post-translationally added to the NCS protein. Its function has been extensively studied in recoverin (14). In recoverin, the myristoyl group is buried in a nonpolar pocket formed by EF1 and the surrounding region in the Ca²⁺-free state (15). When recoverin binds calcium, there is a large conformational change that ejects the myristoyl group from its pocket (16). This exposed hydrophobic tail is then free to interact with the nonpolar cell membranes or hydrophobic protein domains. This exposure of the myristoyl tail on calcium binding is referred to as a "calcium/myristoyl switch" and considered to be primarily a mechanism by which the protein can translocate from the cytosol to intracellular membranes (14, 16). The structural features required for the Ca²⁺/myristoyl switch are apparently conserved in all NCS proteins. The presence of this Ca²⁺-mediated membrane-association process was, therefore, suggested for the other members of the neuronal calcium sensor protein family (16). There are biochemical data from disrupted cells for some of the family members in support of this mechanism (17, 18). A clear understanding of the localization and translocation of these proteins in response to calcium will allow the generation of models of how these proteins modulate and integrate calcium signals. There has, however, been little in vivo work to demonstrate the Ca²⁺/myristoyl switch during elevation of Ca²⁺ concentration within live cells, and so kinetic aspects of the proposed membrane translocation are unknown.

In this study we have examined the potential Ca²⁺-myristoyl switch in three NCS proteins. Hippocalcin is expressed most highly in hippocampal neurons (19) and the closely related neurocalcin  (VILIP-3) in cerebellar Purkinje cells (20, 21). Biochemical studies with these proteins suggest Ca²⁺-dependent membrane interactions (17, 18), but this has not been examined within intact cells. The functions of these two proteins are unknown. In contrast, NCS-1 has been shown to play crucial roles in learning and memory in Caenorhabditis elegans (22), in the release of neurotransmitters (12) and hormones (23, 24), and in the regulation of voltage-gated Ca²⁺ channels (25, 26). In addition, the yeast orthologue of NCS-1 is essential for survival as a regulator of the PI 4-kinase, Pik1 (27). Unlike the other NCS proteins, NCS-1 is found outside of the nervous system (23, 28) and may be a regulator of mammalian Golgi-associated PI 4-kinase  in non-neuronal cells (29). Biochemical data suggest that, in contrast to other NCS proteins, NCS-1 can bind to isolated membranes in the absence of Ca²⁺ (24). We have prepared enhanced GFP-variant constructs of these proteins to compare their Ca²⁺/myristoyl switch mechanisms. We demonstrate different uses of N-terminal myristoylation, which would generate distinct spatio-temporal Ca²⁺ sensing by members of the NCS family and have assessed the kinetics of the Ca²⁺/myristoyl switch-dependent membrane translocation in living cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids-- The EYFP-tagged hippocalcin fusion construct (pHippo-EYFP) was made by the in-frame insertion of a hippocalcin sequence, amplified from rat brain cDNA, into the pEYFP-N1 vector (CLONTECH). The primers contained endonuclease sites (underlined) to facilitate this cloning. The primers were based on the rat nucleotide sequence (GenBank^TM accession no. NM017122). The sense primer was designed to the 5'-untranslated region from 165 to 135 (5'-GGCCGGCTAGCTCTTTTTGGGTCAAATGAG-3'; NheI) and the antisense primer with a mutated stop codon to the region +735 to +765 (5'-CCTCTCACTCGAGAACTGGGAAGCGCTGCT-3'; XhoI). The amplified sequence digested with the endonucleases NheI/XhoI was cloned into the vector pEYFP-N1 digested with the endonucleases NheI/SalI using standard methods. The ECFP-tagged NCS-1 fusion construct (pNCS-1-ECFP) was made by the in-frame insertion of a NCS-1 sequence, amplified from the pNCS-1 plasmid (23), into the pECFP-N1 vector (CLONTECH). The primers contained endonuclease sites (underlined). The sense primer was designed to the region from 0 to +30 (5'-ATACGGTACCATGGGGAAATCCAACAGCAAG-3'; KpnI) and the antisense primer to the region from +492 to +522 (5'-ATGCGGTACCAATACCAGCCCGTCGTAGAGGG-3'; KpnI). The amplified sequence digested with KpnI was inserted into the vector pEYFP-N1 digested with KpnI using standard methods.

The pHippo-(1-14)-EGFP construct was made by the endonuclease digestion of pHippo-EYFP using PstI/NotI. This removed all the hippocalcin and EYFP sequences except for the first 14 codons of the hippocalcin sequence, including the 10 codons required for myristoylation. Into this construct was ligated the coding sequence for EGFP. The nonmyristoylatable hippocalcin and NCS-1 fusion constructs (pHippo(G2A)-EYFP and pNCS-(1(G2A)-ECFP) were both made using the QuikChange^TM site-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands). The primers used for hippocalcin were 5'-TTGGCTCTTCTGCCATGGCCAAGCAGAATAGCAAGCTGCG-3' (sense) and 5'-CGCAGCTTGTATTCTGCTTGGCCATGGCAGAAGAGCCAA-3' (antisense). The primers used for pNCS were 5'-CTGCAGTCGACGGTACCATGGCGAAATCCAACAGCAAGTT-3' (sense) and 5'-AACTTGCTGTTGGATTTCGCCATGGTACCGTCGACTGCAG-3' (antisense).

