Part 3:Magnesium Efflux From Drosophila Kenyon Cells Is Critical For Normal And Diet-enhanced Long-term Memory
Mar 17, 2022
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was restricted to adult flies, suggesting UEX has a more sustained role in neuronal physiology. In contrast, knocking down uex expression in either the abs or a0b0 neurons did not impair LTM. The activity of a0 b0 neurons is required after training to consolidate appetitive LTM (Krashes and Waddell, 2008), whereas abc and abs KC output, together and separately, is required for its expression (Krashes and Waddell, 2008; Perisse et al., 2013). Therefore, observing normal LTM performance in flies with uex loss-of-function in abs and a0b0 neurons argues against a general deficiency of ab neuronal function when manipulating uex.
Dietary Mg2+ could not enhance the defective LTM performance of flies that were constitutively uex mutant or harbored ab KC-restricted uex loss-of-function. However, expressing uex in the ab KCs of uex mutant flies restored the ability of Mg2+ to enhance performance. Therefore, the ab KCs are the cellular locus for Mg2+-enhanced memory in the fly.
It perhaps seems counterintuitive that UEX-directed magnesium efflux is required in KCs to support the memory-enhancing effects of Mg2+ feeding, when dietary Mg2+ elevates KC [Mg2+]i . At this stage, we can only speculate as to why this is the case. We assume that the brain and ab KCs, in particular, have to adapt in a balanced way to the higher levels of intracellular and extracellular Mg2+ that result from dietary supplementation. Our live-imaging of KC [Mg2+]i in wild-type and uex mutant brains suggests that UEX-directed efflux is likely to be an essential factor in the active, and perhaps stimulus-evoked, homeostatic maintenance of these elevated levels.
A number of mammalian cell-types extrude Mg2+ in a cAMP-dependent manner, a few minutes after being exposed to b-adrenergic stimulation (Romani and Scarpa, 2000; Vormann and Gu¨n- ther, 1987; Jakob et al., 1989; Romani and Scarpa, 1990b; Romani and Scarpa, 1990a; Vormann and Gu¨nther, 1987; Gu¨nther et al., 1990; Howarth et al., 1994). The presence of a CNBH domain suggests that UEX and CNNMs could be directly regulated by cAMP. We tested the importance of the CNBH by introducing an R622K amino acid substitution that should block cAMP binding in the UEX CNBH. This subtle mutation abolished the ability of the uexR622K transgene to restore LTM performance to uex mutant flies. We also used CRISPR to mutate the CNBH in the native uex locus. Although deleting the CNBH from CNNM4 abolished Mg2+ efflux activity (Chen et al., 2018), flies homozygous for the uexT626NRR lesion were viable, demonstrating that they retain a sufficient level of UEX function. However, these flies exhibited impaired immediate and long-term memory. In addition, the performance of uexT626NRR flies could not be enhanced by Mg2+ feeding. These data demonstrate that an intact CNBH is a critical element of memory-relevant UEX function. Binding of clathrin adaptor proteins to the CNNM4 CNBH has been implicated in basolateral targeting (Hirata et al., 2014), suggesting that UEXT626NRR might be inappropriately localized in KCs. Furthermore, KC expression of the CNNM2 E122K mutant variant, which retains residual function
but has a trafficking defect (Arjona et al., 2014), did not restore the uex LTM defect.
Although it has been questioned whether the CNNM2/3 CNBH domains bind cyclic nucleotides (Chen et al., 2018), we found that FSK evoked an increase in ab KC [Mg2+]i that was sensitive to uex mutation, and that UEX::HA was mislocalized in rut2080 adenylate cyclase (Han et al., 1992) and dnc1 phosphodiesterase (Dudai et al., 1976) learning defective mutant flies. Whereas UEX::HA label was evenly distributed in g, abc, and abs KCs in wild-type flies, UEX::HA label was diminished in the g and abs KCs and was stronger in abc neurons in rut2080 and dnc1 mutants. The chronic manipulations of cAMP in the mutants are therefore consistent with cAMP impacting UEX localization, per- haps by interacting with the CNBH. In addition, altered UEX localization may contribute to the memory defects of rut2080 and dnc1 flies.
