{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002150","ANALYSIS_ID":"AN003520","VERSION":"1","CREATED_ON":"April 24, 2022, 5:47 pm"},

"PROJECT":{"PROJECT_TITLE":"Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells","PROJECT_SUMMARY":"Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-molecule dynamic approaches to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with neutral sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.","INSTITUTE":"Universidad CEU San Pablo","DEPARTMENT":"Center of Metabolomics and Bioanalysis","LAST_NAME":"Gonzalez-Riano","FIRST_NAME":"Carolina","ADDRESS":"km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501","EMAIL":"carolina.gonzalezriano@ceu.es","PHONE":"646251045"},

"STUDY":{"STUDY_TITLE":"Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed cell migration in human T cells","STUDY_SUMMARY":"Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-molecule dynamic approaches to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with neutral sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration","INSTITUTE":"Universidad CEU San Pablo","LAST_NAME":"Gonzalez-Riano","FIRST_NAME":"Carolina","ADDRESS":"km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501","EMAIL":"carolina.gonzalezriano@ceu.es","PHONE":"646251045"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Blasto_Control_1",
"Sample ID":"Blasto_Control_1",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_Control_1"}
},
{
"Subject ID":"Blasto_Control_4",
"Sample ID":"Blasto_Control_4",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_Control_4"}
},
{
"Subject ID":"Blasto_Control_5",
"Sample ID":"Blasto_Control_5",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_Control_5"}
},
{
"Subject ID":"Blasto_Control_7",
"Sample ID":"Blasto_Control_7",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_Control_7"}
},
{
"Subject ID":"Blasto_Control_8",
"Sample ID":"Blasto_Control_8",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_Control_8"}
},
{
"Subject ID":"Jurkat_Control_2",
"Sample ID":"Jurkat_Control_2",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_Control_2"}
},
{
"Subject ID":"Jurkat_Control_5",
"Sample ID":"Jurkat_Control_5",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_Control_5"}
},
{
"Subject ID":"Jurkat_Control_6",
"Sample ID":"Jurkat_Control_6",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_Control_6"}
},
{
"Subject ID":"Jurkat_Control_8",
"Sample ID":"Jurkat_Control_8",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_Control_8"}
},
{
"Subject ID":"Jurkat_Control_9",
"Sample ID":"Jurkat_Control_9",
"Factors":{"Factor1":"CONTROL"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_Control_9"}
},
{
"Subject ID":"Blasto_SMasa_10",
"Sample ID":"Blasto_SMasa_10",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_SMasa_10"}
},
{
"Subject ID":"Blasto_SMasa_2",
"Sample ID":"Blasto_SMasa_2",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_SMasa_2"}
},
{
"Subject ID":"Blasto_SMasa_3",
"Sample ID":"Blasto_SMasa_3",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_SMasa_3"}
},
{
"Subject ID":"Blasto_SMasa_6",
"Sample ID":"Blasto_SMasa_6",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_SMasa_6"}
},
{
"Subject ID":"Blasto_SMasa_9",
"Sample ID":"Blasto_SMasa_9",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Blasto_SMasa_9"}
},
{
"Subject ID":"Jurkat_SMasa_1",
"Sample ID":"Jurkat_SMasa_1",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_SMasa_1"}
},
{
"Subject ID":"Jurkat_SMase_10",
"Sample ID":"Jurkat_SMase_10",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_SMase_10"}
},
{
"Subject ID":"Jurkat_SMasa_3",
"Sample ID":"Jurkat_SMasa_3",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_SMasa_3"}
},
{
"Subject ID":"Jurkat_SMasa_4",
"Sample ID":"Jurkat_SMasa_4",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_SMasa_4"}
},
{
"Subject ID":"Jurkat_SMasa_7",
"Sample ID":"Jurkat_SMasa_7",
"Factors":{"Factor1":"CASE"},
"Additional sample data":{"RAW_FILE_NAME":"Jurkat_SMasa_7"}
},
{
"Subject ID":"QC_1_B",
"Sample ID":"QC_1_B",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_1_B"}
},
{
"Subject ID":"QC_2_B",
"Sample ID":"QC_2_B",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_2_B"}
},
{
"Subject ID":"QC_3_B",
"Sample ID":"QC_3_B",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_3_B"}
},
{
"Subject ID":"QC_1_J_1",
"Sample ID":"QC_1_J_1",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_1_J_1"}
},
{
"Subject ID":"QC_1_J_2",
"Sample ID":"QC_1_J_2",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_1_J_2"}
},
{
"Subject ID":"QC_2_J",
"Sample ID":"QC_2_J",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_2_J"}
},
{
"Subject ID":"QC_3_J",
"Sample ID":"QC_3_J",
"Factors":{"Factor1":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_3_J"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"HEK-293T cells were obtained from the ATCC (CRL-11268) and human Jurkat leukemia CD4+ cells were kindly donated by Dr. J. Alcamí (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain). When needed, Jurkat cells lacking endogenous CXCR4 expression (Jurkat-/-) were transiently transfected with CXCR4-AcGFP (20 µg; JK-/-X4) using a BioRad electroporator (20 × 106 cells/400 µL RPMI 1640 with 10% fetal calf serum) and analyzed 24 hours later. Human peripheral blood mononuclear cells were isolated from buffy coats by centrifugation through FicollPaque PLUS density gradients (GE Healthcare, Wakuesha, WI) at 760 × g for 30 minutes at room temperature (RT). They were then in vitro activated with 20 U/mL of IL-2 (Teceleukin; Roche, Nutley, NJ) and 5 µg/mL phytohemagglutinin PHA (Roche) to generate T cell blasts.","SAMPLE_TYPE":"HEK cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"For lipid extraction, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For lipid extraction from Jurkat and T cell blasts, cell pellets were mixed with 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for direct injection into the system for LC-MS analyses in positive and negative ionization modes."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"RP-UHPLC-ESI(+)-QTOF MS","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290 Infinity II","COLUMN_NAME":"Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"CEMBIO","OPERATOR_NAME":"Carolina Gonzalez Riano"},

"MS":{"INSTRUMENT_NAME":"Agilent 6546 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The Agilent 6545 QTOF mass spectrometer equipped with a dual AJS ESI ion source was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 50 to 1800 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds were used throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 for the positive and m/z 119.0363 for the negative ionization modes; and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098 for the positive and m/z 980.0163 (HP-0921+acetate) for the negative ionization modes. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. Ten Iterative-MS/MS runs were performed for both ion modes at the end of the analytical run. They were operated with an MS and MS/MS scan rates of 3 spectra/s, 40–1800 m/z mass window, a narrow (∼ 1.3 amu) MS/MS isolation width, 3 precursors per cycle, and 5000 counts and 0.001% of MS/MS threshold. Five iterative-MS/MS runs were set with a collision energy of 20 eV, and the subsequent five runs were performed at 40 eV. References masses and contaminants detected in blank samples were excluded from the analysis to avoid inclusion in the iterative-MS/MS."},

"MS_METABOLITE_DATA":{
"Units":"AREA",

"Data":[{"Metabolite":"C17 Sphinganine 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}