The neurocalcin  plasmid (pNeurocalcin ) was made by the insertion of the bovine neurocalcin  sequence, amplified from the pDL1312 vector (18) (kindly provided by Daniel Ladant, Institut Pasteur, Paris, France), into the pcDNA 3.1() vector (Invitrogen, Groningen, The Netherlands). The primers contained endonuclease sites (underlined) to facilitate this cloning. The sense primer used was 5'-CCAGGATCCATGGGCAAGCAGAACAGCAA-3' (BamHI), and the antisense primer was 5'-CCGAAGCTTTCAGAACTGGCTAGCACT-3' (HindIII). The NCS-1 EF hand mutant construct, pNCS-1^EF2-4, was made using pNCS-1 as a template and the QuikChange^TM site-directed mutagenesis kit (Stratagene Europe). Three pairs of primers were used sequentially, one pair for each EF hand. The primers used for EF2 were 5'-GCAGGATCGAGTTCTCCCAATTCATCCAGGCTC-3' (sense) and 5'-GAGCCTGGATGAATTGGGAGAACTCGATCCTGC-3' (antisense). The primer pair used for EF3 were 5'-CACCAGAAACCAGATGCTGGACATAGTCGACGCCATTTACC-3' (sense) and 5'-GGTAAATGGCGTCGACTATGTCCAGCATCTGGTTTCTGGTG-3' (antisense). The primer pair used for EF4 were 5'-AACTCTTCAGCAGTTCCAGGAAGGATCCAAGGCCG-3' (sense) and 5'-CGGCCTTGGATCCTTCCTGGAACTGCTGAAGAGTAG-3' (antisense).

The sequence of all these constructs were confirmed by automated sequencing by Oswel (Southampton, United Kingdom (UK)).

Culture and Transfection of HeLa Cells-- HeLa cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Paisley, UK) containing 5% fetal calf serum (Invitrogen) and 1% nonessential amino acids (Invitrogen), incubated at 37 °C in an atmosphere of 5% CO₂ and plated onto glass coverslips in a 24-well plate at 40,000 cells/well. The cells were allowed 4-24 h to adhere before they were transfected. The transfection reaction mixture contained 93 µl of Dulbecco's modified Eagle's medium (Invitrogen), 3 µl of FuGENE^TM (Roche), and 4 µl of plasmid DNA (250 µg/µl). This was incubated at room temperature for 30 min before being added dropwise to HeLa cells in a 24-well plate. The cells were maintained for 8-96 h before being used in experiments.

Immunofluoresence Staining-- Anti-NCS-1 was prepared in rabbits as described previously (23). Anti-neurocalcin  was prepared as a rabbit antiserum using a similar protocol in which rabbits were immunized with purified recombinant neurocalcin  expressed in Escherichia coli and affinity-purified on immobilized recombinant neurocalcin . Anti--adaptin was from Sigma (Poole, UK), anti-GFP from CLONTECH (Basingstoke, UK), and anti-transferrin receptor from Roche (East Sussex, UK). Cells attached to coverslips were washed twice in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM NaH₂PO₄), and fixed in PBS containing 4% formaldehyde for 30 min. The cells were washed twice in PBS and incubated in blocking buffer (0.3% bovine serum albumin, 0.5% Triton X-100 in PBS) for 1 h. The blocking buffer was removed and the primary antibody added at the appropriate dilution (anti--adaptin 1 in 100, anti-GFP 1 in 400, anti-transferrin receptor 1 in 400, anti NCS-1 1 in 1,000, and anti neurocalcin  1 in 100). The primary antibody was incubated for 1-2 h and removed, and the cells were washed three times in blocking buffer. The cells were then incubated for an additional 1 h with the appropriate biotinylated secondary antibody diluted to 1 in 100, (anti-rabbit for anti-NCS-1 and anti-neurocalcin  primary antibodies and anti-mouse for the other primary antibodies; Amersham Biosciences, Buckinghamshire, UK). The cells were washed three times and then incubated in streptavidin-Texas Red (Amersham Biosciences) diluted 1 in 50 for 30 min. The cells were washed three times, and the coverslips were dried and mounted on antifade glycerol (glycerol/PBS 9:1, 0.25% diazabicyclo(2-2-2)octane, 0.002% p-phenyldiamine). The cells were examined on a Zeiss Universal microscope and fluorescence imaged using the following standard Zeiss filter sets: ECFP BG3+LP400, FT460, LP495; EYFP, BP450-490, FT510, BP515-565; Texas Red, BP546/12, FT850, LP590.

Polyacrylamide Gel Electrophoresis and Western Blotting-- The cells of each well in a 24-well plate were lysed using 100 µl of Laemmli buffer (Sigma, Dorset, UK)/well and boiled for 10 min. The samples were loaded onto and separated using a 15% SDS-polyacrylamide gel and then blotted onto nitrocellulose by transverse electrophoresis. The nitrocellulose membranes were washed twice in PBS and placed in blocking buffer (PBS containing 5% bovine serum albumin, 5% fetal calf serum, 5% Marvel^TM milk powder, 0.5% Tween 20^TM) for 1 h. The PBT was removed and the primary antibody added at the appropriate dilution in PBS, 3% Marvel^TM milk powder (anti-NCS-1 at 1in 1000, affinity-purified anti-neurocalcin  at 1 in 400, and anti-GFP at 1 in 400 from CLONTECH, Basingstoke, UK). The primary antibody was incubated for 1-2 h and removed, and the membranes were washed three times. The membranes were then incubated for an additional 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody diluted to 1 in 400 with PBT, (anti-rabbit for anti-NCS-1 and anti-neurocalcin  primary antibodies and anti-mouse for anti-GFP primary antibodies; Sigma). The membranes were washed once in PBT, three times in PBS, and once with distilled water. The signal was detected using enhanced chemiluminescence (Amersham Biosciences).