Our physiological data using Magnesium Green in mammalian cell culture and the genetically encoded MagIC reporter in ab KCs demonstrate that fly UEX facilitates Mg2+efflux. Stimulating the fly brain with FSK evoked a greater increase in ab KC [Mg2+]i in uex mutant brains than in wild-type controls which provides the first evidence that UEX limits a rise in [Mg2+]i in Drosophila KCs. Our MagIC recordings also revealed a slow oscillation (centered around 0.015 Hz, approximately once a minute) of ab KC [Mg2+]i that was dependent on UEX. We do not yet understand the physiological function of this [Mg2+]i fluctuation although it likely reflects a homeostatic systems-level property of the cells. Biochemical oscillatory activity plays a crucial role in many aspects of cellular physiology (Nova´k and Tyson, 2008). Most notably, circadian timed fluctuation of [Mg2+]i links dynamic cellular energy metabolism to clock-controlled translation through the Mg2+ sensitive mTOR (mechanistic target of rapamycin) pathway (Feeney et al., 2016). It is, therefore, possible that slow Mg2+ oscillations could unite roles for cAMP, UEX, energy flux (Plac¸ais et al., 2017), and mTOR-dependent translation underlying LTM-relevant synaptic plasticity (Casadio et al., 1999; Huber et al., 2000; Beaumont et al., 2001; Hou and Klann, 2004; Hoeffer et al., 2008).
Contact for reagent and resource sharing
A full list of reagents can be viewed in the Key Resources Table.
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Scott Waddell (scott.waddell@cncb.ox.ac.uk).
Experimental model and subject details
Fly strains
Unless stated otherwise, flies were raised on standard cornmeal food under a 12 hr light-dark cycle at 60% humidity and 25˚C. Test and control flies for GAL80ts experiments were raised at 18˚C. Mixed sex flies 1–7-days-old were used in experiments.
Canton-S was the wild-type strain. The GAL4 driver lines used in this study are c739-GAL4 (McGuire et al., 2001), c305a-GAL4 (Krashes et al., 2007), NP7175-GAL4 (Tanaka et al., 2004), 0770-GAL4 (Gohl et al., 2011), MB247-GAL4 (Zars et al., 2000), nSyb-GAL4 (Bloomington Drosophila Stock Centre, BDSC 51635), elav-GAL4 (BDSC, 8765), and uex-GAL4 (Kvon et al., 2014); Vienna Drosophila Resource Center, VDRC, VT23256-GAL4). The UAS lines obtained from the stock center are UAS-CD8::GFP (BDSC, 5136), UAS-NmdarRNAi (BDSC, 25941), and UAS-uexRNAi (BDSC, 36116). The various mutant and transgenic lines are described, uexMI01943 (Venken et al., 2011; BDSC, 32805), uexNC1 (BDSC, 7167), rut2080 (Han et al., 1992), and dnc1 (Dudai et al., 1976), tubP- GAL80ts (McGuire et al., 2003) and PhsILMiT (BDSC, 24613). The uexMI01943.ex1 and uexMI01943.ex2 Minos excision lines were generated using the procedure described in Arc et al., 1997. The detailed mating scheme is shown in Figure 2—figure supplement 2A. Potential excision lines were established from individual flies exhibiting the yellow body color phenotype. Genomic DNA was extracted from six such lines and DNA flanking the uexMI01943 MiMIC was amplified by PCR and sequenced. The uexMI01943.ex1 and uexMI01943.ex2 lines were identified to harbor precise excisions, having restored the wild-type genomic sequence. See Resource Table for PCR and sequencing primer sequences. Schematic of the sequence detail of the uexMI01943 MiMIC insertion and in the excisions is shown in Figure 2—figure supplement 2B. To construct UAS-uex transgenic flies a full-length uex coding sequence (CDS) was cloned by RT-PCR. Total RNA was isolated from wild-type flies using TRIZOL (Thermo Fisher, 15596018) and reverse transcribed into cDNA using SuperScript III first-strand synthesis system (Invitrogen, 18080400). This total cDNA mix was used as a template to amplify the uex CDS. See Resource Table for primer sequences. The PCR product was digested with SacII and XhoI and then ligated into the complementary sites of pUAST (Brand and Perrimon, 1993). The pUAST cloned uex CDS was fully sequenced and verified to represent the 2505 bp of the wild-type uex cDNA reading frame (note, all four possible uex mRNA isoforms, FlyBase Release 6, encode the same 834 amino acid protein). UAS-uex transgenic flies were generated commercially (Bestgene) by transformation with the pUAST-uex vector. We mapped the UAS-uex chromosome insertion of 10 independent transgenic lines and behaviorally tested three lines, denoted UAS- uex3M, UAS-uex5M and UAS-uex8M, with an insert on the third chromosome. UAS-uex3M flies were those used throughout the study and referred to as UAS-uex in the manuscript.
