Spira, Felix

English

Digitale Hochschulschriften der LMU

Spatio-temporal organisation of plasma membrane proteins in budding yeast

Spira, F.

MPG.PuRe

2 31 4 56MethionineMup1Art1Rsp5PMoutsideinside1)2)3)GPI Proteinliquid disordered domain liquid ordered domainoutsideinsideGlycosylationCan1RFP Pma1GFP merge24756310204060801001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22number of TM segmentsproteinsSensor51Aminoacid4StressMetabolism51Cell Wall2Lipid2EisosomeSignalling62GTPase2Kinase1Bud Site Selection1PhosphataseTransporter264Aminoacid1Anorganic Acid10Ion3Multidrug1Small Carbohydrate3Sugar Vitamin3Organic Acid1Atto488Sag1Sur7GFP01intensity (a.u.)deconvolvedraw TIRF-SIM01posit ion intensity (a.u.)BATef1GFPintensity (a.u.)raw deconvolveddeconvolvedTIRF-SIM01deconvolvedTIRF-SIM01intensity (a.u.)deconvolvedrawSTIRFdeconvolvedPma1GFP raw STIRF overlaySTIRFdeconvolvedConfocal STEDTIRFw/ DeconvolutionRawTop-SectionPil1GFPApatch-likenetwork-likeFet3GFPHxt3GFPMid2GFPSur7GFP Gpa1GFP Ras2GFPBio5GFPMep2GFPBBio5Mep2Hxt3LinescanIntensity(a.u.)*Psr1GFPCLact-C2GFP Gap1GFP Hxt1GFPpatch-likeLinescanIntensity(a.u.)LinescanIntensity(a.u.)D0.00.10.20.3Lact-C2network factorlog(gfp intensity)0.0 0.2 0.4123FET3 wtHXT3 wtMRH1 wtPMA1 wt PMP1 wtSSY1 wtSUR7 wtYLR413W wtintensity histogramarea over  cummulative histogram0.010.090.44network factorSsy1GFPNha1GFPRas2GFP0.2 0.6 1.00.20.61.00.2 0.6 1.00.20.61.00.2 0.6 1.00.20.61.00.0 0.4 0.80.00.20.0 0.4 0.80.00.20.0 0.4 0.80.00.21.0log(GFP intensity)3451 2 3A BC DBio5Mep2Sur7Fet3Hxt3Pma1Ras2CBAl.a. 1 3 9 10 1213 14020406080No. TMSt 1/2 in sLact-C2PlcGpa10.20.2 1.01.0100 20 30 400.20.40.60.81Image Similarity (a.u.)secondsHxt3GFPRas2GFPSur7GFP0 sMerge40 stimeAutomatic Cell DetectionDeconvolutionDeconvolutionChannel Alignmentwith Latex BeadsAdjust ROISur7RFPRas2GFPx-y-shift MergeCorrect Channel ShiftSort OutRaw ImagesCell 1Cell 2......Colocalization CoefficientManders OverlapExpectedManders Overlap18/2016/20 20/2012/2010/208/204/201/202/2010.10 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Linear Colocalization CoefficientExpected(Manders Overlap)2Expected:Fraction of Spot Counts:Channel 1:Channel 2:4/16 6/12 2/4 17/20 19/200.25 0.850.05 0.950 0.2 0.4 0.6 0.8 100.20.40.60.810 0.2 0.4 0.6 0.8 100.20.40.60.81Image Pairy=sqrt(x)Image Pairy=xA B