Stimulation of Cells with Ionomycin and Imaging of Fixed Cells-- Transfected HeLa cells were washed three times in Krebs-Ringer buffer (145 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 1.2 mM NaH₂PO₄, 10 mM glucose, 20 mM HEPES, pH 7.4). The cells were then incubated for 10 min in 1 µM ionomycin in Krebs-Ringer buffer in the presence or absence of 3 mM CaCl₂ at 37 °C. The Krebs-Ringer buffer was removed, and the cells were fixed in 4% formaldehyde for immunofluorescence staining and imaged using a Zeiss Universal microscope fitted with a Nikon Coolpix 995 digital camera.

Confocal Laser Scanning Microscopy on Living Cells-- For confocal laser scanning microscopy, live transfected HeLa cells were examined with a Leica TCS-SP-MP microscope (Leica Microsystems, Heidelberg, Germany) using a 166.27-µm pin-hole and a 63× water immersion objective with a 1.2 numerical aperture. For optimal imaging of the spatial distribution of hippocalcin-EYFP, the cells were excited at 514 nm and light collected at 545-625 nm and fluorescence images were collected every 1.86 s with alternate phase-contrast images to check for changes in cell morphology. For dual imaging of hippocalcin-EYFP and NCS-1-ECFP, the cells were excited at 514 nm with light collected at 560-600 nm for EYFP or excited at 458 nm for and light collected at 465-500 nm for ECFP detection. In experiments on the kinetics of Ca²⁺-dependent translocation, cells were loaded with Fura Red (Molecular Probes) by incubation in 5 µM Fura Red in growth medium for 30 min. The cells were then excited at 488 nm and light collected at 625-725 nm for Fura Red emission and at 525-590 for hippocalcin-EYFP emission and images collected every 137 ms. Ionomycin was added to the bath solution a final concentration of 3 µM.

Permeabilization Experiments-- Transfected HeLa cells were washed three times in Krebs-Ringer buffer and incubated for 15 min in 10 µM digitonin in 300 µl of permeabilization buffer (139 mM potassium glutamate, 20 mM PIPES, 5 mM EGTA, pH 6.5) in the presence or absence of 10 µM free calcium at 37 °C. If the cells were to be fixed, the buffer was removed and 500 µl of 4% formaldehyde was added per well. For the leakage experiments, the permeabilization buffer was removed and centrifuged briefly at 12,000 rpm to sediment any detached cells. The proteins were concentrated by precipitation with cold methanol (30) and resuspended in 100 µl of Laemmli buffer (Sigma). The cells remaining in the wells were solubilized in Laemmli buffer. All samples were boiled for 10 min and then used for polyacrylamide gel electrophoresis and Western blotting.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression and Localization of NCS Proteins in HeLa Cells-- To determine the localization of the neuronal calcium sensing proteins NCS-1 and hippocalcin in living cells, constructs were made that fused the C terminus of these proteins to the fluorescent proteins ECFP and EYFP to produce the plasmids pNCS-1-ECFP and pHippo-EYFP, respectively. Vector-derived sequence produced a 10- or 13-amino acid linker between the NCS-1 protein and hippocalcin, respectively, and the fluorescent protein. These constructs were transfected into HeLa cells, which do not express detectable levels of the proteins endogenously, and the expressed proteins observed in living and fixed cells. HeLa cells were chosen as a convenient cell type to allow examination of the basic localization and translocation mechanism of these proteins within a cellular environment. To demonstrate their correct expression, Western blots were carried out on samples from transfected cells. Fig. 1 shows that the expressed fusion proteins were of the expected size (~60 kDa) and recognized by both anti-GFP and specific antibodies. Anti-NCS-1 was used for cells transfected with pNCS-1-ECFP. Expression of Hippo-EYFP could be demonstrated using an antiserum prepared against the closely related protein neurocalcin  (96% identical (Ref. 7)). The 60-kDa band representing the fusion proteins was not seen in samples from cells transfected with the control vectors pEYFP-N1 and pECFP-N1 but instead a polypeptide band corresponding to the free fluorescent proteins.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Demonstration of fusion protein expression in transfected HeLa cells. Western blots used an anti-GFP antibody (A and C), an anti-NCS-1 antibody (B), or an anti-neurocalcin  antibody with cross-reactivity for hippocalcin (D). The samples were lysates from cells transfected with pNCS-1-ECFP, pHippo-EYFP, pECFP-N1, and pEYFP-N1. A polypeptide was detected with both the anti-GFP and anti-NCS-1 antibodies at the expected size (60 kDa) in cells transfected with pNCS-1-ECFP. A polypeptide was also detected with both the anti-GFP and anti-neurocalcin  antibodies at the expected size (60 kDa) in cells transfected with pHippo-CFP. This demonstrated that both fusion proteins were being expressed correctly. For both the controls, pEYFP-N1 and pECFP-N1, the only band detected with any of the antibodies was a 27-kDa band, corresponding to EYFP or ECFP, using the anti-GFP antibody.