UAS-uexR622K transgenic flies were generated similar to UAS-uex flies. A missense mutation was introduced at codon 622 of UEX within the CNBH domain, mimicking that previously engineered in the cAMP-binding domain of the regulatory subunit of protein kinase A (Bubis et al., 1988). The mutation changes the CGT codon encoding Arg into AAA encoding Lys. The mutation was intro- duced into the wild-type uex CDS using Gibson Assembly Master Mix (New England Biolabs, E2621S) as described in ‘Improved methods for site-directed mutagenesis using Gibson Assembly Master Mix’ (NEB Application Note). The primer sets used are detailed in the Resource Table. The product of Gibson assembly was further amplified by PCR and the resulting product was cloned into the pUAST vector and sequenced. Transgene insertions were mapped as for UAS-uex and one of two insertions mapped to the third chromosome was used in behavior experiments.
UAS-CNNM2, UAS-CNNM2E122K, UAS-CNNM2E357K, UAS-CNNM2S269W, and UAS-CNNM2T568I transgenic fly lines were generated by transformation with pUAST constructs containing wild-type or point mutated versions of a mouse CNNM2 cDNA tagged with HA (mCNNM2::HA), described in Arjona et al., 2014. Wild-type or mutated versions of CNNM2 were amplified from original mCNNM2::HA clones in pCiNEO_IRES_GFP plasmids (Arjona et al., 2014). Primers are detailed in the Resource Table. PCR products were digested with XhoI and XbaI and ligated into the complementary sites in pUAST. Insertions of each construct on the third chromosome were identified by mapping as described above and were used in the behavior experiments. Note that all CNNM2 encoding constructs used in the study are HA-tagged, although the notation is often omitted for brevity.
UAS-MagFRET-1 transgenic fly lines were generated by transformation with pJFRC-MUH constructs containing MagFRET-1 CDS, which was sub-cloned from the pCMVMagFRET-1 plasmid, described in Lindenburg et al., 2013. Primers are detailed in the Resource Table. PCR products were digested with XhoI and XbaI and ligated into the complementary sites in pJFRC-MUH. Insertion of the construct was mediated by the site-specific transgenesis system and the landing site is attP2 (on the third chromosome).
UAS-MagIC and UAS-MARIO transgenic fly lines were generated by transformation with pTW constructs containing the MagIC/MARIO CDS, which were sub-cloned from the plasmids MagIC/ pcDNA3 and MARIO/pcDNA3, kindly provided by T. Nagai: (Maeshima et al., 2018 and Koldenkova et al., 2015). MagIC/MARIO CDS were first PCR amplified from MARIO/pcDNA3 and MagIC/pcDNA3 respectively and were cloned into the pENTR/D-TOPO vector. Primers are detailed in the Resource Table. Note that the MARIO sense primer was designed to overlap with the sequence of pcDNA3 at the insertion site of MARIO. MagIC/MARIO CDS were further cloned into the Gateway destination vector pTW (Drosophila Gateway Vector Collection).
The CRISPR/Cas9 edited uexD locus was generated commercially by GenetiVision. The editing scheme is shown in Figure 2—figure supplement 2C. The uex locus sits in reverse orientation on chromosome 2R, spanning a 49,141 bp region between position 3,900,285 and 3,949,425 (FlyBase, Release 6). The following description relates to these coordinates within the uex locus. To generate uexD, two gRNA plasmids and one double-strand DNA donor (dsDNA) plasmid were constructed and injected into nos-Cas9 embryos (BDSC, 54591). As indicated in Figure 2—figure supplement 2C and detailed in the Resource Table, the upstream gRNA1 lies in Exon 6 and targets sequence 30,930.30,952. The corresponding downstream gRNA2 lies between Exon 7 and Exon 8 between 33,988 and 34,010. Both gRNAs were individually cloned into pCFD3-dU63gRNA (Addgene, 49410). The cut site of gRNA1 should be between 30,946 and 30,947 while gRNA2 should lead to a cut between 33,993 and 33,994. A 795 bp upstream homology arm (30,152.30,946) and 977 bp downstream homology arm (33,994.34,970) were cloned into the donor DNA plasmid. A termination codon (STOP, in all three reading frames) was inserted between the two homology arms and followed by a GFP cassette driven by a 3xP3 promoter. The donor DNA backbone was engineered by GenetiVision and the complete donor sequence for the uexD line is available upon request. Success- ful editing was identified by expression of GFP in the fly eyes and confirmed by genomic PCR and sequencing. In the uexD flies, a 3047 bp fragment from 30,947 to 33,993 was replaced by the sequence between the two homology arms in the donor plasmid, mainly the STOP signal and GFP cassette. The uexD allele truncates the uex ORF. Primers used for genomic PCR verification are detailed in the Resources Table. The nos-Cas9 transgene (on X chromosome) was removed by crossing.
