Calcium and SOL Protease Mediate Temperature Resetting of Circadian Clocks

Summary Circadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. We found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein TIM. This degradation is mechanistically distinct from photic CRY-dependent TIM degradation. Thermal TIM degradation is triggered by cytosolic calcium increase and CALMODULIN binding to TIM and is mediated by the atypical calpain protease SOL. This thermal input pathway and CRY-dependent light input thus converge on TIM, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian SOL homolog SOLH blocks thermal mPER2 degradation and phase shifts. Thus, we propose that circadian thermosensation in insects and mammals share common principles.

Resulting product was confirmed by sequencing and was used as template for sitedirected mutagenesis and truncations of TIM-LUC. Putative CaM binding sites were predicted using Calmodulin Target Database program from the University of Toronto (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html), Full length tim promoter (4.6kbp) with TIM-LUC or TIM-LUC 5C were cloned into pUASTattb plasmid using SphI and EcoRI sites. These sites allowed for removal of the endogenous 5xUAS-HSP70 promoter in the plasmid. Constructs were verified by sequencing and then introduced into flies by Genetic Services, Inc (Cambridge, MA) using site-directed insertion into 68A4 locus on 3L chromosome. tim 0 ;TIM-LUC and tim 0 ;TIM-LUC 5C were made by combining ptim-TIM-LUC and ptim-TIM-LUC 5C with y w;tim 0 stock, respectively. TD2;TIM-LUC were made by combining tim-GAL4,UAS-dcr2/CyO with ptim-TIM-LUC and were used for luciferase experiments.

Behavioral and luciferase analysis in flies
The locomotor activity of individual male flies were monitored in Trikinetics Activity Monitors (Waltham, MA) using I-36LL Percival incubators (Percival Scientific, Perry IA). Data analysis was done with FaasX software (Grima et al., 2002) and represented with error bars indicating standard deviation from mean. Statistical significance was determined by One-way ANOVA, Tukey's multiple comparison test and indicated as *= P< 0.05, **= P< 0.01 or ***= P< 0.001.
The luciferase activity of isolated heads in culture (M3 medium, SIGMA, 5mM luciferin) or whole flies on Luciferin (Gold-biotech) containing agar/sucrose medium (100µl volume, 1% agar, 2% sucrose, 5mM luciferin), was monitored in Berthold LB960 plate reader (Berthold technologies, USA) in I-36LL Percival incubators with 90% humidity (Percival Scientific, Perry IA). Flies in 96-well white plates were covered with needle-poked Pattern Adhesive PTFE Sealing Film (Analytical sales & services 961801). The distance between the agar and film was such that the flies were not able to move vertically.
Cells were seeded in 96-well plates. At 70-90% confluence, they were transfected with 200ng/well DNA using Cellfectin II (Invitrogen) as detailed in the manufacturer's instructions. Luciferin (200µM final) was added to the SFX medium during transfection.
Two days after transfection, luciferase activity in living cells was measured using a microtiter plate luminometer.

dsRNA synthesis
Traditional PCR methods were used to generate a T7 promoter flanking (both sides) template for dsRNAs from cDNA of Calpains A,B,C and sol. Calmodulin immunoprecipitation, fly head extracts and western blots 2-7 days old male flies (n=80) from LUC, TIM-LUC and TIM-LUC 5C were entrained under 12/12 LD at 20 o C and frozen heads were collected at ZT18 on the third day. Extracts were made using mechanical homogenization in lysis buffer (50mM TRIS-HCl, 150mM NaCl, 5% glycerol, 1mM DTT, 1% Triton-X, 1X Roche Complete protease inhibitor cocktail) under native conditions and were then divided into two equal volumes and 3mM CaCl 2 or 3mM EGTA were added. An aliquot for "input" was collected immediately and remaining lysates were incubated with calmodulin sepharose 4B (GE Healthcare) for two hours at room temperature. Two wash steps using lysis buffer with 3mM CaCl 2 or EGTA were performed and calcium-dependent binding was assayed by 30 minute room temperature elution with lysis buffer containing 25mM EGTA for both conditions. Measurement of luciferase activity was performed using 5mM luciferin (final) in a Berthold LB960 plate reader (Berthold technologies, USA). Ratio of the signal of CaCl 2 to EGTA samples for each extract was used for calcium-dependent binding to CaM. LUC flies were used as background control for potential binding of LUCIFERASE enzyme to CaM beads and were significantly in excess, compared to TIM-LUC or TIM-LUC 5C. Western blots for temperature shift experiments were made using lysis buffer with 15 heads per time point and were quantified with ImageJ.

Isolated brain culture and PER immunohistochemistry
Brains were dissected in M3 Medium [Shields and Sang M3 insect medium (Sigma) + 10% Fetal Bovine Serum+ Penicillin-Streptomycin+ Insulin-Transferrin-Selenium] and distributed to 4 or 6 cell-culture inserts (MilliCell) placed in a 24-well plate with M3 medium. Microporous sealing film (USA Scientific) was used to reduce evaporation during the 5-6 days of incubation. At each desired time point, one insert was transferred to fixation buffer and at least 5 brains within were fixed. Whole-mount immunohistochemistry for PER was performed and imaged essentially as previously described (Zhang et al., 2010). Anti-PER (generous gifts from Dr. M. Rosbash) was diluted at 1:1500. Secondary antibody was anti-rabbbit-Cy3 (1:200) from Jackson ImmunoResearch Inc. Mounted brains were scanned using a Zeiss LSM5 Pascal confocal microscope. Images are digitally projected Z stacks.

Culture and manipulation of mammalian cells, Western Blots and RT-PCR
Primary fibroblasts homozygous for PERIOD2::LUCIFERASE (Yoo et al., 2004) were isolated from the lung tissue of adult males (Seluanov et al., 2010) and cultured as described previously (O'Neill and Hastings, 2008) School RNAi core facility. Solh shRNA plasmids were transfected to 293T cells (4 parts transfer vector: 3 parts lentivirus packaging plasmid HIV GAG-POL :1 part envelope VSV-G) using TransIT-LT1 (Mirus) transfection reagent, according to manufacturer's protocol. The viral supernatant was collected 48 hours after transfection, filtered through a sterile 0.45 µm syringe filter (Millipore), and stored in 1mL aliquots at -80 °C. Solh knockdown viruses were added to the liver cells followed by selection with 2mg/mL puromycin for one week. Bioluminescence assays were performed with cells that were seeded into 96-well plates, sealed with standard PCR film and entrained to 36/38.5 o C temperature cycles for two days in puromycin containing complete Williams'E medium with Luciferin (400µM final). Data was recorded using a Berthold LB960 plate reader