CSur7RFPPma1RFPFet3GFPMep2GFPSur7RFP Pma1RFP (others)RFPhexosetransportertest set GFPidenticalproteinsPmp1GFPTpo1GFP Vht1GFPMep2GFP Mss4GFPHxt6GFPHxt2RFPPma1GFPPma1RFPSur7RFPSur7GFP Hxt1GFPHxt3RFPMep2RFPBio5RFPHxt3GFPHxt3RFPFet3RFPBEC DA00.20.40.60.8ColocalizationAbundance(ProteinGFP) * Abundance(ProteinRFP)00.20.40.60.81e+06 1e+07 1e+08 1e+09Hxt1Hxt3GFPRFPHxt6Hxt2Hxt6GFPHxt2GFPHxt1GFPHxt3GFP00.40.800.40.8low affinity high affinity00.40.800.40.80 0.2 0.4 0.600.20.40.6overlap coefficientdecoy coefficient better than randomworse than randomrandomA BC Doverlapcell1 cell2 cell3cell1cell2cell3......green channelred channeldecoy0.1 0.3 0.500.20.40.6joint network factoroverlap coefficient4 6 8 10 120.00.20.40.6joint expression (a.u.)overlap coefficientR: 0.44p: 1e-6correlation coefficient (R)decoy divergence1 20.40.60.81.00 3overlap coefficient0.0 0.2 0.4 0.600.20.40.6decoy coefficientR: 0.92p: 00.0 0.2 0.4 0.600.20.40.6decoy coefficientR: 0.36p: 0overlap coefficientA BC+ LatB+ myriocin+ lyso-PSHxt3Mid2 Pmp1Ras2Sur7 Bio5 Fet3 Pma1patch-likenetwork-likep-valueAB0 0.05n.d. n.d. n.d. n.d.n.d.+lyso-PS -lyso-PSC Sur7 Bio5 Mid2 Hxt3Pma1Fet3DBio5GFPPmp1GFPlyso-PS for: 1 h 12 h0.30.400.11 h 12 hnetwork factornetwork factorTMMid2TMPmp1 TMFet3TMFet3RFP46%57% 51%81%RFP RFPFet3Fet3RFPPmp1GFPjoint network factorFet3TMFet3TMMid2TMPmp1Pmp1TMPmp1TMMid2ADBC0.4 0.5 0.60.40.50.60.7decoy coefficientoverlap coefficientPmp1FetPmpFet3 Pmp1FetPmpRFPFet3RFPFet3GFP Pmp1GFP Fet3GFP Pmp1GFP0.2.4.6.80.2.4.6.8ColocalizationColocalization******0 10 200.20.61.01.4time (h)OD(600)wtfet3fet3fet310**Cell Death *Cell SurvivalCanavanineSur7 domainPma1domainCan1Artificial PPISur7RFPPma1GBCan1GFPwtCan1GFPPma1CBSur7CB, Can1GFPPma1CB, Can1GFP[Canavanine]ABBio5GFPHxt3GFPMep2GFPMid2GFPPma1GFPA Bpil1 control Mup1GFP  Abp1RFP-met+met- met +metSur7RFPMup1GFPCMup1GFP -met +met10min-met40minABD Mup1GFP Abp1RFP Art1C     Art1GFP   Mup1RFP+ LatB/Met  Mup1GFP Abp1RFP + LatB/met Mup1GFP Sur7RFP Art1+ LatB/met0min 10min 20min 30min 40minMup1 T1Mup1 T2Mup1 T3ABT1 T2 T3C TIRFM Mup1GFP T1 +metPMProteinLipid Environment                           DomainsPossible Lipid-TMS“Patchwork”0.00.10.20.3SSY1PIL1SUR7BIT61FPS1SHO1ATR1MNR2ENA1RAX2DNF1ITR2MSS4BIO5MEP2ENA2SLN1RSN1TCB3FET4YOR1PDR12FLC1NHA1FUI1THI7YLR413WGAS1VHT1MID2FTR1FAA3TPO1HNM1GPA1GAP1BAP2FET3PDR5PSR1HXT2MRH1HXT6PMA1HXT3HXT1PMP1RAS2network