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Reticulocyte and red blood cell deformation triggers specific phosphorylation events

Pedro L. Moura, Maria A. Lizarralde Iragorri, Olivier Français, Bruno Le Pioufle, Johannes G. G. Dobbe, Geert J. Streekstra, Wassim El Nemer, Ashley M. Toye and Timothy J. Satchwell

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Red blood cell deformation induces measurable changes in the phosphoproteome. (A) Experimental design for proteomic comparison: RBCs and cultured reticulocytes were subjected to flow in a microsphiltration system and lysed before, during, and after passage. The lysates were equalized through hemoglobin quantification using Drabkin’s reagent and analyzed through NanoLC-MS/MS. Cells were fixed and imaged under brightfield at all 3 stages, showing the extent of their deformation within the system and their recovery after passage. Scale bars, 10 μm. (Microsphiltration schematic adapted from Duez et al.48) (B) Venn diagram of differentially phosphorylated proteins with ≥50% detection across all samples in RBCs (left, n = 63 with 33 modifications detected exclusively in cells mid- and postdeformation) and reticulocytes (right, n = 226 with 55 modifications detected exclusively in cells mid- and postdeformation). (C) Top-scoring protein-protein interaction network from the joint dataset of mid- and postdeformation exclusive phosphoproteins in reticulocytes, as predicted by STRING.49 Two main subsets of proteins were present in the network, kinases (marked in red) and vesicle transport-related proteins (marked in dark blue). (D) List of phosphoproteins exclusive to the RBC mid-deformation and mid-/postintersection datasets. Count indicates the number of observed instances of the phosphopeptide (n = 3); the phosphopeptides were sorted by the sum of their counts in both datasets and filtered for their presence in at least 2 samples per condition. PhosphoSite location indicates the predicted location of the phosphorylation site in the protein sequence (as predicted by SEQUEST and/or PhosphoSitePlus data50), as well as the effect of its phosphorylation if described in low-throughput studies according to PhosphoSitePlus.50 (E) List of phosphoproteins exclusive to the reticulocyte mid-deformation and mid-/postintersection datasets (n = 4). Dataset analysis, filtering, and annotation were performed as previously described. (F) List of phosphoproteins present among both reticulocyte and RBC mid-/postdeformation datasets, with no significant detection in the predeformation dataset; dataset analysis, filtering, and annotation were performed as previously described.

  • Figure 2.

    GSK3 and Lyn inhibition impacts capillary traversal of reticulocytes and red blood cells. (A) Percentage recovery of reticulocytes treated with the respective reagent after microsphiltration. After treatment, the cells were added to an untreated RBC suspension in a 5:95 ratio and the mixture then subjected to microsphiltration. Anti-GPA antibody was used as a positive control for negative effects on cell deformability. Bafetinib (3 µM) was used as a Lyn inhibitor and CHIR-98014 (10 nM) was used as a GSK3 inhibitor. Error bars correspond to the standard deviation (n = 3). All comparisons were performed with a paired 2-tailed Student t test. The P values for each comparison are shown below the bar graph. *P < .05, **P < 0.01. (B) Image of the microfluidic PDMS biochip used for the capillary traversal experiment. The magnified image shows a section of the microcapillary channels, through which reticulocytes and red blood cells were subjected to successive deformations. Frame sequences were obtained through the use of a high-speed camera and then subjected to image analysis for automated cell tracking with the use of TrackMate.51 Scale bars, 50 μm. (C) Percentage microcapillary traversal velocity of reticulocytes and red blood cells treated with the respective reagents. The cells were treated for a minimum period of 1 hour and subjected to passage through the microfluidic biochip. The graph shows the average cell microcapillary traversal velocity of each treated sample as a percentage of the respective DMSO control sample traversal velocity. All comparisons were performed with a 1-sample Student t test. The error bars correspond to the standard deviation (n = 3). The P values for each comparison are shown below the dot plot. *P < .05, **P < .01. A minimum of 1000 cell tracks were analyzed per sample. n.s.s., not statistically significant.

Tables

  • Table 1.

