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Lecture 08 Membrane Fluidity Polarity
Cell Biology (ZOOLOGY 570)
University of Wisconsin-Madison
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Membrane Fluidity & Polarity_Lecture_08 Cell Fusion Ce l lf us i oni s at e c hni quei nwhi c ht wodi ffer entt ypesofc e l l sorc e l l sf r o mt wodi ffer ents pec i es ar ef us ed t o pr oduc e one c e l lwi t hac ommon c yt opl as m and a s i ngl e ,c ont i nuous pl as ma membr ane . I nt hi st e c hni que ,c e l l sar ei nduc edt of us ewi t ho neano t herbymaki ngt heout ers ur f ac eo ft he c e l l s“ s t i c ky”s ot hatt hei rpl as mamembr anesadher et ooneano t her .Ce l l sc anal s obei nduc edt o f us ebyaddi t i onofc er t ai ni nac t i vat edvi r us est hatat t ac ht ot hes ur f ac eme mbr ane ,byaddi ngt he c ompoundpol ye t hyl enegl yc o l ,orbyami l de l ec t r i cs ho c k. Ce l lf us i on pl aysan i mpor t antr ol ei n pr epar at i on o fs pec i fic ant i bodi e s . Frye and Edidin Experiment: Experimental Approach I nt hi se xper i ment ,mous e and human c e l l s wer ef us e d by addi t i ono fs endaivi r us ,andt hel o c at i onofs pec i ficpr o t e i nsof t he pl as ma membr ane wer e f ol l o wed onc e t he pl as ma membr anebec amec ont i nuous . To f o l l o w t he di s t r i but i on o fei t her t he mous e me mbr ane pr o t ei no rt hehumanme mbr anepr o t ei natvar i oust i mesaf t er f us i on,ant i bodi esagai ns toneort heo t hert ypeofpr o t ei nwer e pr epar ed and c o val ent l yl i nked t o fluor es c ent dye s . The ant i bodi esagai ns tt hemous epr o t e i nswer ec ompl e xe dwi t ha dyet hatfluor es c esgr een and t heant i bodi esagai ns thuman pr o t ei nswi t honet hatfluor es c esr ed. Whent heant i bodi eswer eaddedt of us edc e l l s ,t he yboundt o human or mous e pr o t e i ns and c oul d be l oc at ed under a fluor es c entl i ghtmi c r os c ope . Att het i meoff us i on,t hepl as ma membr ane appe ar ed hal f humanandhal fmous e;i . e . ;t het wot ypesofpr o t ei nr emai ned s e gr egat edi nt hei ro wnhemi s pher e . Ast het i meoff us i oni nc r e as ed,t heme mbr anepr o t ei nswer es eent omo vewi t hi nt hemembr ane i nt ot he oppos i t e hemi s pher e .By about 40 mi nut es ,e ac hs pec i es ’pr o t ei ns wer e uni f o r ml y di s t r i but edar oundt heent i r ehybr i dc e l lmembr ane . Immunofluorescence o I ti so ft wot ypes :( i . )Di r e c tI mmunofluor e s c e nc e ;and( i i . )I ndi r e c tI mmunofluor e s c e nc e . ( i . ) Di r e c tI mmunofluor e s c e nc e I ti nvo l vesi mmunol oc al i z at i onofpr o t ei nswi t hi nac e l lt hr oughant i bodi est hathave beens pec i fic al l ypr epar edagai ns tt hatpar t i c ul arpr o t ei n. Thepr epar edant i bo dymol ec ul e sar el i nked/ c onj ugat edt oas ubs t anc e( ant i gen)t hat makest hem vi s i bl eundert hemi c r os c ope ,butdoes n’ ti nt er f er ewi t ht hes pec i fic i t yof t hei ri nt er ac t i ons . Ant i bodi esar ec ompl e xedwi t hs mal lfluor es c entmol ec ul es ,s uc hasr hodami neand fluor es c ei nt ogener at eder i vat i vest hatar et heni nc ubat edwi t ht hec e l l sors ec t i ono f c e l l s . ( i i . ) Thebi ndi ngs i t esar et henvi s ual i z edwi t ht hefluor es c entmi c r os c ope . I ndi r e c tI mmunofluor e s c e nc e Ant i bodi e sf r om di ffe r e nts pe c i e sar edi s t i ngui s he dbyt he i rdi ffe r e ntc ons t antr e gi ons I ni ndi r ec ti mmunofluor es c enc e ,c e l l s ar ei nc ubat ed wi t h s e c ondar y ant i body mol e c ul e sw/fluor oc hr omet agswhi c hi sal l o wedt of or mc ompl e xwi t ht hec ons t ant r e gi on( s e c ondar yant i ge n)o ft hepr i mar yant i body .Se c ondar yant i bodi e se . g .FI TC goatant i r abbi tI e e n Fl uor ophor e Fl uo r e s c e i n;Rd goatant i mous eI d gG:Gr gG:Re Fl uor ophor e Rhodami ne . Thepr i mar yant i bodymol ec ul esbi ndt hec or r es pondi ngpr o t ei n ant i gen i mar y ant i bodi e se . g .Rabbi tant i mous eH2;Mous eant i humanHLA. I ndi r ec ti mmunofluor es c