factor123BAFRAP Analysis020406080 After 5 minutes After 1 minutepatch-likenetwork-likePma1GFP Sur7GFP Pma1GFP Sur7GFPcolocalization coefficient Pma1RFP Sur7RFPABintensityintensity00.40.8positionintensity (a.u.)01CPma1GFP Pma1RFPColocalizationColocalizationSSY1FPS1RAX2SHO1TCB3ENA1BAP2FET3ENA2PMA1MEP2BIT61ATR1RSN1PDR12MSS4NHA1YOR1SLN1FLC1TPO1MID2MRH1BIO5HXT2THI7VHT1HNM1GAP1PMP1FTR1PDR5FET4ITR2DNF1MNR2FUI1HXT3GPA1PSR1FAA3RAS2GAS1YLR413W PIL1SUR70.2.4.6.8GFPGFP0.2.4.6.8Pma1RFPSur7RFPPIL1SUR7SSY1VHT1TCB3RSN1FPS1ITR2ENA1BIO5BIT61MSS4HNM1MEP2ATR1MNR2YLR413WSLN1SHO1FET3FUI1RAX2MID2NHA1FET4YOR1PDR12DNF1ENA2HXT3FLC1TPO1FTR1HXT2GAP1FAA3GAS1PSR1THI7BAP2PMP1PDR5MRH1RAS2GPA1PMA1ABAHnm1GFP Mid2GFP Tpo1GFP Vht1GFPHxt3GFPFet3GFPAtr1GFP Flc1GFP Mep2GFPMss4GFPBit61GFPBio5RFPBio5GFPMep2RFP0.2.4.6.8Mep2ColocalizationAtr1 Bit61 Flc1 Mss40.2.4.6.8Bio5Atr1 Bit61 Flc1 Mss4Bio5RFP Mep2RFP0.2.4.6.8Hnm1 Mid2 Tpo1 Vht10.2.4.6.8Hnm1 Mid2 Tpo1 Vht1Hxt3RFP Fet3RFPBProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSAtr1/Snq1GFPTransporterMultidrug 18.59±1.54n: 440.04±0n: 75(44)RecoveryT/2: 45.6±14 .4Mf: 32±4n: 7 0.06±0.02n: 100.16±0.02n: 1914Bap2GFPTransporterAmino Acids 181.17±6.79n: 950.16±0.02n: 14(10)RecoveryT/2: 40.1±5.2Mf: 29±0n: 30.03±0.01n: 260.5±0.03n: 1412Bio5GFPTransporterVitamin 546.01±16.45n: 640.05±0n: 40(20)NoRecovery0.06±0.01n: 210.09±0.02n: 1312Bit61GFPSignallingKinase Activity 7.76±0.21n: 740.03±0n: 92 (s ignal too low)0.03±0.01n: 200.11±0.01n: 28-Dnf1GFPMetabolismLipid Metabolism 24.36±1.43n: 380.05±0n: 25(s ignal too low) 0.08±0.01n: 40.23±0.02n: 2110Ena1GFPTransporterIon 17.12±0.33n: 1560.04±0n: 38(12)Techn. Limiting(no recovery ) 0.03±0.01n: 240.09±0.01n: 149Ena2GFPTransporterIon 18.67±0.51n: 630.05±0n: 28(15)Techn. Limiting(no recovery ) 0.04±0.01n: 200.26±0.03n: 89Faa3GFPMetabolismLipid Metabolism 62.97±2.01n: 940.13±0.01n: 22(22)*Techn. Limiting(no recovery ) 0.14±0.02n: 60.36±0.02n: 16-ProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSFet3GFPMetabolismOxidoreductase 293.98±12.23n: 850.16±0.01n: 78(24)RecoveryT/2: 35.3±4.4Mf: 24±2n: 60.03±0.01n: 190.17±0.02n: 101Fet4GFPTransporterIon 22.58±1.16n: 700.07±0.01n: 29 (s ignal too low)0.1±0.02n: 180.22±0.04n: 117Flc1/ Bop1GFPTransporterIon 715.93±18.69n: 890.08±0.01n: 31(7)Techn. Limiting(no recovery ) 0.06±0.02n: 