CRISPR/Cas9-edited uex::HA flies were generated by WellGenetics using the ScarlessDsRed system developed by Kate O’Connor-Giles’ lab (unpublished, original plasmid donated to Addgene, #80822). A 6XHA tag was fused in frame to the carboxy-terminus of UEX by inserting the 6XHA-coding sequence immediately prior to the native STOP codon in the uex locus (Figure 3—figure supplement 1A). The process involved two main steps. In step 1, a 6XHA tag together with a pBAC transposon containing a DsRed cassette were inserted in frame with the STOP codon of uex using CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) using 1 gRNA and one dsDNA plasmid donor. The gRNA lies 50 bp from the uex STOP codon and should direct a cut between 48,587 and 48,588. The gRNA was cloned into a pCFD3-dU63gRNA plasmid. A 1,200 bp upstream arm (47,438.48,637) and 1,033 bp downstream arm (48,641.49,673) were cloned into the donor DNA plasmid with the pUC57-Kan (2579 bp) backbone. See Resource Table for gRNA and primer sequences. A Protospacer Adjacent Motif (PAM) mutation (TCC to TCG, 48,581.48,583) was introduced in the donor to promote HDR. A 6XHA tag, followed by a pBAC transposon containing a 3XP3 promoter-driven DsRed cassette, was inserted between the two homology arms. A pBAC recognition motif TTAA is embedded in the STOP codon of 6XHA. The complete donor sequence is available upon request. Donor and gRNA plasmids were injected into nos-Cas9 embryos (NIG-FLY, CAS0002). Successful editing was identified by expression of DsRed in the fly eyes and confirmed by genomic PCR and sequencing. Six independent positive lines were identified and four passed PCR validation. Of these four lines, one further passed sequencing validation and is the intermediate line represented in Figure 3—figure supplement 1A. Four isogenized and balanced stocks were established from this line. In step 2, the DsRed selection marker was excised by PiggyBac (PBac) transposition with the helper line Tub-PBac (BDSC, 8285). Five homozygous viable lines with successful excision were validated by genomic PCR and sequencing. One designated uex::HA was used in experiments in the manuscript.
To construct the CRISPR/Cas9-edited uexT626NRR flies, we designed and cloned a gRNA and designed and ordered (Sigma) a single-stranded oligo-deoxynucleotide (ssODN). gRNA and ssODN sequences are detailed in the Resource Table. As we planned to make a single amino acid substitution R622K in the UEX CNBH domain, the 120 bp ssODN donor was centered on codon R622 and carries the codon change CGT to AAA (at 31,179.31,181) corresponding to R622K. The expected cut site of the gRNA (between 31,192 and 31,193) is only 11 bp away from the expected mutation point. To enhance the likelihood of HDR, which is reportedly low using ssODN as donor, we commercially (GenetiVision) injected editing material into 250 lig4 KO vasa-Cas embryos (Zimmer et al., 2016). We obtained 37 viable G0 flies from the injected embryos. A total of 224 G1 flies were subjected to single fly genomic PCR and sequencing to screen for the expected mutation. Primers detailed in Resource Table. We identified 59 putative edited lines from first-round screening, and of these 12 were confirmed. Despite using lig4 KO vasa-Cas9, we detected only non-homologous end joining (NHEJ) events instead of HDR-mediated point mutations. Of the 12 edited lines, six were homozygous lethal and the other six were viable. In four of the homozygous viable lines, we found a replacement of G with T at position 31,192 together with a 6 bp in-frame insertion of ATCTTC between 31,192 and 31,193. This NHEJ editing corresponds to the T626 ! NRR change in the pro- tein sequence of UEX (Figure 5A). The X chromosome vasa-Cas9 was removed from these lines by crossing and one line referred to as uexT626NRR was used in the behavior experiments in the manuscript.
Method details
Behavioral experiments
For behavioral T-maze experiments, 1–7-day-old mixed sex flies were used. Odors were 4-methylcy- cyclohexanol (MCH) and 3-octanol (OCT), diluted ~1:103 (specifically, 9 ml MCH or 7 ml OCT in 8 ml mineral oil). All experiments were performed at 23˚C and 55–65% relative humidity.
Appetitive immediate and later memory experiments were performed essentially as described (Krashes and Waddell, 2008; Perisse et al., 2013). Batches of 100–120 flies were starved for 21–23 hr before training in 35 ml starvation vials containing ~2 ml 1% agar (as a water source) and a 2 cm 4 cm filter paper. Sugar papers (5 cm 7.5 cm) for training were prepared by soaking with 4 ml of 2 M sucrose and drying overnight. Water papers of the same size were soaked with water and left overnight. For appetitive training, flies were transferred from a starvation tube to a training tube with a dry ‘water’ paper, and immediately attached to the training arm of the T-maze and exposed to the CS odor for 2 min, followed by 30 s of clean air. Flies were then transferred to another training tube with dry sugar paper, attached to the T-maze and exposed to the CS+ odor for 2 min. Immediate memory was tested by transporting flies to the T-choice point and allowing them 2 min to choose between the two odor streams. To assay 24 hr memory, flies were removed from the training tube and transferred to standard cornmeal food vials for 1 hr, then transferred back into starvation vials for 23 hr until testing. Performance Index was calculated as the number of flies in the CS+ arm minus the number in the CS arm, divided by the total number of flies. MCH and OCT were alternately used as CS+ or CS and a single sample, or n, represents the average Performance Index from two reciprocally trained groups.
