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Gdje nestaju proteini membrane nakon egzocitoze?

Gdje nestaju proteini membrane nakon egzocitoze?


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Egzocitotični mjehurići oduzimaju membranske proteine ​​i glikokaliks na površini stanične plazma membrane. Kad se te vezikule puste u intersticijsku tekućinu i gdje god idu, kamo odlaze?

Da li se svuda drže vezikule? Ili ih se uklanja iz vezikula? Ako se vezikula spoji u drugu ćeliju, da li se ti proteini i vezani ugljikohidrati lijepe za novu ćeliju?


U procesu egzocitoze materijali koji će se uskoro osloboditi transportuju se u malim vezikulama do plazma membrane. Plazma membrana se stapa s tim vezikulama i to oslobađa tvari s vanjske strane ćelije. Pogledajte sliku (odavde):

Druga mogućnost za transportne vezikule je da stignu u svoju ciljnu stanicu i da se ili spoje s membranom i oslobode transportiranu tvar u stanicu ili da budu unesene u drugu vezikulu koja je zatim usmjerena prema endosomu/lizosomu. Pogledajte sliku (ovde):

Nije bitno koji je način odabran, membrana vezikule se reciklira ili integracijom u drugu membranu ili prolaskom kroz lizozom.


Ćelijska biologija 04: Tajni put

Sekretorni put Odnosi se na endoplazmatski retikulum, Golgijev aparat i vezikule koji putuju između njih, kao i na staničnu membranu i lizosome. Nazvana je ‘sekretorna’ jer je put kojim ćelija luči proteine ​​u ekstracelularno okruženje. Ali kao i obično, etimologija govori samo delić priče. Ovaj put također obrađuje proteine ​​koji će biti vezani za membranu (bilo u staničnoj membrani ili u samim membranama ER ili Golgi), kao i lizosomske enzime, kao i sve proteine ​​koji će živjeti svoj život u samom sekretornom putu. Takođe radi i neke stvari osim prerade proteina.

Citosol i ‘lumen ’ (tečnost koja ispunjava sekretorni put) različita su hemijska okruženja i obično se nikada ne miješaju. Citosol je reduktivan (kada ste u citosolu, stalno se susrećete s molekulima koji vam žele ponuditi elektrone), a ER, Golgi i ekstracelularno okruženje su oksidativni (molekuli stalno prilaze do vas tražeći elektrone). Pogledajte redoks ako ste još uvijek zbunjeni. Ovo dovodi do različitih uslova savijanja proteina: na primjer, disulfidne veze obično nastaju samo u oksidativnim uvjetima. Štaviše, različiti proteini mogu živjeti samo na sekretornom putu ili samo u citosolu. Sekretorni put omogućava ćeliji put da rukuje stvarima koje možda nije dobro imati u citoplazmi, i/ili su najkorisnije kada se drže koncentrisane u specijaliziranom odjeljku sa svojim željenim partnerima u interakciji. Hepatociti (u jetri) sekvestriraju lijekove i toksine u glatki ER i razgrađuju ih za izlučivanje iz tijela. Sekretorni put nije susjedan, ali svako kretanje između njegovih komponenti nalazi se u malim mjehurićima ispunjenim mikrokosmosima vlastitog kemijskog svijeta, koji se zovu vezikule.

Mnogi proteini koji prolaze kroz sekretorni put nikada ne dodiruju citosol – osim dijelova membranskih proteina koji strše na citosolnoj strani. Mnogima od njih potrebni su pratitelji kako bi pomogli pri presavijanju i/ili čitavom nizu post-translacijskih izmjena kako bi bili spremni za svoju izvornu funkciju, a sekrecijski put specijaliziran je za pružanje svega toga.

Današnje predavanje će se fokusirati na to kako se proteini prevode u ER i kako putuju (u vezikulama) između ER, Golgija i drugih destinacija. Ovo je lijepo prikazano u videu Život ćelije:

