Egészségügy | Biofizika » The role of mosaicism in the genetically influenced topography of cell surface receptors

The role of mosaicism in the genetically influenced topography of cell surface receptors Sándor Damjanovich Department of Biophysics and Cell Biology Center of Medical and Health Sciences University of Debrecen, Hungary Contributors Cell Biophysics Res. Gr Dr. Andrea Bodnár Dr. György Vámosi Miklós Bagdány Dept. of Biophysics Prof. Rezső Gáspár jr Prof. János Szöllősi Dr. György Panyi Dr. Péter Nagy Dr. László Mátyus Dr. Attila Jenei Dr. György Vereb Dr. László Bene Gergely Szentesi Univ. of Debrecen Dr. Péter Bagossi Dr. József Tőzsér Prof. Sándor Varga Dr. Tibor Farkas (Szeged), Dr János Matkó (Budapest), Prof Michael Edidin (Baltimore), Dr. Thomas Jovin (Göttingen), Dr Thomas Waldmann (NIH), Carolyn Goldman (NIH) THE CELL SURFACE IS THE PLATFORM FOR COMMUNICATION, THROUGH WHICH THE CELL EXCHANGES MATTER, ENERGY AND INFORMATION WITH THE ENVIRONMENT. FOR A SINGLE CELL EVEN THE NEIGHBORING CELL BELONGS TO THE ENVIRONMENT. Ig superfamily

LFA-3 CD58 LFA-2 CD2 ICAM-1 ICAM-1 VCAM-1 CD54 TCR-CD3 complex CD4 CD8 MHC II MHC I - Role of K+ ion channels in lymphocyte activation - Polarized localization of Na+ and K+ channels in neuronal membranes - Role of the TCR-CD3 and IL2 receptor complexes in lymphocyte activation We wanted to answer the following questions in our experiments: 1. Are TCR/CD3 receptor complexes associated with voltage dependent Kv1.3 channels in the membrane of lymphocytes? 2. How are Kv13 ion channels distributed in the lymphocyte membrane? Experimental strategy, PCR mutagenesis PCR primer (61 bases, 24 bases coding the FLAG epitope) Aat II pRc/CMV/Kv1.3 Amp DYKDDDDK PCR products WT S1 S2 + +S4 + + S3 NH2 S6 HOOC α α S5 α α extracellular space intracellular space 1 +2 3 + 4 5 + 6 7 + 8 9 Sequence analysis Mutation at the Aat II recognition site FLAG Functional test I.: Kv1.3/FLAG and Kv13/WT ion currents are similar Rf Kv1.3/WT Ip

− + elektroporation CTLL-2 Vout Vm Kv1.3/FLAG 10 Kv1.3/FLAG 10 τ = 214.6 ms Current (nA) áram (nA) áram (nA) Current (nA) 12 8 6 4 Kv1.3/WT 8 τ = 234.6 ms 6 4 2 2 0 0 0 500 1000 1500 idõ (m s) time (ms) 2000 0 500 1000 time (ms) 1500 2000 Functional test II.: Specific expression of the FLAG epitope Kv1.3/WT effectene Jurkat Jurkat anti-FLAG M2 FITC-RAMIG flow cytometer Jurkat Kv1.3/FLAG Kv1.3/FLAG control 200 F-RAMIG 100 anti-FLAG + F-RAMIG 50 cells 250 150 Kv1.3/WT 300 frekvencia Number of frekvenciaof cells Number 300 250 200 control 150 F-RAMIG anti-FLAG + F-RAMIG 100 50 0 0 0 100 200 300 csatorna szám Channel# 400 500 0 100 200 300 csatorna szám Channel# 400 500 Specific labeling of the FLAG epitope by 10 nm immunogold beads Kv1.3/WT effectene Jurkat Jurkat anti-FLAG M2 aurogamig G10 Jurkat electron microscopy Kv1.3/FLAG Kv1.3/FLAG A 200 nm Kv1.3/WT B 200 nm Distribution of Kv1.3