Fig. 2 shows the localization of proteins encoded by pNCS-1-ECFP, pHippo-EYFP, and constructs expressing the free fluorescent proteins ECFP and EYFP. The location patterns were not dependent upon expression levels of the proteins and were indistinguishable in cells transfected for between 8 and 96 h and with varying levels of fluorescence intensity at each time point. The free fluorescent proteins were found in the cytoplasm but were particularly concentrated in the nucleus (Fig. 2, A and B). The hippocalcin-EYFP fusion protein was present in the cytoplasm and also in the nucleus but to a lesser extent than ECFP or EYFP (Fig. 2C). The NCS-1 fusion protein, in contrast, had a radically different distribution. The fluorescence of NCS-1-ECFP was at its most intense in a perinuclear region to one side of the nucleus, whereas the nucleus was devoid of fluorescence (Fig. 2D). Additionally, NCS-1 ECFP was also associated with plasma membrane, usually in a patchy distribution. The presence of the ECFP and EYFP tags did not influence the localization of the NCS proteins, as similar localization patterns were seen with wild-type protein detected by immunofluorescence (shown below). In addition, similar localization patterns were seen in living and fixed cells. Previous work has shown that endogenous NCS-1 in COS cells is also localized to a perinuclear compartment that overlaps partially with the trans-Golgi network marker -adaptin (28). Fig. 3 (A-C) shows that ECFP-NCS-1 also partially co-localizes with -adaptin. In contrast, ECFP-NCS-1 did not significantly co-localize with the transferrin receptor used as a marker of endosomal compartments (Fig. 3, D-F).

View larger version (74K): [in this window] [in a new window]   Fig. 2.   Differences in localization of fluorescent proteins in HeLa cells. The cells were transfected with pEYFP-N1 (A), pECFP-N1 (B), pHippo-EYFP (C), or pNCS-1-ECFP (D). Expressed ECFP-N1 and YFP-N1 was found diffusely throughout the cell but with an accumulation of fluorescence in the nucleus. Expressed hippocalcin-EYFP had a similar pattern of fluorescence but at lower levels in the nucleus. The expression of NCS-1-ECFP differed radically from the other proteins with fluorescence being concentrated in a perinuclear region of the cell. The plasmids used are shown schematically below each panel. The scale bar represents 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Comparison of the intracellular localization of NCS-1-ECFP (B and E) with the trans-Golgi marker -adaptin (A) and the endosomal marker transferrin receptor (D). Partial co-localization can be seen between the -adaptin and NCS-1-ECFP, shown by the yellow in the overlay (C). The cell on the left in A is a nontransfected cell in which the -adaptin localization was similar to those cells expressing NCS-1-ECFP. There was little co-localization between NCS-1-ECFP expression and the transferrin receptor (F), showing that NCS-1 was not present at significant levels on early endosomes. The scale bar represents 20 µm.

Effect of Ca²⁺ Elevation on Localization of NCS Proteins-- The effect of a global elevation in intracellular Ca²⁺ concentration on the localization of the NCS fusion proteins was assessed by treating cells with 1 µM ionomycin in the presence of external Ca²⁺. The NCS-1 fusion protein did not change its localization in response to an increase in intracellular Ca²⁺ levels (Fig. 4, A and B). This suggests that, at both resting and raised levels of Ca²⁺, NCS-1 has its myristoyl tail exposed allowing membrane association. In contrast, following Ca²⁺ elevation, the hippocalcin fusion protein translocated from its apparent diffuse cytosolic localization to a predominantly perinuclear pattern (Fig. 4, C and D). This perinuclear localization appeared to be very similar to that seen with the NCS-1 fusion protein at resting Ca²⁺ levels. A more diffuse localization was also present, spreading out to the cell periphery consistent with an additional patchy plasma membrane localization. The Ca²⁺-dependent translocation of hippocalcin-EYFP was rapidly reversed when the external buffer was exchanged for a buffer without added Ca²⁺ (data not shown). In cells transfected with a construct expressing an untagged version of neurocalcin , another member of the NCS family that is closely related to hippocalcin (7), a similar diffuse localization was seen in unstimulated cells using immunofluorescence to detect the protein in fixed cells (Fig. 4E). Translocation of neurocalcin  to the perinuclear region plasma membrane structures was also observed following an increase in intracellular Ca²⁺ by ionomycin treatment (Fig. 4F). The apparent translocation of hippocalcin and neurocalcin  from the cytosol to membranes is consistent with the proposed Ca²⁺/myristoyl switch mechanism and suggests that these proteins only become membrane-associated at cytosolic Ca²⁺ concentrations above those in resting cells.

View larger version (65K): [in this window] [in a new window]   Fig. 4.   The effect of raising the intracellular calcium concentration using the calcium ionophore ionomycin on the localization of NCS proteins. A and B, cells expressing hippo-EYFP; C and D, cells expressing NCS-1-ECFP; E and F, cells expressing neurocalcin  and stained with anti-neurocalcin  antiserum. Cells were incubated in the absence (control) or presence of 1 µM ionomycin. Both hippocalcin-EYFP and neurocalcin  translocated to the perinuclear compartment in cells treated with ionomycin. NCS-1-ECFP localization was unaffected by ionomycin treatment. The plasmids used are shown schematically below each panel. The scale bar represents 20 µm.