For behavior tests after Mg2+ feeding, 1–2-day-old flies were housed in vials with Mg2+ supplemented food for 1–5 days before being starved for appetitive training and testing, as described above. To make 80 mM [Mg2+] food, 40 ml of 1 M MgCl2 solution was added to 460 ml of normal liquid fly food; 1 mM [Mg2+] food was made by diluting 0.5 ml 1 M MgCl2 in 39.5 ml MilliQ water and adding it to 460 ml liquid food. Food was aliquoted and cooled to solidify. MgSO4 and CaCl2 supplemented food was prepared the same way.
Aversive immediate and 24 hr memory experiments were conducted as previously described (Hirano et al., 2013; Perisse et al., 2016; Tully and Quinn, 1985). Groups of 100–120 flies were trained with either one cycle of aversive training, or five cycles spaced by 15 min inter-trial intervals (spaced training). For aversive immediate memory, flies were tested after one-cycle training. Aver- sive 24 hr memory was tested using two different protocols. In the fasting-facilitated protocol, flies were starved for 16 hr before one-cycle training (Hirano et al., 2013). For spaced training, flies were not starved before training. Flies were fed on normal fly food for 24 hr after fasting-facilitated and spaced training, before being tested for memory performance. During each aversive training cycle, flies were exposed for 1 min to a first odor (CS+) paired with twelve 90 V electric shocks at 5 s inter- vals. Following 45 s of clean air, a second odor (CS ) was presented for 1 min without shock. Performance Index was calculated as the number of flies in the CS arm minus the number in the CS+
arm, divided by the total number of flies. MCH and OCT were alternately used as CS+ or CS and a single sample, or n, represents the average Performance Index from two reciprocally trained groups.
Sensory acuity tests (Figure 2—source data 1) were performed as described (Keene et al., 2004; Keene et al., 2006; Schwaerzel et al., 2003) with modifications. To test olfactory acuity, untrained flies were given 2 min to choose between a diluted odor as used in conditioning and air bubbled through mineral oil in the T maze. An Avoidance Index was calculated as the number of flies in the air arm minus the number in the odor arm, divided by the total number of flies. Electric shock avoidance was performed and calculated similarly. Untrained flies chose for 1 min between two tubes containing electric grids, but only one was connected to the power source. An avoidance index was calculated as the number of flies in the non-electrified arm minus the number in the electrified arm, divided by the total number of flies. To assess sugar acuity, starved flies were given 2 min to choose between an arm of the T-maze containing a dried sugar paper and the other contain- ing a dried ‘water’ filter paper. Both papers were prepared as in the appetitive memory assays. A Preference Index was calculated as the number of flies in the sugar arm minus that in the other arm, divided by the total number of flies. We found that keeping the light on in the behavioral room and having airflow running through the testing tubes greatly enhanced the Preference Index in wild-type flies and therefore applied those conditions for all sugar preference testing.
Anti-UEX antibody and western blot
A polyclonal UEX antibody was developed commercially by Eurogentec. Two peptides were synthesized as antigens: Peptide 1 H-CLPKLDDKFESKQSKP-OH (16aa) and Peptide 2 H-CVDNRTK TRRNRYKKA-NH2 (16aa) and injected into rabbits. Only Peptide 2 induced a robust immune response and was processed further. The final serum was purified against Peptide 2 and used for western blot analysis as a 1:2000 dilution.
