ࡱ> KMJ@$CbjbjFF.N,,nbbbb <......../111111$(RzUuw ..w w U..*W W W w ../W w /W W W ." :<by W /H<W ` F`W `W .hJW < [...UU~A ~ CHOLERA TOXIN USES DIFFERENT TRAFFICKING PATHWAYS ON IN Balb3T3 AND HeLa CELL LINES Blagojevi G, Mahmutefendi H, Ku i N and Lu in P. Department of Physiology and Immunology, Medical faculty, University of Rijeka Problem It is well known that cholera toxin uses different mechanisms to enter the host cells. The aim of our study was to compare endocytotic pathway(s) and intracellular trafficking of cholera toxin binding subunit (CTB) between murine fibroblasts (Balb3T3) and human epithelial cells (HeLa). Materials and methods In order to investigate the endocytotic pathway of CTB, we used different chemical inhibitors known to impair clathrin-mediated endocytosis (chlorpromazine) and endocytosis mediated by caveolae (filipin and -cyclodextrin). Cells were analyzed by flow cytometry and confocal microscopy . To dissect intracellular pathway of CTB we used markers of subcellular compartments, GM-130, Lamp-1, EEA-1, Rab 5A and cCaveolin-1. We also used inhibitors of vesicular acidification (monensin, bafilomycin A1 and ammonium chloride) to distinguish contribution involvement of acidic compartments in CTB transport from plasma membrane to Golgi compartmentse apparatus (GA). Kinetics of degradation , was followed by flow cytometry and western blot analysis. In our study we used CTB Alexa Fluor 555 or CTB Alexa Fluor 488 in concentration of 1g/ml. Results Following 30 minutes of incubation at 37 C CTB was found in Golgi apparatus (GA) on HeLa cell line and after 45 minutes on Balb3T3 cell line. The majority of CTB was colocalizated with marker of lysosomes after 75 minutes on Balb3T3 cell line .Following After 30 minutes of incubation at 37 C CTB was found in GA ofn HeLa cells line, and after 45 minutes ion Balb3T3 cells line. The A majority of CTB was colocalized with marker of lysosomes after 75 minutes ion Balb3T3 cells, line but no colocalization coul d be found in t on HeLa cells line. On In both cell lines CTB was colocalized with caveolin-1 but following different kinetics, whereaswhile colocalization with CTB is colocalizated with early endosomal markers was foundes only oin HeLa cells line. Filipin (cholesterol binding agent) prevented internalization of CTB only oin Balb3T3 cells line, butwhile in on HeLa cells line had no effect. -cyclodextrin, a drug that sequesters cholesterol from membranes, had partial effect on in both cell lines. Chlorpromazine, an inhibitor of clathrin endocytosis, partially prevented transport of CTB from plasma membrane to GA only on in HeLa cells line. Cytochalazin D (the agents that disturbs actin microfilaments) had no effect, whereas . Nnocodazol (the agents that disruptsturbs microtubules) inhibited transport to GA ion both cell lines inhibited only vesicular