In fixed cells the localization of hippocalcin-EYFP after ionomycin treatment was similar to the perinuclear localization of NCS-1-ECFP. The extent of co-localization of the two proteins in living cells was examined directly using laser scanning confocal microscopy of cells transfected to express both hippocalcin-EYFP and NCS-1-ECFP. In resting cells, hippocalcin-EYFP showed a diffuse localization (Fig. 5A). In contrast, NCS-1-ECFP was associated with the perinuclear compartment and also had a patchy distribution on the plasma membrane (Fig. 5B). Following treatment with ionomycin for 2 min, hippocalcin-EFYP was observed to have translocated (Fig. 5D). A proportion of hippocalcin-EYFP remained in the nucleus but in the rest of the cell showed almost complete co-localization with NCS-1-CFP (Fig. 5, D-F).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Hippocalcin-EYFP and NCS-1-ECFP show similar localizations in co-transfected HeLa cells stimulated with ionomycin. HeLa cell were co-transfected to express both hippocalcin-EYFP and NCS-1-ECFP and living cells examined using confocal laser scanning microscopy. Before ionomycin treatment hippocalcin-EYFP (A) was diffusely distributed, whereas NCS-1-ECFP (B) was concentrated in the perinuclear compartment and on plasma membrane patches with little overlap with hippocalcin-EYFP (C). After treatment with ionomycin for 2 min, hippocalcin-ECFP (D) and NCS-1-ECFP (E) were both localized to the same regions except that hippocalcin-EYFP was also seen in the nucleus. Extensive co-localization was evident in the overlapped image (F). The scale bar represents 20 µm.

The data from intact cells on the effects of raising Ca²⁺ with ionomycin are consistent with membrane association of NCS-1-ECFP even at low Ca²⁺ concentration and Ca²⁺-dependent translocation of cytosolic hippocalcin-EYFP and neurocalcin  to membranes. To confirm this interpretation, the localization of the proteins was examined after digitonin permeabilization to allow soluble proteins to leak out of the cells. Transfected cells were permeabilized with 10 µM digitonin in a buffer containing either 0 or 10 µM free Ca²⁺. At 10 µM free Ca²⁺ concentration, the hippocalcin fusion protein translocated to the perinuclear compartment as seen with ionomycin treatment (Fig. 6). Cells transfected with the pNCS-1-ECFP also retained their perinuclear fluorescence after permeabilization in 10 µM Ca²⁺. These results showed that the perinuclear localization was because of the fusion proteins associating with intracellular structures. When transfected cells were permeabilized in a buffer containing 0 Ca²⁺, essentially all fluorescence was rapidly lost from pHippo-EYFP-transfected cells except a small amount that remained trapped in the nucleus. The hippocalcin fusion protein was, therefore, essentially completely cytosolic at 0 Ca²⁺ levels and free to diffuse from the permeabilized cells. Permeabilization of pNCS-1-CFP-transfected cells in 0 Ca²⁺ buffer did not affect the fluorescence pattern. The NCS-1 fusion protein remained attached to the perinuclear membrane construct regardless of the presence or absence of Ca²⁺ (Fig. 6). The data from examination of fluorescence were confirmed by biochemical analysis using Western blotting of cells and medium containing leaked proteins (Fig. 7). The majority of the NCS-1-ECFP was retained in the permeabilized cells irrespective of Ca²⁺ concentration (Fig. 7). In contrast, the majority of hippocalcin-YFP leaked into the medium at 0 Ca²⁺ but the protein was fully retained in cells at 10 µM free Ca²⁺ (Fig. 7). Retention was half-maximal at ~0.2-0.3 µM free Ca²⁺ (data not shown). Similar Ca²⁺-dependent translocation to the perinuclear region (Fig. 6, E and F) and Ca²⁺-dependent retention in permeabilized cells (Fig. 7) was seen in cells transfected with untagged neurocalcin  with expression detected using anti-neurocalcin  antiserum. The extent of retention of the NCS proteins in the presence of Ca²⁺ (Fig. 7) suggested that the majority of the expressed proteins were myristoylated.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Localization of NCS proteins in transfected HeLa cells permeabilized with digitonin in the presence or absence of 10 µM Ca2+. The localization pattern of NCS-1-ECFP was unaffected by the permeabilization in digitonin for 15 min and the presence (A) or absence (B) of calcium. The localization of hippocalcin-EYFP was affected dramatically by the presence or absence of calcium during permeabilization. Hippocalcin-EYFP rapidly translocated to the perinuclear region in the presence of high calcium (C). When calcium was absent hippocalcin-EYFP leaked from the cells (D), although in some cells the fluorescent protein remained trapped in the nucleus. Neurocalcin  in transfected cells was detected using anti-neurocalcin  antiserum. It was also translocated to the perinuclear compartment in the presence of 10 µM Ca2+ (E) and was lost from the cells in the absence of Ca2+ (F). The scale bar represents 20 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Detection by Western blotting of retained and leaked proteins from transfected HeLa cells after digitonin permeabilization. Transfected HeLa cells were permeabilized for 15 min in the absence or presence of 10 µM Ca2+, and samples of the cells and medium probed with anti-GFP for pNCS-1-ECFP and pHippo-EYFP-transfected cells and with anti-neurocalcin  for pNeurocalcin -transfected cells.

Requirement for Ca²⁺ Binding and Myristoylation for Perinuclear Localization of NCS Proteins-- The data so far showed that localization of NCS-1-ECFP to the perinuclear compartment was independent of Ca²⁺ concentration in intact and permeabilized cells. To confirm that Ca²⁺ binding was not required for the localization of NCS-1, use was made of constructs encoding untagged wild-type NCS-1 and a version with all three functional EF hands (EF2-4) mutated to eliminate Ca²⁺ binding. These mutations (E84Q, E120Q, and E168Q) replaced conserved acidic residues in the EF hands required for Ca²⁺ coordination (18). Cells were fixed after permeabilization, and NCS-1 localization was determined by immunofluorescence staining. The untagged wild-type protein was found to be localized to the perinuclear compartment in the presence or absence of Ca²⁺ (Fig. 8, A and B) as previously seen for NCS-1-ECFP. The same localization pattern was seen with the NCS-1(EF2-4) mutant, indicating a complete lack of dependence on Ca²⁺ binding for the localization of NCS-1 (Fig. 8, C and D).