channels is non-random λ = 5 .7 0 .2 0 relatív gyakoriság Relative frequency Relative frequency relatív gyakoriság 0 .2 5 0 .1 5 0 .1 0 0 .0 5 0 .0 0 λ = 5 .2 0 .2 0 0 .1 5 0 .1 0 0 .0 5 0 .0 0 0 2 4 6 8 10 12 14 16 18 0 a beads b e a d -per e k unit s z á area m a # of 0 .1 0 0 .0 5 0 .0 0 6 8 10 12 14 16 18 te r ü le t e g y s é g e n k é n t relatív gyakoriság Relative frequency Relative frequency relatív gyakoriság λ = 5 .8 0 .1 5 4 b e a dper - e kunit s z áarea m a # ofabeads te r ü le t e g y s é g e n k é n t 0 .2 0 2 0 .2 0 λ=5 0 .1 5 0 .1 0 0 .0 5 0 .0 0 0 2 4 6 8 10 12 14 16 a b e a d -e k s z á m a # of beads per unit area 18 te r ü le t e g y s é g e n k é n t 0 2 4 6 8 10 12 14 16 18 b e a d -e k s z á m a # ofabeads per unit area te r ü le t e g y s é g e n k é n t Co-localization of CD3 and Kv1.3/FLAG Kv1.3/FLAG Jurkat Jurkat effectene CD3 anti-FLAG M2 + Alexa 546-GAMIG

Alexa 633-anti-CD3 Jurkat CLSM Numerical analysis of the co-localization of CD3 and Kv1.3/FLAG (calculation of the cross correlation coefficient) A B ∑( x ij C= ij − y ) i, j ∑(x ij i, j C )( y − x − x ) ∑( y 2 ij − y ) 2 = 0.745 i, j D -xij and yij : fluorescence signals at individual pixels with coordinates i and j of the two channels x and y of the picture - <x> and <y> : mean pixel intensities in the corresponding channels Fluorescence Resonance Energy Transfer (FRET) hν’ FRET hν D A A D kT ∝ JR κ −6 2 R06 E= 6 R0 + R 6 Molecular vicinity of CD3 and Kv1.3/FLAG: fluorescence resonance energy transfer energy transfer anti-FLAG M2 + F-RAMIG+Cy5-RAMIG Kv1.3/FLAG Jurkat Jurkat flow cytometer Jurkat Cy3-anti-CD3 effectene CD3 E = 1− aFLAG+Cy5-RAMIG Cy5-RAMIG transfer 150 100 50 felső 15% 1 10 100 1000 fluorescence intensity Cy5-channel:selection 200 number of cells number of cells FL-1

(fluorescein) FL-4 (Cy5) 200 0 FDA − B ; E = 51 ± 29% (SD, n=3) FD − B aFlag+F-RAMIG F-RAMIG transfer 150 100 50 10000 0 1 10 100 1000 10000 fluorescence intensity Fluorescein channel: FRET measurement Kv1.3 ion channels on CTLs are present in the immunological synapse MHC I Kv1.3 CTL CD8 JY (target) Transmission image Confocal section Summary • We produced a Kv1.3 gene coding for the FLAG epitope • We proved that the mutant protein is functional (patch- clamp technique, K+ current) and can be labeled specifically (flow cytometry). • Electron microscopy and confocal microscopy showed that Kv1.3 proteins have a non-random distribution In addition, Kv1.3 channels and CD3 molecules are significantly colocalized (CLSM), and are in molecular vicinity (FRET) in the plasma membrane of T cells. • Kv1.3 channels are often enriched at the immunological synapse Number of cells FRET efficiency (%) IN PRINCIPLE THE CELL SURFACE

CONTAINS ALL DATA ON THE IMMEDIATE PAST OF THE CELL THE PLASMA MEMBRANE IS THE DESTINATION OF THE MAJORITY OF SYNTHESIZED PROTEINS. THE MICRODOMAIN STRUCTURE OF THE PLASMA MEMBRANE IS BUILT OF PROTEINS AND LIPIDS PROTEINS MAY ALSO CONTRIBUTE TO THE MAINTENANCE OF THE DOMAIN STRUCTURE THE MICRODOMAIN STRUCTURE – LIPID RAFTS – AND THE ASSOCIATION PATTERNS OF PROTEINS MAY ALSO BE GENETICALLY DETERMINED A REASON FOR THIS CAN BE THAT TRANSMEMBRANE α-HELICAL SEGMENTS DETERMINED BY THE GENETICALLY CODED PRIMARY STRUCURE MAY BE LOCALIZED PREFERENTIALLY IN LIPID MICRODOMAINS. THEREFORE CELL SURFACE RECEPTOR PATTERNS MAY CONTAIN INFORMATION ON THE GENETIC MAKE-UP AND ON THE IMMEDIATE PAST OF THE CELL. THE PLASMA MEMBRANE HOSTS DETERMINISTIC AND INDETERMINISTIC ELEMENTS SIMULTANEOUSLY Change of receptor patterns Epitope mapping by triangulation 3 2 4’ 3’ 2 2 r12 1 r23 ? 1 r14 3 r24 ? 4 r13 1 3 2 r24 1 r14 4 r34 ! Kit225 K6 Kit225 IG3 Hut102B2