    Integrative list of deformation-induced phosphorylations in the reticulocyte

    Accession no.Gene symbolProtein nameSequenceCountSite
    E9PIJ1AMPD2AMP deaminase 2TDSDSDLQLYK7*S190/S197
    A0A0U1RQT1ACAP2Arf-GAP with coiled-coil, ANK repeat, and PH domain-containing protein 2SSPSTGSLDSGNESK7*S384
    B7Z3I9ALADΔ-aminolevulinic acid dehydrataseSSPAFGDR7S215
    P49840GSK3AGlycogen synthase kinase-3 αGEPNVSYICSR7*Y279
    P49840GSK3AGlycogen synthase kinase-3 αTSSFAEPGGGGGGGGGGPGGSASGPGGTGGGK7*S21/S39
    E9PK89RAB3IL1Guanine nucleotide exchange factor for Rab-3ATLVITSTPASPNR7S168
    B4DMA2HSP90AB1Heat shock protein HSP 90-βIEDVGSDEEDDSGKDK7*S255
    Q9H1E3NUCKS1Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1VVDYSQFQESDDADEDYGR7*S19
    S4R2Y4CBX3Chromobox protein homolog 3SLSDSESDDSK6S93
    Q08495DMTNDematinDSSVPGSPSSIVAK6S26
    Q9UG54MAP3K7Mitogen-activated protein kinase kinase kinase 7SIQDLTVTGTEPGQVSSR6S439
    P61006RAB8ARas-related protein Rab-8AKLEGNSPQGSNQGVK6S181/S185
    B3KVN0SLC2A1Solute carrier family 2, facilitated glucose transporter member 1TPEELFHPLGADSQV6S490
    A0A087WYB4STOML2Stomatin-like protein 2, mitochondrialAPVPGTPDSLSSGSSR6T327
    Q8IV54TSC22D4TSC22D4 proteinNGSPPPGAPSSR6S62
    P16157ANK1Ankyrin-1NGASPNEVSSDGTTPLAIAK5S759
    A0A0U1RQT1ACAP2Arf-GAP with coiled-coil, ANK repeat, and PH domain-containing protein 2YSISLSPPEQQK5S521
    P27824CANXCalnexinQKSDAEEDGGTVSQEEEDR5S564
    G3V238EEF1AKMT2EEF1A lysine methyltransferase 2SDKGSPGEDGFVPSALGTR5S21
    P05198EIF2S1Eukaryotic translation initiation factor 2 subunit 1VVTDTDETELAR5T279
    F8WAE5EIF2AEukaryotic translation initiation factor 2ASDKSPDLAPTPAPQSTPR5S506
    C9JII4GCFC2GC-rich sequence DNA-binding factor 2ELPVPGSAEEEPPSGGGR5S40
    Q2VPJ6HSP90AA1HSP90AA1 proteinDKEVSDDEAEEK5S231
    D3DWY2IKBKGInhibitor of κ light polypeptide gene enhancer in B-cells, kinase γSPPEEPPDFCCPK5S387
    O60307MAST3Microtubule-associated serine/threonine-protein kinase 3SSENVLDEEGGR5S134
    F2Z2K0NSFL1CNSFL1 cofactor p47SPNELVDDLFK5S114
    B7Z8Y1NFATC2Nuclear factor of activated T-cells, cytoplasmic 2TSPDPSPVSAAPSK5S330
    H0Y5Q9INPP5DPhosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1GESPPTPPGQPPISPK5T963
    D6RC77PGM3Phosphoacetylglucosamine mutaseSTIGVMVTASHNPEEDNGVK5S64
    M4QHP2PIEZO1Piezo-type mechanosensitive ion channel componentSGSEEAVTDPGER5S1621
    Q9BVG4PBDC1Protein PBDC1GADSGEEKEEGINR5S197
    B4E3V2SQSTM1Sequestosome-1LTPVSPESSSTEEK5T269/S272
    Q6PJD5SHARPINSHARPIN proteinSPGNLTER5S165
    B2RMN7SPTBSpectrin β chainTSPVSLWSR5S2114/S2117
    C9JWF0SMC4Structural maintenance of chromosomes protein 4REEGPPPPSPDGASSDAEPEPPSGR5S22
    H0YN01TLN2Talin-2LDEGTPPEPK5T1843
    C9J3F6TBC1D5TBC1 domain family member 5NISSSPSVESLPGGR5S541/S544
    E9PJJ0TBCELTubulin-specific chaperone cofactor E-like proteinYSPENFPYR5S18
    Q8IXQ3C9orf40Uncharacterized protein C9orf40RDSGDNSAPSGQER5S76
    Q5JSH3WDR44WD repeat-containing protein 44VGNESPVQELK5S50
    F8WBS8PSMD226S proteasome non-ATPase regulatory subunit 2DKAPVQPQQSPAAAPGGTDEKPSGK4S16
    Q92625ANKS1AAnkyrin repeat and SAM domain-containing protein 1ASESLSNCSIGK4S647