View larger version (68K): [in this window] [in a new window]   Fig. 8.   Localization of NCS-1 to the perinuclear compartment does not require Ca2+ binding. HeLa cells were transfected with plasmids encoding wild-type untagged NCS-1 (A and B) or NCS-1(EF2-4) with mutations in all three functional EF hands (C and D). Cells were permeabilized with digitonin in the presence or absence of 10 µM Ca2+ for 15 min and fixed for immunocytochemical staining with anti-NCS-1. Both wild-type and the NCS-1(EF2-4) mutant were associated with the perinuclear compartment irrespective of Ca2+ concentration. The scale bar represents 20 µm.

To determine whether myristoylation of the NCS proteins was required for their localization and translocation, constructs were made with the second glycine residue, essential for myristoylation (31), mutated to an alanine in both the NCS-1 and hippocalcin fusion proteins. Fig. 9 shows that intact cells transfected either with pHippo(G2A)-EYFP or pNCS-1(G2A)-ECFP showed a diffuse fluorescence pattern compatible with cytosolic localization of the proteins (Fig. 9, A and C). No indication of translocation was seen in response to elevated intracellular Ca²⁺ levels. In addition, when the cells were permeabilized, even in the presence of 10 µM free Ca²⁺, the unmyristoylated fusion proteins rapidly leaked out of the cells with only low levels of fluorescence remaining in the nuclei (Fig. 9, B and D). These data indicate that myristoylation was essential for the perinuclear localization of NCS-1 and for Ca²⁺-dependent translocation of hippocalcin.

View larger version (78K): [in this window] [in a new window]   Fig. 9.   Localization and translocation of NCS proteins requires myristoylation. HeLa cells were transfected with plasmids encoding hippocalcin-EYFP (A and B) and NCS-1-ECFP (C and D), each with a mutation (G2A) to remove the myristoylated glycine residue or with a construct consisting of the minimal myristoylation sequence (residues 1-14 of hippocalcin). In intact cells hippocalcin (G2A)-EYFP and NCS-1(G2A)-ECFP had a diffuse location, and both proteins were lost from the cells following permeabilization with digitonin for 15 min in the presence of 10 µM Ca2+. Hippocalcin-(1-14)-EGFP was associated with a perinuclear compartment in both intact and permeabilized cells. The scale bar represents 20 µm.

To determine the degree to which the exposure of the myristoyl tail was sufficient for perinuclear membrane localization, a construct was made that fused the myristoylation sequence taken from hippocalcin (hippocalcin-(1-14)) to EGFP. This was expressed in HeLa cells, and its localization is shown in Fig. 9E. The distribution of the hippocalcin-(1-14)-EGFP protein appeared to be much the same as the perinuclear localization of the NCS proteins. There was strong perinuclear fluorescence together with some possible plasma membrane localization. This indicates that the myristoyl tail can be the sole determinant for intracellular targeting of these proteins. Using the permeabilization assay in 0 Ca²⁺ solution, it was found that the hippocalcin-(1-14)-EGFP remained in the cells, concentrated in the perinuclear region (Fig. 9F). This demonstrated that the myristoyl group accounted for all the intracellular targeting seen with the NCS-1 and hippocalcin fusion proteins but not the Ca²⁺ dependence of hippocalcin localization.

Kinetics of the Ca²⁺/Myristoyl Switch in Living Cells-- To examine the kinetics of the Ca²⁺-dependent translocation of hippocalcin-EYFP, live cells were imaged using confocal laser scanning microscopy and fluorescence was monitored following addition of ionomycin (Fig. 10). Cells initially showed a diffuse fluorescence indicative of cytosolic and nuclear localization. Within a few seconds of ionomycin addition, translocation of hippocalcin-EYFP occurred with the appearance of a bright punctate spots with a perinuclear localization. In addition, hippocalcin-EYFP also appeared to a lesser extent associated with the plasma membrane in distinct patches (arrows in the last frame of Fig. 10), confirming the earlier interpretation from epifluorescence microscopy. The time course of translocation was similar to that previously seen for the Ca²⁺-dependent C₂ domain of PKC (6) or the pH domain of PLC (32). Translocation of hippocalcin-EYFP was also observed under more physiological conditions following stimulation with histamine and was reversible several minutes after the initial translocation.²

View larger version (79K): [in this window] [in a new window]   Fig. 10.   Confocal laser scanning microscopy showing the time course of translocation of hippocalcin-EYFP in response to Ca2+ elevation. HeLa cells transfected with pHippo-EYFP were imaged using confocal microscopy, and the panels show a compilation of time-lapse images at the times indicated from 0 to 68.4 s. In this series, frames were taken every 1.9 s to give optimal spatial resolution. Addition of ionomycin at time 0 was followed by translocation of hippocalcin-EYFP to perinuclear vesicular structures and to patches on the plasma membrane (arrows in the last frame). The scale bar represents 20 µm.