The endoplazmatski retikulum je prvi korak u sekretornom putu. Njegova membrana je kontinuirana s vanjskom jezgrom, iako nije jasno zašto je to važno, budući da ne poput proteina započinje svoj život u jezgri. Umjesto toga, mRNA se kreću po citoplazmi sve dok ih ne pokupi ribosom zainteresiran za njihovo prevođenje. U ‘posttranslacijskoj translokaciji ’ novi protein se premješta u hitnu nakon što je preveden ’s. U zanimljivijoj pojavi koja se naziva ‘kotranslacijska translokacija ’ ribosom započinje translaciju kao i svaki drugi protein, ali negdje u prvih 16 do 30 aminokiselina pogađa signalni peptid (poznat i kao signalni niz). Taj signalni motiv često je 1 pozitivno nabijena aminokiselina nakon koje slijedi 6-12 hidrofobnih aminokiselina. Ovaj motiv prepoznaje čestica za prepoznavanje signala (SRP, ‘ribonukleoprotein ’ ili hibridna molekula RNA/proteina) koja se veže za nju i sprječava nastavak translacije ribosoma. Translacija se zaustavlja sve dok kompleks ribosom/SRP ne naiđe na SRP receptor na ER membrani. Kad se sretnu, svaki SRP i njegov receptor vežu po jedan GTP molekul u membrani ER, što očigledno jača njihovu interakciju. Na sreću, sve se to događa u blizini Sec61 translokona i proteinskog kompleksa koji formira kanal koji prelazi membranu ER. Translokon je zapravo kompleks tri različita proteina (geni: SEC61A1 ili SEC61A2, SEC61B, SEC61G), od kojih podjedinica Sec61a ima 10 a-heliksa koji se protežu kroz membranu i koji formiraju kanal. Kada se ribosom pričvrsti za membranu, nastavlja translaciju, potiskujući signalni peptid i na kraju cijeli protein kroz kanal u lumen ER. Kad prevođenje prestane, SRP i receptor SRP -a hidroliziraju svoj GTP kako bi oslobodili jedni druge i teret ribosoma (za to je potrebna energija GTP -a, budući da je prvobitno vezivanje bilo nizbrdo), signalna peptidaza cijepa signalni peptid s novonastalog proteina , a protein se može slobodno početi savijati u hitnoj pomoći.

Nekoliko drugih igrača uključeno je u neke ER proteine. Oligosaharid transferaza, koja dodaje glikozilne grupe asparaginima u novonastalom proteinu, dio je kompleksa translokona i zapravo vrši glikozilaciju dok novi protein se još uvijek prevodi. Pa iako glikozilaciju nazivamo ‘post-translacijska modifikacija ’, ona se u ovom slučaju zapravo vrši tijekom prevođenja. Također, da bi se postigla njihova pravilna struktura, neki proteini moraju biti u potpunosti prevedeni prije nego što im se dozvoli da se počnu savijati – ako se N-terminalnom dijelu omogući da se počne presavijati čim uđe u lumen, završiće sa pogrešna ukupna struktura. Da bi se to spriječilo, ponekad BiP pratilja veže protein kako bi ga zadržao neko vrijeme rasklopljenim. Zamislite BiP kao još jednog Pac-Mana koji grize protein kako bi ga održao linearnim, poput Hsc70 u procesu ciljanja mitohondrija (vidi prošle sedmice).

Prvih nekoliko minuta prikazuje osnovni scenarij opisan gore. Zatim se prelazi na složeniji scenarij koji ću predstaviti za minutu. Za informaciju, video prikazuje dvije ‘kontroverzne ’ stvari koje nisu uključene u gornji opis: (1) signalni peptid se razgrađuje u membrani, i (2) ‘proteinski protein ’ koji zaustavlja kanal prije/poslije prevod. Još se ne slažu svi naučnici u ove dvije stvari.

Svi proteini za koje znamo da prolaze kroz sekretorni put bili su tamo precizirani od strane ljudi koji su radili eksperimente lokalizacije kako bi vidjeli gdje u ćeliji leži protein. Čudna činjenica u vezi s ER -om je da možete staviti ćeliju u blender, a nakon toga će se ER samo početi ponovo povezivati ​​sa sobom, formirajući male ‘mikrosome ’ koji nisu vezani za jezgru, ali tvore susjedne mjehuriće ER. Tada možete početi igrati s proteazama – koje razgrađuju proteine ​​– i deterdžentima – koje otapaju membranu ER. Pod pretpostavkom da je vaš protein od interesa preveden, možete provjeriti da li (1) preživi tretman proteazom, ali (2) ne ’t preživjeti tretman proteazom + deterdžentom, onda je to protein sekretornog puta. Logika je da je u slučaju (1) bio zaštićen unutar ER-a, ali u slučaju (2) ste rastvorili ER, pa ga je pojela proteaza. Sve ovo pretpostavlja da imate antitijelo ili neki drugi način da otkrijete postoji li protein koji vas zanima nakon ovih tretmana.

Ljudi su također koristili takve tehnike kako bi shvatili da se samo 70 aminokiselina novog proteina može prevesti prije nego što postane prekasno da taj protein završi u hitnoj medicinskoj pomoći. Zapamtite, signalni peptid je u prvih 16-30 aminokiselina, a translokacija u ER ovisi o prisutnosti SRP-a. Ribozomi se prevode predvidljivom brzinom, tako da su ljudi započeli sa prevođenjem neke mRNA, a zatim su čekali određeno vrijeme prije dodavanja SRP-a, da vide koliko bi se translacija moglo dogoditi prije nego što SRP više ne može obavljati svoj posao.

SRP receptor i Sec61 proteini su proteini ER membrane –, a tu su i mnogi drugi proteini ER membrane, Golgi membrane i membrane lizosoma. U stvari, čak se i membranski proteini (vidi klasu 02) ćelijske membrane obrađuju u sekretornom putu. Mnogi od njih imaju nekoliko ili desetine transmembranskih domena (svaka po 20-25 hidrofobnih aminokiselina) koje je potrebno umetnuti pravilnim redoslijedom i orijentacijom (na primjer, zaista želite da vaši ionski kanali i transporteri budu usmjereni u pravom smjeru, u van ćelije). Shodno tome, postoji gomila fantastičnih bioloških mehanizama za ispravno umetanje ovih proteina u membranu. Ovo je ono što prikazuje druga polovina gornjeg videa.