For each sample in western blot, proteins were extracted from 20 fly heads by homogenizing thoroughly in 120 ml of protein sample buffer containing a mixture of 30 ml 2-mercaptoethanol (Bio-Rad), 270 ml 4 Laemmli sample buffer (BioRad), and 900 ml Nuclease Free Water (Invitrogen). Samples were then boiled on a 100˚C heat block for 3 min and centrifuged for 10 min before loading. A sample volume equivalent to four heads was loaded into each SDS-PAGE gel lane. Proteins were transferred to PVDF membrane and blocked in 5% skim milk for 1 hr at 25˚C with 35 rpm agitation. The membrane was then incubated in anti-UEX solution (1:2000 rabbit anti-UEX in 5% skim milk) overnight at 4˚C with 35 rpm agitation. The membrane was washed quickly three times followed by 3 10 min washes in TBST solution (100 ml of TBS 10 solution, BioRad, diluted in 900 ml of MilliQ water, with 0.1% Tween 20) and then incubated with HRP-conjugated secondary antibody solution (1:5000 of goat anti-rabbit in 5% skim milk) for 1–2 hr at 25˚C with 35 rpm agitation. The membrane was again washed quickly for three times followed by 3 10 min washes in TBST. Protein bands were visualized using Pierce ECL western blotting substrate (Life technologies, 32134). The membrane was then stripped using Millipore ReBlot Plus Mild solution (Merck, 2502), blocked again in 5% skim milk, and probed with mouse anti-Tubulin primary antibody (1:2000, Sigma, T6199) and corresponding HRP conjugated goat anti-mouse secondary antibody (1:5000) following the protocol detailed above.
Immunostaining
Immunostaining was performed as described (Wu and Luo, 2006). Brains from 1- to 5-day-old adult flies were dissected in PBS and fixed for 20 min in PBS with 4% paraformaldehyde at room temperature. They were then washed twice briefly in 0.5% PBT (2.5 ml Triton-X100 in 497.5 ml PBS) and three 20 min washes. Brains were then blocked for 30 min at room temperature in PBT containing 5% normal goat serum and then incubated with primary and secondary antibodies with mild rotation (35 rpm) at 4˚C for 1 or 2 days. Primary antibodies were rabbit anti-GFP (1:250; Invitrogen A11122) and rabbit anti-HA (1:250, NEB 3724T). Alexa 488–conjugated goat anti-rabbit (1:250; Invitrogen, A11034) was the secondary antibody. Before and after the secondary antibody incubation, brains were subjected to two quick washes followed by three 20 min washes in 0.5% PBT. Stained brains were mounted on glass slides in Vectashield (Vector Labs H1000) and imaged using a Leica TCS SP5 confocal microscope at 40 magnification (HCX PL APO 40 , 1.3 CS oil immersion objective, Leica). Image stacks were collected at 1024 1024 resolution with 1 mm steps and processed using Fiji (Schindelin et al., 2012). For quantification in Figure 3G and H, rectangular ROIs of approximately 40 25 mm for the for the globe, or round ROIs with a diameter of 15 mm for ab, a’b’, and EB were manually drawn on a single section of a z-stack scan of the fly brain. Corresponding ROIs were also drawn on the superior medial protocerebrum (SMP) as a background control region, and the mean fluorescence was calculated using ImageJ. ROI intensity of the MB lobes and the EB was normalized to that of the respective SMP intensity. An average between left and right brains was used for a single data point. For quantification in Figures 7C and D, ROIs are indicated in the figures and ROI intensity was calculated similarly to results in Figure 3H. In Figure 7C, a line was drawn through the widest part of the tip of the a lobe. The intensity profile of this line was obtained through ImageJ. Thirty data points in the middle of such a profile spanning about a 15 mm line were extracted for each line profile. The profile was further normalized to the mean value of the first five data points (F0) and calculated as (F F0)/F0. Mean values of these normalized profiles from different brains were plotted (Figure 7C, middle panel). Left and right profiles of brains were calculated and are separately displayed. In Figure 7D, the relative intensities from different ROIs representing different regions are added together to generate a total intensity measure for the MB.
The human CNNM4 cDNA expression construct used to investigate Mg2+ efflux in cell culture is that described previously (Yamazaki et al., 2013). A construct expressing Drosophila uex was generated by inserting a FLAG tag in front of the STOP codon of the uex CDS. FLAG-tagged CNNM4 and uex cDNAs were subsequently inserted into pCMV tag-4A (Agilent) for expression in HEK293 cells. HEK293 cells were cultured in Dulbecco’s modified Eagle medium (Nissui) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics. Expression plasmids were transfected with Lipofectamine 2000 (Invitrogen).
For immunostaining, cells were fixed with 3.7% formaldehyde in PBS for 20 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min, both at room temperature. They were next blocked with PBS containing 3% FBS and 10% bovine serum albumin (blocking buffer) for 1 hr at room temperature. Cells were then incubated overnight at 4˚C with rabbit anti-FLAG antibody (F7425, Sigma- Aldrich) diluted in blocking buffer, washed 3 with PBS, and incubated for 1 hr at room temperature with Alexa 488-conjugated anti-rabbit IgG (Invitrogen) and rhodamine-phalloidin (for F-actin visualization, Invitrogen) diluted in blocking buffer. After three washes with PBS, coverslips were mounted on slides and imaged with a confocal microscope (FluoView FV1000; Olympus).