transport to GA but had not effect on internalization of CTB. Inhibitors of vesicular acidification prevented, inhibited  2Dnpvz| = Y ` 𽫛yfWH9hjhhCJaJmH sH hjhFCJaJmH sH hjh CJaJmH sH %hjha 56>*CJaJmH sH %hjh 56>*CJaJmH sH hLhFCJaJmH sH hHhF5CJaJmH sH "hHhF5>*CJaJmH sH hhF5 h5Hhݢ&hz.55 hc%5(ha hz.55cHdhdhdhݢ& ha 5 hA5 h85   8H@@B!C#C$C dh7$8$H$gdl $dha$gdl dh7$8$H$gd dhgdl $dha$gda $C` b h j u v w x    A ⳤ񤕆wwdQ%hjhF56>*CJaJmH sH %hjhA56>*CJaJmH sH hjhHYCJaJmH sH hjhhCJaJmH sH hjh 2VCJaJmH sH hjhHCJaJmH sH hjhRACJaJmH sH ?hjhFhc%CJaJcHcHdhdhݢ&dh%FmH sH hjhFCJaJmH sH hjhACJaJmH sH A C R S V Y ] ^ g l { | } ~  ôҥҖxj\PD5P5hjh CJaJmH sH hjh CJ\aJhjhlCJ\aJhjhl5CJ\aJhjh;A5CJ\aJhjh;ACJaJmH sH hjhHCJaJmH sH hjh>CJaJmH sH hjhlCJaJmH sH hjhZ.CJaJmH sH hjhACJaJmH sH hjhFCJaJmH sH ;hjhFhz.5CJaJcHdhdhdhݢ&mH sH    " $ D P v   J Ǹ|ǩmǚ^L#Hhݢ&hz.5CJaJmH sH hjh CJaJmH sH hjhRACJaJmH sH hjhACJaJmH sH hjh}CJaJmH sH hjhFCJaJmH sH hjh>CJaJmH sH hjh;ACJaJmH sH hjh!7CJaJmH sH hjh CJaJmH sH hjhlCJaJmH sH hCJaJmH sH  IVbjlñññÃtbD;hjh;Ahz.5CJaJcHdhdhdhݢ&mH sH #Hhݢ&hz.5CJaJmH sH hjhFCJaJmH sH hjh) CJ\aJmH sH ;hjh hz.5CJaJcHdhdhdhݢ&mH sH #Hhݢ&hz.5CJaJmH sH hjh CJaJmH sH hjh!7CJaJmH sH ;hjh!7hz.5CJaJcHdhdhdhݢ&mH sH 468H#ôzk_k??hjh}hc%CJaJcHcHdhdhݢ&dh%FmH sH hv?CJaJmH sH hjhv?CJaJmH sH %hjhF56>*CJaJmH sH hjh!7CJ\aJhjh!7CJaJmH sH hCJaJmH sH hjhFCJaJmH sH ;hjhRAhz.5CJaJcHdhdhdhݢ&mH sH ;hjhFhz.5CJaJcHdhdhdhݢ&mH sH #()4KX\^_޹{]?$5hOhc%CJaJcHdhdhdh%FmH sH ;hjhFhc%CJaJcHdhdhdh%FmH sH ;hjhRAhc%CJaJcHdhdhdh%FmH sH ?hjh}hc%CJaJcHcHdhdhݢ&dh%FmH sH ;hjh}hc%CJaJcHdhdhdh%FmH sH HHhݢ&hjhz.5hc%CJaJcHdhdhdh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH _`ablmrs῟_?$5hOhc%CJaJcHdhdhdh%FmH sH ?hjhRAhc%CJaJcHcHdhdhݢ&dh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH ;hjhRAhc%CJaJcHdhdhdh%FmH sH ?hjhG8hc%CJaJcHcHdhdhݢ&dh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH ;hjhG8hc%CJaJcHdhdhdh%FmH sH  ޾b@bHHhݢ&hjhz.5hc%CJaJcHdhdhdh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH ?hjhG8hc%CJaJcHcHdhdhݢ&dh%FmH sH ;hjhG8hc%CJaJcHdhdhdh%FmH sH ;hjhRAhc%CJaJcHdhdhdh%FmH sH ?hjhRAhc%CJaJcHcHdhdhݢ&dh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH   #ácCc4"#Hhݢ&hz.5CJaJmH sH hjh CJaJmH sH ?hjh hc%CJaJcHcHdhdhݢ&dh%FmH sH ;hjh hc%CJaJcHdhdhdh%FmH sH ?hjhG8hc%CJaJcHcHdhdhݢ&dh%FmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH ;hjhG8hc%CJaJcHdhdhdh%FmH sH ;hjhFhc%CJaJcHdhdhdh%FmH sH  #$27@AFHKLN^be̮ooZK-;hjh}hz.5CJaJcHdhdhdhݢ&mH sH hjh}CJaJmH sH )Hhݢ&hjhz.5CJaJmH sH ;hjh hz.5CJaJcHdhdhdhݢ&mH sH #Hhݢ&hz.5CJaJmH sH hjh CJaJmH sH ;hjh hz.5CJaJcHdhdhdhݢ&mH sH #Hhݢ&hz.5CJaJmH sH BHhݢ&hz.5hc%CJaJcHdhdhdh%FmH sH  efrðsPs9,Hhݢ&hz.5B*CJaJmH phsH DhjhNchz.5B*CJaJcHdhdhdhݢ&mH phsH ,Hhݢ&hz.5B*CJaJmH phsH %hjh) B*CJaJmH phsH %hjhNcB*CJaJmH phsH %hjh}B*CJaJmH phsH hjh}CJaJmH sH hjhNcCJaJmH sH ;hjhNchz.5CJaJcHdhdhdhݢ&mH sH  ,.8<Nʳݠ}}jWW4WDhjh5hz.5B*CJaJcHdhdhdhݢ&mH