The relationship of kinetics of the Ca²⁺/myristoyl switch for membrane translocation to changes in cytosolic Ca²⁺ concentration was examined in cells expressing hippocalcin-EYFP and loaded with the fluorescence Ca²⁺ indicator Fura Red. Dual imaging using confocal microscopy indicated that changes in hippocalcin-EYFP localization could be measured in parallel with an expected decrease in Fura Red emission because of Ca²⁺ elevation following ionomycin addition. Measurement at high time resolution (1 frame/137 ms) allowed a comparison of the time course of hippocalcin-EYFP translocation and changes in cytosolic Ca²⁺ concentration (Fig. 11). Cytosolic Ca²⁺ concentration was monitored over the whole cell. Translocation was assessed by monitoring the fluorescence of 9 spots/cell where translocation occurred and averaging the time courses for each cell. Cytosolic Ca²⁺ concentration began to increase in a homogeneous manner over the whole cell immediately upon ionomycin addition. In contrast, hippocalcin-EYFP translocation began only after a lag period of ~2 s (2.17 ± 0.13 s from 4 cells). The time course of translocation, which then followed, could be fitted with a single hyperbolic function, indicating a first order reaction, with a half-time of ~12 s.

View larger version (26K): [in this window] [in a new window]   Fig. 11.   Kinetics of changes in cytosolic Ca2+ concentration and hippocalcin-EYFP translocation. HeLa cells transfected to express hippocalcin-EYFP were loaded with Fura Red and imaged by confocal microscopy. To provide optimal time resolution, frames were taken every 137 ms. The decrease in Fura Red fluorescence (top) began as soon as ionomycin was added. The lower panel shows averaged data from 9 regions of translocation in the same cell. The standard errors of the means are not shown for clarity but did not exceed ±4% of the mean values. Translocation began after a 2-s lag and then followed first order kinetics (as shown by the fitted curve) with a half-time of 11.6 s.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It has become increasingly apparent that the control of various cellular activities involves proteins that either are tightly co-localized in signaling complexes or can translocate in a dynamic way into such complexes (33, 34). Studies on protein dynamics have been complemented by increasing information on the differential use of local versus global Ca²⁺ signals in the regulation of cell function (2). In contrast, relatively little is known about the dynamics and localization of Ca²⁺ sensor proteins apart from calmodulin and protein kinase C (3-6). Here we have examined the localization of three members of the NCS family of Ca²⁺ sensors and demonstrate that, under the same conditions in the same cellular context, their N-terminal myristoyl groups are used in quite different ways to determine their localization. We also provide data on the kinetics of the Ca²⁺-myristoyl switch mechanism in living cells. The classic example of the NCS proteins has been the well characterized retinal protein recoverin, which is believed to show Ca²⁺-dependent translocation to membranes via a Ca²⁺/myristoyl switch mechanism based on biochemical and structural analyses (14-16), although this has not been confirmed in living cells. The differences in behavior we observed for other NCS proteins are surprising, as the features within the sequence of recoverin that determine the Ca²⁺/myristoyl switch mechanism including the inactivated first EF hand and hydrophobic residues that cradle the myristoyl group are highly conserved in all of the NCS proteins (15). It has recently been shown, however, that residues within EF1 of GCAP-2 are crucial for binding to guanylyl cyclase (35). The inactivated EF hand may therefore be more generally important in NCS proteins for target protein interactions.

In HeLa cells, NCS-1 was constitutively associated with, and hippocalcin and neurocalcin  could become associated with, membranes of a perinuclear compartment overlapping the trans-Golgi network (shown using  adaptin as the marker). All three proteins also appeared to associate with the plasma membrane. This localization was not dependent on the presence of the fluorescent tags and is likely to be the result of the preference of the myristoyl group for cholesterol- and sphingolipid-rich domains (36). Although hippocalcin and neurocalcin  are expressed only in neurons, NCS-1 is expressed in many non-neuronal cell types (23, 28, 37). Association with a perinuclear compartment overlapping the trans-Golgi was reported for endogenous NCS-1 in COS-7 cells (28). Importantly, studies on NCS-1 in fixed brain slices indicated by the use of electron microscopy that NCS-1 was present on cisternae of the trans-Golgi complex, in addition to its presence in both pre- and post-synaptic structures (38). The Ca²⁺ dependence of the localization of NCS-1 to the trans-Golgi was not determined in these studies, and so it remained possible that resting cytosolic Ca²⁺concentration was sufficient to activate a Ca²⁺/myristoyl switch in NCS-1 as it has a high affinity for Ca²⁺ (24, 39). We have demonstrated here that NCS-1 association with the perinuclear compartment is independent of free Ca²⁺and that NCS-1 does not need functional Ca²⁺-binding motifs for this localization. The presence of NCS-1 on the trans-Golgi network may be related to its regulation of PI 4-kinase  (28, 29), which has the same localization. The ability of other NCS proteins such as hippocalcin and neurocalcin  to interact with PI 4-kinase  has not been examined, and their localization in neurons has only been investigated after fixation without elevation of intracellular Ca²⁺. The presence of NCS-1 on the plasma membrane that we have observed would be consistent with the known involvement of NCS-1 in Ca²⁺ channel regulation (25, 26).