Mg2+-imaging with Magnesium Green was performed as described (Yamazaki et al., 2013), with slight modifications. To avoid potentially decreasing [Mg2+]i with the expressed proteins, transfected HEK293 cells were cultured in growth media supplemented with 40 mM MgCl2 until imaging. Cells were then incubated with Mg2+-loading buffer (78.1 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 40 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-KOH [pH 7.4]), including 2 mM Magnesium Green-AM (Invitrogen), for 30 min at 37˚C. Cells were then rinsed once with loading buffer and viewed with an Olympus IX81 microscope equipped with an ORCA-Flash 4.0 CMOS camera (Hamamatsu) and a SHI-1300L mercury lamp (Olympus). Fluorescence was measured every 20 s (excitation at 470–490 nm and emission at 505–545 nm) under the control of Metamorph software (Molecular Devices). Buffer was then changed to Mg2+free buffer (MgCl2 in the loading buffer was replaced with 60 mM NaCl). Data are presented as line plots (mean of 10 cells). After imaging, cells were fixed with PBS containing 3.7% formaldehyde and subjected to immunofluorescence microscopy to confirm protein expression.
FRET-based Mg2+ concentration measurements in fixed fly brains
One- to two-day-old flies with genotype c739; UAS-MagFRET-1 were housed in vials with 1 mM or 80 mM [Mg2+] food for 4 days before being collected. Fly brains were dissected in PBS and fixed for 20 min in PBS with 4% paraformaldehyde at room temperature. They were then washed twice briefly in 0.5% PBT (2.5 ml Triton-X100 in 497.5 ml PBS) and three 10 min washes. Brains were then mounted on glass slides in Vectashield (Vector Labs H1000) and imaged using a wide-field Scientifica Slicescope with a 40 , 0.8 NA water-immersion objective and an Andor Zyla sCMOS camera with Andor Solis software (v4.27). In order to get the FRET ratio that indicates the Mg2+ concentration of the ab neuron, time series were acquired alternatively between the cerulean channel and the citrine channel at 3 Hz with 512 512 pixels and 16 bit. The excitation wavelength for both channels is 436 nm, while the emission filter for cerulean is 460–500 nm and that for citrine is 520–550 nm. Series acquisition starts from the cerulean channel and lasts for 5 s, then switches to the citrine channel and last for another 5 s, and this cycle is repeated for two more times. A total of 30 s (90 frames) image stack was therefore acquired for each brain. Image stacks were subsequently analyzed using ImageJ and custom-written Matlab scripts. In brief, rectangle ROIs (Figure 1E, left panel) were manually drawn on the ab lobes (one on a lobe and one on b lobe for each hemisphere), and outside the ab lobes (one for each hemisphere) as background control. Fluorescence intensity from the cerulean channel was calculated by dividing each vertical or horizontal lobe ROI by the background ROI, and averaged between the two hemispheres for each lobe, and averaged over the 15 frames for each cycle. That from the citrine channel was obtained similarly. A FRET ratio was obtained from the above intensities, further averaged among the three cycles of acquisition, depicted as one data point in Figure 1E (right panel).
Confocal Mg2+ imaging in explant fly brain
Explant brains expressing c739-GAL4 driven UAS-MagIC were placed at the bottom of a 35 mm glass-bottom microwell dish (Part No. P35G-1.5–14 C, MatTek Corporation), beneath extracellular saline buffer solution (103 mM NaCl, 3 mM KCl, 5 mM N-Tris, 10 mM trehalose, 10 mM glucose, 7 mM sucrose, 26 mM NaHCO3, 1 mM NaH2PO4, 1.5 mM CaCl2, 4 mM MgCl2, osmolarity 275 mOsm [pH 7.3]) following dissection in calcium-free buffer (Barnstedt et al., 2016). To determine the Mg2+ sensitivity of UAS-MagIC as well as the response of UAS-MagIC to other chemicals such as EDTA, EGTA, and CaCl2 (Figure 8B), brains were incubated in the saline buffer solution with 20 mg/ml digitonin for 6 min before imaging (Koldenkova et al., 2015). To investigate the Mg2+ fluctuation in response to Forskolin (FSK) application (Figure 8C–I), brains were put in the saline buffer solution without digitonin or incubation. In both situations, saline refers to the buffer (either with or without digitonin) in which the brain is submerged.
Imaging was carried out in an LSM780 confocal microscope (Zeiss) with a 20 air objective using the ZEN 2011 software. The Venus part of MagIC was excited with a 488 nm laser and its emission was collected in the 520–560 nm range. mCherry was excited with a 561 nm laser and its emission was collected in the 600–640 nm range. Time series were acquired at 0.5 Hz with 512 512 pixels and 16 bit. Following 60 s of baseline Venus/mCherry measurement, 2–20 ml of saline or other rele- vant chemical solution was added via a micropipette to the dish with constant image capture. The effects of applied agents on Venus/mCherry emission were then recorded for 15–20 min.
Image stacks were subsequently analyzed using ImageJ and custom-written Python scripts. In brief, rectangle ROIs were manually drawn on the ab neurons (one for each hemisphere, Figure 8A), and another ROI of the same size was drawn in the middle but outside the MBs as background control. Fluorescence intensity from the Venus (or mCherry) channel was calculated by subtracting the background ROI from the calyx ROIs, respectively, and averaged between the two hemispheres. This is referred as ‘Rel. Intensity (a.u.)’ in Figure 8D and E. The ratio between Venus and mCherry intensity was calculated as ‘MagIC Ratio’ in Figure 8B and C and Figure 8F and G. For Figure 8H, the intensity for the two channels was calculated separately. In this case, ‘Rel. Intensity (DF/F0)’ refers to the relative fluorescence intensity normalized to the mean intensity from the baseline period F0, calculated as (F F0)/F0. The relative intensity DF/F0 of Venus was used to calculate the PSD (Figure 8I) through python function psd (under matplotlib.pyplot), which adopted Welch’s average periodogram method (Bendat et al., 2000).
Reverse transcription and quantitative real-time PCR
For each sample, 120 flies were frozen in liquid nitrogen and their heads were homogenized completely in TRIzol reagent (Invitrogen). Total RNA was extracted using a Direct-zol RNA MiniPrep (R2050) kit following the manufacturer’s instructions. cDNA was synthesized using the SuperScript III First-Strand synthesis system (Invitrogen). Five independent samples were prepared for each different treatment or genotype. Quantitative PCR was performed in triplicate for each cDNA sample on a LightCycler 480 Instrument (Roche) using SYBR Green I Master Mix (Roche). Melting curves were analyzed after amplification, and amplicons were visualized by agarose gel electrophoresis to confirm primer specificity. Relative transcript levels were calculated by the 2-DDCt method (Livak and Schmittgen, 2001), and the geometric mean of the Ct values of three reference genes (Gapdh, Tbp, and Ef1a 100E) was used for normalization. Primers are detailed in the Resource Table.
Inverse PCR
Inverse PCR was used to map the MiMIC insertion position in uexMI01943 flies. Genomic DNA was prepared from 15 adult flies. DNA equivalent to two flies was then digested in a 25 ml restriction reaction with Mbo I and 10 ml of the product was ligated overnight at 4˚C overnight to circularize the fragments; 5 ml of the ligation product was used for inverse PCR. PCR product was purified using Exo/SAP reaction (Thermo Fisher, 78201) before being sequenced. The sequence was compared to the D. melanogaster genome (FlyBase, Release 6) by BLAST and matched uniformly to the region 3,882,886.3,882,641 on 2R, consistent with the reported uexMI01943 insertion on FlyBase. Primers are detailed in the Resource Table.
Protein domain prediction and alignment
Protein sequence alignment was carried out using Geneious R10.2.2. Protein domain prediction was performed with InterPro (Finn et al., 2017; Jones et al., 2014) and Phyre2 (Kelley et al., 2015). Pro- tein domain and structure alignment was performed using TM-align (Zhang and Skolnick, 2005). Protein structure visualization was rendered in Chimera 1.11.2 (Pettersen et al., 2004).
Quantification and statistical analyses
Behavior data were analyzed using Excel and Prism 6. Imaging data were analyzed using ImageJ and custom-written MATLAB or Python scripts. Unpaired two-tailed t-tests were used for comparing two groups, and one-way ANOVA followed by a Tukey’s post-hoc test was used for comparing multiple groups. The threshold of statistical significance was set at p<0.05.
Acknowledgements
We thank F J Arjona and J G J Hoenderop for the murine CNNM2 clones and comments on the manuscript. We are grateful to T Nagai for clones of MagIC and MARIO and to the Bloomington Stock Center and VDRC for flies. We thank P Cognigni, Y Huang, R Brain, R Szoke-Kovacs, and M Goodwin for technical support and other members of the Waddell group for discussion. We acknowledge N Halidi, C Monico, and the Micron Advanced Bioimaging Unit (supported by Well- come Strategic Awards 091911/B/10/Z and 107457/Z/15/Z) for their support and assistance in this work. E M was funded by an EMBO long-term fellowship (ALTF 184-2109). K D J acknowledges support from the Rhodes Trust. S W was funded by a Wellcome Principal Research Fellowship (200846/ Z/16/Z) and an ERC Advanced Grant (789274).