phsH %hjh5B*CJaJmH phsH %hjhFB*CJaJmH phsH DhjhNchz.5B*CJaJcHdhdhdhݢ&mH phsH %hjhNcB*CJaJmH phsH ,Hhݢ&hz.5B*CJaJmH phsH %hjh}B*CJaJmH phsH Dhjh}hz.5B*CJaJcHdhdhdhݢ&mH phsH 02<\^f  ,36QtƳƳƳvcPc=%hjh=^B*CJaJmH phsH %hjh;AB*CJaJmH phsH %hjhNcB*CJaJmH phsH 2Hhݢ&hjhz.5B*CJaJmH phsH hOB*CJaJmH phsH %hjhFB*CJaJmH phsH %hjh5B*CJaJmH phsH Dhjh5hz.5B*CJaJcHdhdhdhݢ&mH phsH ,Hhݢ&hz.5B*CJaJmH phsH ٶnK8%n%hjhFB*CJaJmH phsH %hjhNcB*CJaJmH phsH Dhjh5hz.5B*CJaJcHdhdhdhݢ&mH phsH ,Hhݢ&hz.5B*CJaJmH phsH 2Hhݢ&hjhz.5B*CJaJmH phsH ,Hhݢ&hz.5B*CJaJmH phsH Dhjh5hz.5B*CJaJcHdhdhdhݢ&mH phsH %hjh5B*CJaJmH phsH %hjh;AB*CJaJmH phsH  %*/>XYʻmmmXF#Hhݢ&h.?CJaJmH sH )Hhݢ&hjh.?CJaJmH sH ;hjhNchz.5CJaJcHdhdhdhݢ&mH sH #Hhݢ&hz.5CJaJmH sH hjhNcCJaJmH sH hjh=^CJaJmH sH hjhFCJaJmH sH %hjhFB*CJaJmH phsH DhjhFhz.5B*CJaJcHdhdhdhݢ&mH phsH YZl@@@ҴxZ*CJaJmH sH hjhFCJaJmH sH hjh) CJaJmH sH hjh CJaJmH sH ;hjhFh.?CJaJcHdhdhdhݢ&mH sH ;hjhNch.?CJaJcHdhdhdhݢ&mH sH hjhNcCJaJmH sH  AAMAPAQASA\A]AbAgAAAAAAAAAҙ{oQ<-Qhjh]$CJaJmH sH )Hhݢ&hjh.?CJaJmH sH ;hjh]$h.?CJaJcHdhdhdhݢ&mH sH hOCJaJmH sH ;hjh5h.?CJaJcHdhdhdhݢ&mH sH #Hhݢ&h.?CJaJmH sH )Hhݢ&hjh.?CJaJmH sH #Hhݢ&h.?CJaJmH sH hjh5CJaJmH sH ;hjh5h.?CJaJcHdhdhdhݢ&mH sH AAAABBBBBBB0ByB~BBBBBBBBBBBBBBB񲠲s^ss^RhOCJaJmH sH )Hhݢ&hjh.?CJaJmH sH ;hjh]$h.?CJaJcHdhdhdhݢ&mH sH hjh CJaJmH sH #Hhݢ&h.?CJaJmH sH hjhHYCJaJmH sH ;hjh]$h.?CJaJcHdhdhdhݢ&mH sH #Hhݢ&h.?CJaJmH sH hjh]$CJaJmH sH B!C"C#C$Chjh|CJaJhjh!7CJaJhjh CJ\aJhjhFCJaJmH sH ,1h. A!"#$% @@@ FNormalCJ_HaJmHsHtH>A@> Zadani font odlomkaVi@V Obi na tablica4 l4a .k@. Bez popisaZ@Z ! Popisna tablica 1:Vj%@ jj#j0 j|44$a$QCJ@B*`JphB*`Jph56B* \]`J ph5\N@N z.5Tekst balon iaCJOJQJ^JaJnN[go  kmp^>0\>0\>0\>0\>0\>0\>0\>0^>0\>0\>0\>0\>0\>0[go  kmp00000000000000[go p\>0\>0p\>0\>0\>0m \>0p` A  #_#eY@@ AAB$C "#$%&$C $Ci2jkl "mn4olGCCOpMKVVpC*urn:schemas-microsoft-com:office:smarttagsmetricconverter9*urn:schemas-microsoft-com:office:smarttagsplace=*urn:schemas-microsoft-com:office:smarttags PlaceName=*urn:schemas-microsoft-com:office:smarttags PlaceType 488 in ProductIDJNY[ebm~*2|$)*/;@AF#3<cW[i X\ip  " ' R [ i m pZ[YhCE3?^dTW ? F p333333333[T c kmpcWp0/8 ) G8>]$c%[(-X/z.5JEHO 2V=^Ncu|2} ! 5RAHY j} @Al\!7 .?v?F a LZ.h;AH@ccccpKKKK J n``@` `@``$@``,@`@UnknownPeroGogaGz Times New Roman5Symbol3& z Arial5& zaTahoma"qcڢ&+Fڢ&'G 'G !24dgg 3KP)?8uV CHOLERA TOXIN USES MULTIPLE TRAFFICKING PATHWAYS FROM PLASMA MEMBRANE TO GOLGIGOGAGogaOh+'0 (4D T` |   W CHOLERA TOXIN USES MULTIPLE TRAFFICKING PATHWAYS FROM PLASMA MEMBRANE TO GOLGI GOGA CHOGAOGANormalHGogalH22aMicrosoft Word 10.0@`@ ~;@Jx;@Z :<'G ՜.+,0D hp  #C15 DHg W CHOLERA TOXIN USES MULTIPLE TRAFFICKING PATHWAYS FROM PLASMA MEMBRANE TO GOLGI Naslov  !"#$%&')*+,-./123456789;<=>?@ACDEFGHILRoot Entry F:<NData (1Table0`WordDocument.NSummaryInformation(:DocumentSummaryInformation8BCompObjk  FDokument Microsoft Worda MSWordDocWord.Document.89q