120.3±0.04n: 611Fps1GFPTransporterSmall Carbohydrates 28.52±0.91n: 910.03±0n: 37(15)Techn. Limiting(no recovery ) 0.02±0n: 140.09±0.01n: 236Ftr1GFPTransporterIon 160.52±7.01n: 890.13±0.01n: 39(24)Techn. Limiting(no recovery ) 0.09±0.01n: 160.31±0.02n: 237Fui1GFPTransporterOrganic acid 90.82±2.82n: 890.1±0.01n: 34(21)RecoveryT/2: 19.0±4.9Mf: 21±2n: 50.12±0.02n: 80.18±0.02n: 2612Gap1GFPTransporterAmino Acids 60.74±1.76n: 960.16±0.01n: 35(13)RecoveryT/2: 19.9±2.9Mf: 38±5n: 40.09±0.01n: 260.36±0.03n: 912Gas1GFPMetabolismCell Wall Metabolism 117.67±19.42n: 490.12±0.01n: 20(6)RecoveryT/2: 28.9±10 .9Mf: 49±9n: 30.14±0.03n: 90.42±0.04n: 111ProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSGpa1GFPSignallingGTPase 84.62±2.56n: 810.16±0.01n: 10(23)*No Recovery0.13±0.02n: 90.53±0.03n: 10l.a.Hnm1GFPTransporterAmino Acids 192.41±7.68n: 820.16±0.01n: 73(17)RecoveryT/2: 25.4±11.0Mf: 21±3n: 40.08±0.01n: 150.17±0.03n: 1112Hxt2GFPTransporterSugar 161.69±7.36n: 1320.18±0.01n: 127(28)NoRecovery0.06±0.01n: 160.37±0.03n: 2712Hxt3GFPTransporterSugar 716±19.62n: 860.22±0.01n: 133(18)RecoveryT/2: 45.5±5.3Mf: 74±4n: 30.12±0.01n: 450.25±0.02n: 1912Itr2GFPTransporterSugar 55.89±3.43n: 500.05±0.01n: 29(s ignal too low) 0.1±0.02n: 100.09±0.01n: 1912Mep2GFPTransporterAnorganic Acid 9.43±1.19n: 820.05±0n: 46(18)Techn. Limiting(no recovery ) 0.03±0.01n: 100.14±0.03n: 1410Mid2GFPSensorsStress 94.4±2.58n: 1110.13±0.01n: 70(33)RecoveryT/2: 58.3±17 .2Mf: 35±4n: 50.04±0n: 240.18±0.03n: 91Mnr2GFPTransporterIon 14.57±0.41n: 690.04±0n: 47 (s ignal too low)0.1±0.01n: 160.15±0.02n: 312ProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSMrh1GFPUnknown799.36±32.51n: 700.18±0.01n: 43(22)No Reco-very 0.05±0.01n: 150.48±0.02n: 287Mss4GFPSignallingKinase Activity 14.46±0.45n: 660.05±0n: 69(9)Techn. Limitation(no recovery ) 0.04±0.01n: 160.12±0.02n: 14-Nha1GFPTransporterIon 35.14±1.43n: 1030.08±0n: 50(4)Techn. Limitation(no recovery ) 0.05±0.01n: 230.19±0.02n: 2713Pdr5GFPTransporterMultidrug 360.21±56.54n: 600.17±0.01n: 21(25)RecoveryT/2: 36.7±9.9Mf: 32±2n: 50.05±0.01n: 210.2±0.04n: 713Pdr12GFPTransporterMultidrug 105.96±4.36n: 1200.07±0.01n: 28(6)Techn. Limiting(no recovery ) 0.06±0.01n: 80.52±0.03n: 1313Pil1GFPMetabolismEisosome 595.25±18.7n: 730.02±0n: 37(12)No Recovery0.59±0.03n: 220.02±0.01n: 15-Pma1GFPTransporterIon 