Kit225 IG3 Sequential labeling with 30 and 10 nm gold beads IL-2Rα on Kit225 K6 cells labeled with 30 nm colloidal gold AA + AB = YZ AA BB + BA X = BB Z BA X ⋅ − 1 ≈ 0.61 BB Z AB = YZ − 1 ≈ 0.24 AA The IL-2 and IL-15 Receptor System IL-2Rα IL-15Rα γc IL-2/15Rβ γc IL-2/15Rβ The structure of human IL-15 The IL-15 protein is a 14-15 kDa member of the 4α helix bundle cytokine family. IL-2 HLA-DR CD48 phospholipid sphingolipid α β γc JAK3 JAK1 STAT3 STAT5 ”lipid raft” αβ cholesterol Co-localization of IL-2Rα with raft and non-raft proteins IL-2Rα: green MHC-II: red c = 0.37 IL-2Rα: green transferrin receptor: red c = 0.05 Co-localization of GM1 ganglioside (lipid raft marker), ILRAP and IL-2Rα detected by CLSM Pictures in the individual channels Raft (F-CTX) ILRAP (Cy3-5F7) IL-2Rα (Cy5-αTac) Raft + ILRAP Raft + IL-2Rα ILRAP + IL-2Rα Combined pictures

Co-localization of GM1 ganglioside (lipid raft marker), IL-15Rα and IL-2Rα detected by CLSM IL-2Rα + IL-15Rα Raft + IL-15Rα (A488-aTac + (A488-CTx + A568-Garig-anti-IL-15Rα) A568Garig-anti-IL15Rα) IL-15Rα (A488Garig-anti-IL15Rα) The life and death of the T cell: the IL-15 and IL-2 story Functions shared by IL-2 and IL-15 • Stimulate the proliferation of CTLL T-cell lines, activated CD4-8-, CD4+8+, CD4+, CD8+ and γδ subsets of T-cells. • Facilitate the induction of cytosolic effector T-cells including CTL and LAK cells. • Stimulate the generation, proliferation and activation of NK cells. Synergise with IL-12 to facilitate their synthesis of IFN-γ and TNF-α. • Induce the proliferation and immunoglobulin synthesis by B-cells stimulated by anti-IgM or CD40 ligand. IL-2 and IL-15 have complementary and conflicting actions in different systems • IL-2 and IL-15 have complementary actions supporting innate immunity. They play a pivotal role in development,

survival and activation of NK cells. •IL-2 is involved in peripheral tolerance by inducing suicide of self-reactive T-cells. In particular, IL-2 provides the cytokine signal for activation induced cell death (AICD) and inhibits the maintenance of CD8+ T-cells of memory phenotype. • In contrast, IL-15 favors the survival of memory cells. It is required for the division of CD8+ memory T-cells and it inhibits the cytokine signal provided by IL-2 that induces AICD. Kinetics of intermixing of F-W6/32 and R-W6/32 Fablabeled MHC-I clusters on JY cells fused with PEG 0 min, 15 µm 20 min, 15 µm 40 min, 15 µm 60 min, 10 µm 80 min, 10 µm 100 40 80 30 60 20 40 10 20 0 0 0 20 40 60 80 time after cell fusion (min) FRET efficiency (F- and R-W6/32 Fabs ), fused JY cells overlap of fluorescein and rhodamine clusters, fused JY cells FRET efficiency on a double-labeled, non-fused JY cell sample overlap of clusters, non-fused JY cells overlap of clusters (%) energy transfer