    E9PJP4EPB41L2Band 4.1-like protein 2EVAENQQNQSSDPEEEKGSQPPPAAESQSSLR4S39/S58
    A8MZ77BSGBasiginRKPEDVLDDDDAGSAPLK4S362
    Q9NSY1BMP2KBMP-2-inducible protein kinaseDSQSSNEFLTISDSK4S1029
    A0A0C4DGD1CASTCalpastatinDTSQSDKDLDDALDK4S563
    Q9Y5K6CD2APCD2-associated proteinKNSLDELR4S582
    Q8TEH3DENND1ADENN domain-containing protein 1ATGGTLSDPEVQR4T24
    B4DU91EPN1Epsin-1SPGAFDMSGVR4S435
    M0QXW7KHSRPFar upstream element-binding protein 2VQISPDSGGLPER4S181
    F5H1C6FERMT3Fermitin family homolog 3TASGDYIDSSWELR4Y11
    P04075ALDOAFructose-bisphosphate aldolase AGILAADESTGSIAKR4S36
    H0YJ30GPHNGephyrinASHSAVDITK4S303/S305
    Q2Q9B7G6PDGlucose-6-phosphate 1-dehydrogenaseVQPNEAVYTK4Y401
    E9PK89RAB3IL1Guanine nucleotide exchange factor for Rab-3AELHPQLLSPTK4S179
    O75633GNASGuanine nucleotide-binding protein Gs α subunit isoform L3ISTASGDGR4S352
    Q5H924HUWE1HECT, UBA, and WWE domain containing 1REESPMDVDQPSPSAQDTQSIASDGTPQGEK4S3816
    A0A0F7NGI8LRRFIP1Leucine rich repeatIDGATQSSPAEPK4S714
    E9PJF4CLNS1AMethylosome subunit pIClnEPVADEEEEDSDDDVEPITEFR4S102
    S4R3D6APIPMethylthioribulose-1-phosphate dehydrataseDISGPSPSKK4S87
    Q6NT16SLC18B1MFS-type transporter SLC18B1SKSQNILSTEEER4S438
    B4DS42OATL1Ornithine aminotransferase-like 1QASLDGLQQLR4S506
    P30041PRDX6Peroxiredoxin-6DFTPVCTTELGR4T44
    I3L1U0RILPRab-interacting lysosomal proteinAESSEDETSSPAPSK4S354
    Q9P227ARHGAP23ρ GTPase-activating protein 23SAEALGPGALVSPR4S361/S372
    Q16513PKN2Serine/threonine-protein kinase N2ASSLGEIDESSELR4S583
    C9JIG9OXSR1Serine/threonine-protein kinase OSR1TEDGGWEWSDDEFDEESEEGK4S339
    P02549SPTA1Spectrin α chain, erythrocytic 1QDTLDASLQSFQQER4S1976
    B2RMN7SPTBSpectrin β chainLSSSWESLQPEPSHPY4S2123/S2125/S2128
    B4DZC9SLKSTE20-like serine/threonine-protein kinaseVDEDSAEDTQSNDGK4S571
    P17987TCP1T-complex protein 1 subunit αHGSYEDAVHSGALND4S544/Y545
    B4DUI5TPI1Triosephosphate isomeraseIIYGGSVTGATCK4Y246
    Q8IV54TSC22D4TSC22D4 proteinVEAEAGGSGARTPPLSR4T229
    B4DQ79LYNTyrosine-protein kinaseVIEDNEYTAR4Y397
    A0A024R8I2UBADC1Ubiquitin associated domain containing 1, isoform CRA_cAPSPLPK4S98
    Q5VVQ6YOD1Ubiquitin thioesterase OTU1SSPAFTK4S130
    B3KW31VPS13AVacuolar protein sorting 13AQASFTDVRDPSLK4S1416
    A0A0S2Z3K9CRKV-crk sarcoma virus CT10 oncogene-like protein isoform 2DSSTSPGDYVLSVSENSR4S41
    • List of significantly enriched or exclusive phosphopeptides in reticulocytes after deformation, as assayed through microsphiltration and microcapillary traversal. Generally, only phosphopeptides present in at least 4 of the 7 postdeformation samples (4 microsphiltration + 3 microfluidics) and not present in at least 4 of the 7 predeformation samples were included. The exception to this analysis consists of the phosphopeptides counted as 7*, which were present in all postdeformation samples but also in 4 of 7 of the predeformation samples. The prediction of the most probable phosphosites in the phosphopeptide was included according to the count of occurrences in previous low-throughput and high-throughput studies, as assayed through PhosphoSitePlus.50