540.29±15.64n: 1180.21±0.01n: 41(19)RecoveryT/2: 12.5±3.3Mf: 27±6n: 40.04±0.01n: 230.75±0.02n: 910Pmp1GFPTransporterIon 582.7±17.32n: 1090.28±0.01n: 73(19)RecoveryT/2: 5.8±1.2Mf: 21±2n: 70.07±0.01n: 310.58±0.05n: 71ProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSPsr1GFPSignallingPhosphatase Activity 33.7±0.76n: 1020.17±0.01n: 28(19)*Techn. Limiting(no recovery ) 0.15±0.02n: 160.45±0.02n: 12l.a.Ras2GFPSignallingGTPase 251.28±5.49n: 1570.29±0.01n: 35(29)*RecoveryT/2: 1.6±.3Mf: 67±5n: 40.12±0.01n: 180.54±0.02n: 17l.a.Rax2GFPSignallingBud Site Selection 40.6±1.36n: 710.05±0n: 29(21)Techn. Limiting(no recovery ) 0.03±0.01n: 190.15±0.02n: 101Rsn1GFPUnknown70.01±1.84n: 1290.06±0n: 27(18)RecoveryT/2: 15.2±1.2Mf: 27±4n: 40.04±0.01n: 170.1±0.01n: 1010Sho1GFPSensorsStress 23.43±1.6n: 420.04±0n: 25(s ignal too low) 0.02±0n: 140.15±0.02n: 114Sln1GFPSensorsStress 15.96±0.39n: 940.06±0n: 32(9)Techn. Limiting(no recovery ) 0.06±0.01n: 240.16±0.01n: 82Ssy1GFPSensorsAmino Acids 8.56±0.19n: 720.01±0n: 43 (s ignal too low)0.01±0n: 160.04±0n: 2712Sur7GFPMetabolismEisosomes 256.62±9.31n: 750.03±0n: 42(7)No Recovery0.72±0.02n: 200.02±0.01n: 124ProteinClass TIRFMMembraneAbundanceNetworkFactorEquator FRAP5min. RecoveryColocalization with Sur7RFPColocalization with Pma1RFPNo. TMSTcb3GFPUnknown119.99±3.78n: 920.06±0.01n: 23(9)RecoveryT/2: 49.3±8.4Mf: 57±7n: 70.02±0n: 110.07±0.01n: 121Thi7GFPTransporterVitamin 42.49±4.85n: 650.11±0.01n: 36(35)RecoveryT/2: 20.3±3.5Mf: 38±7n: 30.06±0.01n: 120.48±0.04n: 812Tpo1GFPTransporterAmino Acids 156.09±5.56n: 950.13±0n: 73(49)RecoveryT/2: 31.2±4.1Mf: 24±3n: 110.05±0.01n: 230.3±0.02n: 2712Vht1GFPTransporterVitamin 40.63±2.63n: 720.12±0.01n: 64(15)RecoveryT/2: 24.1±10 .5Mf: 25±3n: 40.08±0.01n: 140.05±0.01n: 149YLR413WGFPUnknown449.99±14.2n: 910.11±0.01n: 21(13)RecoveryT/2: 50.2±12 .2Mf: 29±2n: 50.17±0.03n: 100.16±0.02n: 113Yor1GFPSensorsStress 49.61±2.24n: 77.07±0n: 32(18)Techn. Limiting(no recovery )0.04±0.01n: 90.19±0.01n: 2311

Ludwig-Maximilians-Universität München Dissertationen

https://edoc.ub.uni-muenchen.de/15496/1/Spira_Felix.pdf

Spatio-temporal organisation of plasma membrane proteins in budding yeast

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Digitale Hochschulschriften der LMU

MPG.PuRe

MPG.PuRe

Ludwig-Maximilians-Universität München Dissertationen