effieciency (%) Intermixing of small- and large-scale MHC I clusters follows a different time course Mixing of MHC-I and MHC-II protein clusters on PEG-fused JY cells energy transfer measurements 1.2 0.3 0.2 1.0 0 min 80 min non-fused, double-labeled cells 0.8 0.6 0.4 0.1 0.2 0.0 0.0 M H CIM H CI M H C(M II H M CH I& CII M H C(M II )/M H CH I& CI M H CII )/M H CII 0.4 FM H CI, RM H CFI M H CII ,R -M H CFII M H CII ,R -M H CI energy transfer efficiency 0.5 0 min 80 min non-fused, double-labeled cells ratio of co-cluster area 0.6 NSOM measurements green red remark A Cy3-IL-2R Cy5-IL-2R K6, 0 min B Cy3-IL-2R Cy5-IL-2R C F-MHC-I D F-MHC-I green red remark E Cy3-CD48 Cy5-CD48 K6, 80 min K6, 80 min F Cy3-CD48 Cy5-TrfR K6, 80 min R-MHC-I JY, 80 min G Cy3-IL-2R Cy5-CD48 K6, 80 min R-MHC-I JY, 80 min, on ice H Cy3-TrfR Cy5-TrfR K6, 80 min Raft and non-raft proteins home to their native cluster environment after fusion of

Kit225 K6 human T cells NSOM A 100 overlap of clusters (%) energy transfer efficiency (%) pbFRET 40 30 20 10 B 80 60 40 20 0 0 A B C D E 0 min (A) (B) (C) (D) (E) (F) A F 80 min B C double-labeled CD48 and CD48 TrfR and TrfR IL-2Rα and IL-2Rα IL-2Rα and IL-2Rα with AlF3 MHC-I and MHC-I CD48 and TrfR D E F Fluorescence correlation spectroscopy measures local diffusion properties by detecting the rate of fluorescence intensity fluctuations in a subfemtoliter laserilluminated detection volume ω xy ~ 0.34 µm Carl Zeiss Jena . Fluorescence intensity fluctuations may be due to. . diffusion of fluorescently labeled molecules across the detection volume F(t) time Fluorescence intensity fluctuations may be due to. . photophysical processes like photobleaching, triplet state formation, dark state formation due to protonation. F(t) time Fluorescence intensity fluctuations may be due to. . or any other molecular process affecting the

fluorescence quantum yield. F(t) time . From the fluorescence count trace. δF(t) τD time . which is determined by the dynamic parameters of the molecular processes affecting the fluorescence intensity: .the autocorrelation function G(τ) is calculated G(τ) G(0) ~1/N • the average number of molecules residing in the detection volume, N • the diffusion correlation time, τD, which is inversely proportional to the diffusion coefficient • aggregate size (fluorescence per particle F/N) . • photophysical rate constants, etc. τD = 2 ω xy 4D τ Multicomponent system: Determination of the amount and diffusion coefficient of free and bound molecules 1,9 1/N’ 1,7 1,5 G(τ) 1,3 1,1 0,9 0,01 τfree 0,1 1 τbound 10 100 1000 10000 τ [ms] Cross correlation: The joint fluctuation of two molecules labeled with distinct types of dye indicates co-mobility – demonstration of molecular association a+b ab G (τ ) = × δFa (t ) ⋅ δFb (t + τ )

Fa Fb What kind of interactions can be studied? Protein - DNA Nucleic acid hybridization Antigen - antibody Ligand binding to receptor Diffusion of IL-2 receptor α subunit in the membrane of FT 7.10 cells τD=200 ms D~12×10-10 cm2/s Pixel-by-pixel FRET measurement using acceptor photobleaching Donor: Cy3-L368 (β2m) Acceptor: Cy5-W6/32 (HLA I) Donor fluorescence increases after bleaching the acceptor Difference of donor intensity after and before acceptor photobleaching E = 1 − FDA / FD SNOM Scanning NearField Optical Microscopy SCHEMATIC REPRESENTATION OF THE PRINCIPLE SNOM block diagram Ar or He-Ne laser optical fiber coupler generator ditherpiezo laser XYZ-piezo photodetector (APD) photodiode AFM XY scanning, Z feed-back computer optical and topographical information Topography and SNOM fluorescence image of R-4D5 labeled unstimulated SKBR3 cells topography fluorescence combined