The membrane association of NCS-1 and hippocalcin in HeLa cells was entirely dependent upon myristoylation. In addition, a minimal myristoylation sequence (hippocalcin-(1-14)) was sufficient for localization to the perinuclear compartment. The latter result is compatible with previous work on the targeting of constructs bearing the myristoylation sequence of Src family kinases (40). The difference between the NCS proteins that we investigated, however, was that NCS-1 was membrane-associated even at nominally 0 Ca²⁺, whereas hippocalcin and neurocalcin  required Ca²⁺ elevation above resting levels to translocate from the cytosol to a membrane-associated pool. The membrane association of NCS-1 additionally did not require the presence of any functional EF hand motifs. These data suggest that, in NCS-1, the myristoyl group is exposed even in the Ca²⁺ free form but that hippocalcin and neurocalcin  have a classic recoverin-like Ca²⁺/myristoyl switch. Their myristoyl group would, therefore, be sequestered in the Ca²⁺-free form and then exposed in the Ca²⁺-bound form to allow the proteins to become membrane-associated. The findings on NCS-1 are consistent with biochemical data showing that recombinant myristoylated NCS-1 became bound to membranes in the absence of Ca²⁺ (24). In addition, analysis of the yeast orthologue FRQ1 by NMR suggested that the myristoyl group might always be solvent-exposed (41). This idea could be directly confirmed by x-ray crystallography, but so far the only structure solved is for nonmyristoylated NCS-1 in its Ca²⁺-bound form (28). The study of yeast FRQ also showed, from a biochemical analysis of cell fractions, that some Ca²⁺-dependent membrane association occurred even in the absence of myristoylation (41). In contrast, in intact or permeabilized HeLa cells, NCS-1 localization to the perinuclear membrane compartment was completely dependent upon its myristoylation. This may reflect differences in behavior of the proteins when examining them in a cellular context or in cell extracts and demonstrates the importance of carrying out these studies on proteins within cells.

The different usage of the myristoyl group in the NCS proteins is likely to be important for differential aspects of Ca²⁺ signal transduction. EF hand motifs in Ca²⁺ sensors bind Ca²⁺ very rapidly with binding limited only by the rate of Ca²⁺ diffusion (42). The off-rate is also rapid in Ca²⁺ sensors but can be slowed in Ca²⁺-sensor and Ca²⁺-buffer proteins by changes in the residue at position 9 of the EF hand (42). This allows fine-tuning of the kinetics of activation and time scale of activity of EF hand proteins. The NCS proteins show the properties of fast Ca²⁺ sensors with rapid on- and off-rates of Ca²⁺ binding and inactivation occurring on a millisecond time scale (43, 44). It would be predicted that NCS-1, with an affinity of 0.3 µM, would be able to react to Ca²⁺ concentration changes in a time course of no more than milliseconds and retain its bound Ca²⁺ with a time constant of ~20 ms when Ca²⁺ concentration falls (see similar calculations for GCAP1 (Ref. 44)). In contrast, hippocalcin and neurocalcin  would respond to more global Ca²⁺ signals and only be able to exert their regulatory effects at target membranes after translocation from the cytosol. This would also require the Ca²⁺ elevation to last over seconds and could preclude activation of hippocalcin and neurocalcin  by brief localized Ca²⁺ pulses. NCS-1 appears, therefore, to be a protein that would be able to sense and transduce transient and local Ca²⁺ signals occurring close to the membranes to which it is attached on a millisecond time scale, whereas other NCS proteins such as hippocalcin and neurocalcin  would have a different spatio-temporal pattern of activation requiring longer lasting Ca²⁺ elevations.

The dual imaging of both hippocalcin-EYFP localization and cytosolic Ca²⁺ concentration in living cells provides information on the kinetics of the Ca²⁺/myristoyl switch and membrane translocation in vivo. Hippocalcin and neurocalcin  have high affinities for Ca²⁺ when assayed in vitro (17, 18), and it is conceivable that membrane association could occur already at resting cytosolic Ca²⁺ concentration. We have shown, however, that membrane association of hippocalcin-EYFP does require Ca²⁺ elevation above resting levels, and that this occurs with a half-time of ~12s and follows a short (2 s) lag period after Ca²⁺ elevation. The translocation kinetics were consistent with a first-order reaction. These results are consistent with a rapid conformational change in the protein on Ca²⁺ binding followed by a diffusion-limited translocation to membranes. The time course over which hippocalcin-EYFP accumulates indicates that it would function in Ca²⁺ signal transduction over seconds and thus be a considerably slower Ca²⁺ sensor in vivo than NCS-1.

NCS-1 is the evolutionarily most ancient of the NCS proteins being represented in yeast (27). An additional form most similar to neurocalcin  is present in nematodes, and other NCS proteins appeared later in evolution (7). It seems that an event must have occurred during evolution whereby a membrane-anchored protein NCS-1 gave rise to new forms in which the myristoyl group is sequestered in the Ca²⁺-free form of the protein and thus used in a radically different way. It is likely that further alterations in the use of the myristoyl group of NCS protein has arisen; it is known, for example, from biochemical assays that the photoreceptor protein GCAP-2 shows a reversed Ca²⁺-dependent membrane association and dissociates from membranes as Ca²⁺ is elevated (45). This family of Ca²⁺ sensors, therefore, provides an intriguing example of how a single post-translational modification, N-terminal myristoylation, has been adapted during evolution to allow the appearance of a diverse family of proteins able to mediate distinct spatial and temporal patterns of Ca²⁺ signal transduction.

	FOOTNOTES

^* This work was supported in part by a grant from the Wellcome Trust (to R. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Wellcome Trust Prize studentships.

^§ To whom correspondence should be addressed. Tel.: 44-151-794-5305; Fax: 44-151-794-5337; E-mail: burgoyne@liverpool.ac.uk.

Published, JBC Papers in Press, February 8, 2002, DOI 10.1074/jbc.M111750200

² D. W. O'Callaghan, L. Ivings, J. L. Weiss, M. C. Ashby, A. V. Tepikin, and R. D. Burgoyne, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NCS, neuronal calcium sensor; GCAP, guanylyl cyclase-activating protein; VILIP, visinin-like protein; PI, phosphatidylinositol; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES