The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae

Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner.

2 Supplement Materials and Methods:

FLP-FRT-based JhI-21 deletion
The DrosDel isogenic deficiency kit was used to design a null allele for JhI-21 1 .
Transposons containing FRT compatible sites and covering JhI-21 gene were selected using the following web site: http://www.drosdel.org.uk/fdd/del_hunter.php Because of several misannotations of transposon insertions in this website, the smallest deletion we were able to generate, which could lead accurately to a null allele, was obtained using the following strains: -w 1118; , inserted at 12045.952kb (DGRC#125824) -w 1118 ; PBac{RB}e00136, inserted at 12063.132kb (Exelixis stock e00136).
After 72 hours, parents and progeny (including w,hsFLP/w; P{RS5}CG6770[5-HA-5095]/ PBac{RB}e00136; +/+) of this second cross were placed at 37 °C for 1hr to start the expression of the Flippase (FLP) necessary to rearrange the chromosome and to eliminate JhI-21. Parents were then removed. The following day, eggs and larvae were placed again at 37 °C for 1hr. This heat shock was repeated once a day for 4 days. Emerging adult females were collected and balanced over CyOGFP.

Anti-JhI-21 labeling of embryos:
Flies were allowed to lay eggs for three hours. Embryos were collected at 13-16 h after egg laying, briefly rinsed with water and dechorionized with 4 % bleach for 2-3 minutes, and rinsed with embryo wash solution (7 % NaCl, 0.5 % Triton-X-100). Embryos were transferred (1:100) in 1 % NGS for 2 h and washed six times 10 min using PBST. Embryos were blocked for 1 h and incubated with secondary antibodies for 2 h. After 6 washes of 10 minutes embryos were mount in Vectashield 1000.

NMJ electrophysiology for eEJCs
Electrophysiological recordings were obtained at 19 °C from third instar (110-120 hr after egg laying) larval ventral longitudinal muscle 6 (A3-A4) at -60 mV holding potential using two-electrode voltage clamp technique (TEVC). Dissections and electrophysiology were performed under glutamate free Drosophila HL-3 saline. Electrodes for TEVC were filled with 3M KCl yielding a resistance of 30-40 MOhm. Synaptic current were evoked at 1 mM extracellular Ca 2+ and average single eEJC amplitudes (stimulus: 0.1ms, 1-5V) are based on the mean peak eEJC amplitude in response to ten presynaptic stimuli (recorded at 0.2Hz).
Nerve stimulation was performed with an isolated stimulator (DS2A, Digitimer). All data were digitized at 10kHz.

Other locomotor parameters:
Perti dishes were filled with 2 % agar-agar in ddH 2 O. Larvae of the appropriate genotype and stage were placed on the agar-agar surface and allowed to adapt to their new environment for 30 seconds. Larvae were observed using a bionocular (Brand) and peristaltic waves as well as the number of stops were manually counted for 2 minutes. minutes in the same buffer. Glutamate uptake into Schneider 2 cells was realized for 5 minutes at room temperature (25 °C) and at 4°C, using concentrations varying from 50 µm to 500 µm and different ratios between non-radiolabeled glutamate and radiolabeled glutamate (L-[3,4-3H]-glutamic acid; 1 mCi.mL-1; 47.5 Ci.mmol-1; Perkin Elmer) (1:250 -1:5000), added in 500 µL of sodium-free buffer. The uptake was stopped by washing cells 3 times with cold sodium-free buffer in which 20 mM glutamate was added to avoid release of previously up-taken glutamate. Cell lysis was obtained by adding 1 mL of lysis buffer (NaOH 0.1%; SDS 0.1%). 5 ml of scintillation medium (Ultima Gold XR, Perkin Elmer) were added to 500 µL of lysate and dpi per minute were counted by a scintillation counter for 3 minutes (Tri-Carb 2900 TR, Perkin Elmer).
As a positive control for amino acid net uptake leucine at a concentration of 50 µm and at a ratio of 1:2000 between cold and radiolabeled leucine (L-[3,4,5-3H(N)]-Leucine; 5 mCi.mL-1; 112 Ci.mmol-1; Perkin Elmer) was used. Uptake experiments followed the same 5 protocol, except that leucine uptake was measured for 1 min and 20 mM of leucine were added in sodium-free buffer to wash cells before lysis.
For the inhibition experiments, the uptake of 20 µM Leucine (with a ratio of 1:1500 between cold and radiolabeled Leucine) was measured in the presence or absence of 2 or 10 mM nonlabeled test amino acids. Uptake experiments followed the same protocol as described before for leucine uptake. Experiments were realized at 25° and at 4°C for the background and final results represent net leucine uptake.

1.
Ryder   Data were compared using a Kruskal-Wallis test between controls and mutants. ns: not significant; **: p<0.01 ***: p<0.001. N = 7-10 animals, 800-3,400 events measured per phenotype.  KG and JhI-21 KG/Df1). The data are identical to the data shown in Figure 7a and b, but comparing differences between genotypes instead of stages. Data were compared using a 2way ANOVA test followed by a Bonferroni post hoc test. **: p < 0.01; *: p < 0.05; ns: not significant. N=30 for each genotypes.  concentrations of glutamate and various ratio of cold/radiolabeled glutamate were used to test the possibility to uptake this amino acid into S2 cells at 25 °C within 5 minutes. Each value is compared to the background signal at 4 °C corresponding to glutamate unspecifically bound to the cell surface. Since glutamate uptake between 25 °C and 4 °C does not differ in any tested condition, we conclude that glutamate is not imported into S2 cells and hence the impact of JhI-21 on glutamate translocation cannot be proven. b, As a positive control we tested if radiolabelled leucine could be loaded in S2 cells [20]. Since the values for leucine uptake are drastically higher at 25 °C than at 4 °C, we conclude that in contrast to glutamate Table S1. ERC Values of putative amino acid transporters and glutamate receptors.