enc er es ul t si n a br i ght er i mage bec aus e numer ous s ec ondar yant i bodymo l ec ul esc anbi ndt oas i ngl epr i mar yant i bo dy . Chemi c alfixat i vei saddedt ot hec e l l st opr es er vec e l ls t r uc t ur ebec aus ec e l l sar e “ f r o z en” at t hat moment pr i or t o t he addi t i on o f ant i bodi es i n i ndi r ec t i mmunofluor es c enc e . Thi s s t e p was no tdone i n Fr y e & Edi di n e xpe r i me nt . Frye and Edidin Experiment: Result I nt hegr aph:mos ai cr e f e r st omi x i ngand nonmos ai cr e f e r st onomi xi ng . I nt hef us edc e l l s ,mi xi ng( mos ai c )wasobs er ved af t er t he addi t i on of ant i bodi es at di ffer ent i nt er val satac ons t antt emper at ur eo f37•C. At ,T=5mi nut es ,t wopr o t ei nsf r om mous eand human di dn’ te xhi bi tany mi xi ng,e ac h pr o t ei n r es i dedi nt hei rr es pec t i vehemi s pher e . E. g os o phi l ame l ano gas t e r o I nf r ui tfli esCr umbspr o t e i n( an i nt e gr almembr anepr o t e i n)i s es s ent i alf orepi t he l i alc e l lpol ar i t y . o I nf r ui tfli eshavi ngmut at i oni nCr umbspr o t e i n,noapi c alr e gi on i smade .Henc e ,Crumbsprot ei ni snecessaryf orf ormat i onof api calregi oni nepi t hel i alcel l . o Api c alr e gi onl ar ge r t h a n n o r ma l I nf r ui t fli es c l oni ng of Cr umbs ge ne l e ads t oo ver e xpr es s i on of Cr umbspr o t e i nt han nor mal .Asar es ul t ,t heapi c alr e gi on i sl ar gert han nor mal .Hence, Crumbsprot ei ni ss uffici entf orf ormat i onofapi calre gi oni nepi t he l i alce l l . o Cr umbspr o t e i ni snor mal l ye xpr es s edi n api c alr e gi on oft hec e l latt hej unc t i onalc ompl e x c o mpos ed of Adhe r e ns Junc t i on and Se pt at e Junc t i on tc an be f ound us i ng I ndi r ec t I mmunofluor es c enc ew/ ant c r umbs . o I ft hej unc t i onalc ompl e xi sdi s r upt ed,Cr umbspr o t e i ni smi s l oc al i z edr es ul t i ngi nl os sofc e l l pol ar i t y .Hence,Crumbsprot ei ni ss uffici entt oe s t abl i s hpol ari t y,butnott omai nt ai n pol ari t y . o Tode t er mi ne ,i fa geneand t hec or r es pondi ng pr o t ei n ar e NECESSARY f ora par t i c ul ar pr oc es s ,de l e t et hegeneori nhi bi tt hef unc t i on o ft hepr o t ei n and moni t ort hepar t i c ul ar pr oc es s . I ft hepr oc es soc c ur snor mal l yaf t ert hedi s r upt i on o fpr o t ei ns ,t hen t hatpr o t ei ni sno t nec es s ar yf ort hepr oc es s . I ft hepr oc es si sdys f unc t i onalaf t ert hedi s r upt i o no fpr o t ei ns ,t hent hedi s r upt edpr o t e i n i snec es s ar yf ort hepr oc es s . o Tode t er mi ne ,i fa geneand t hec o r r es pondi ngpr o t ei n ar e SUFFI CI ENT f o ra par t i c ul ar pr oc es s ,o ver e xpr es st hegene / pr o t ei nandmoni t ort hepar t i c ul arpr oc es s . I ft hepr oc es si smor eeffic i entore xagger at ed,t hen t hatpr o t ei ni ss uffic i entf o rt he pr oc es s . I ft hepr oc es soc c ur snor mal l y ,t hent hatpr o t ei ni sno ts uffic i entf ort hepr oc es s . Microscopy Normal Microscopy Confocal Microscopy Nor malmi c r os c opesr es ol vet hei mageonl yi n Conf oc almi c r os c opes r es o l ve t he i mage i n XY Pl ane but no t Z, and pr oduc es 2D XYZpl ane ,andpr oduc es3Di mages . i mages . Conf oc al mi c r os c opy i l l umi nat es s pec i men Nor mal( Br i ght fie l d)mi c r os c opyi l l umi nat es wi t hnar r o w pi npoi ntofl i ght . t hewho l edept ho fs pec i menwi t hl i ght . A p i c al r e g i o n n o t dl e fi n di I nc onf oc almi c r os c o p y , o u t o f f o c u s i g he t s I n nor mal mi c r os c opy , out of f oc us l i ght bl oc ked by pi nhol e aper t ur e and pr o vi des c ont r i but est ot hei mageand al l o wst os ee be t t e rr es o l ut i onasi tal l o wst os eeonl yt he t hef oc usi magei nf o r mat i onal ongwi t ho t her i magei nf or mat i onwhi c happearbl ur . f oc usi nf o r mat i on.
Lecture 08 Membrane Fluidity Polarity
Course: Cell Biology (ZOOLOGY 570)
University: University of Wisconsin-Madison
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