Dakle, ovdje je tautologija: neki proteini imaju topogenu sekvencu koja određuje njihovu orijentaciju u membrani. Ova sekvenca se sastoji od dvije vrste signalnih sekvenci:

  • a stop-transfer sekvenca (skraćeno STA iz nekog razloga) je 22-25 hidrofobna aminokiselinska sekvenca negdje u sredini proteina koja formira alfa heliks. Kada se naiđe, on se gura u membranu, a zatim se prevođenje ostatka proteina nastavlja u citosolu. Dakle, ova vrsta ‘ poništava ’ translokaciju u ER koju je započeo signalni peptid na početku (N termin) proteina.
  • a niz sidra signala (skraćeno SA) je također 22-25aa hidrofobna alfa spirala, ali sa nizom

Sa ta dva signala kao građevnim blokovima, možete zamisliti protein sa nizom sekvenci zaustavljanja i sidrenja signala kako bi se stvorio čitav niz napred-nazad transmembranskih domena ušivenih u membranu kao šivaćom mašinom. Ljudi su klasificirali membranske proteine ​​u pet kategorija:

  1. Tip I ima samo signalni peptid, a zatim transfer sa jednim zaustavljanjem u sredini. Stoga završava sa svojim (hidrofilnim) N terminusom u lumenu, svojom (hidrofobnom) sredinom u membrani i (hidrofilnim) C krajem u citosolu.
  2. Tip II ne počinje sa signalnim peptidom. Počinje kao i svaki drugi protein, ali u sredini ima niz sidra signalnog sidra s +++ aminokiselinama koje dolaze prve, a nakon njih hidrofobnim nizom. Ovo čini da se protein translocira na sredini translacije, pri čemu već prevedeni N-terminalni dio strši u citosol (pošto +++ mora ostati citosolan) i C-terminalni dio koji sada počinje da se prevodi prevođenje direktno u hitnu. Tako završava transmembranski sa svojim C terminusom u ER i N terminusu u citosolu – suprotno od tipa I.
  3. Tip III je poput tipa II – bez signalnog peptida, samo signalno sidro u sredini, ali u ovom slučaju +++ dolazi nakon hidrofobne sekvence, koja mijenja orijentaciju. Dakle, ovo završava sa svojim N terminusom u ER i svojim C završetkom u citosolu. Nasuprot tipu II i, na kraju, istom kao i tipu I, iako je tamo stigao na drugačiji način – nema signalni peptid koji se odvaja u ER.
  4. Proteini tipa IV ili ’multipass ’ imaju naizmjeničnu seriju signalnih sekvenci i sekvence zaustavljanja prijenosa. Očigledno je da je ovo više od jednog tipa ‘type ’, ali ipak ni približno toliko raznoliko koliko bi vaša kombinatorička mašta mogla dopustiti. Orijentacija prve signalne sekvence određuje da li će N završetak završiti u citosolu ili ER, a ukupan broj sekvenci zaustavljanja prijenosa + sidrenih sekvenci signala određuje gdje će završiti C terminus: paran broj = ista strana kao N kraj, neparan broj = suprotna strana kao N termin. STA i SA sekvence moraju se strogo izmjenjivati, s izuzetkom da možete započeti s dvije sekvence sidra signala ako je prva orijentirana s N terminusom u citosol. Samo da se rugaju ovoj šemi kategorizacije, ljudi su definisali neke nepotpuno definisane podtipove tipa IV, gde je tip IVa N-terminalni u citosolu (dakle počinje kao protein tipa II), a tip IVb je N-terminalni u citosolu. lumen (počinje kao protein tipa III, ali zatim ima drugu SA sekvencu koja ga vraća u ER). GLUT1 iz klase 02 je tip IVa. -usidreni proteini, koji su peti tip, ali se ne nazivaju Tip V, počinju signalnim peptidom, a završavaju hidrofobnim C-krajem koji ostaje ugrađen u membranu. Taj hidrofobni kraj se otcjepljuje i zamjenjuje GPI -jem, koji također ostaje ugrađen u membranu. PrP je jedan od ovih i#8211 o tome kasnije.

Do sada smo raspravljali o tome kako proteini mogu završiti u lumenu ER-a ili pokrivati ​​ER membranu. Većina proteina napušta ER u roku od nekoliko minuta, transportira se u vezikulama vezanim za Golgi, a zatim kasnije radi izlučivanja, lizosoma ili stanične membrane. Taj smjer kretanja prema naprijed naziva se anterogradni, ide unatrag od Golgija do ER -a je retrogradni transport.

Obje vrste transporta odvijaju se u vezikulama vezanim za membranu. Ovi pupoljci od membrane odakle god da dolaze, a kasnije se spajaju sa membranom odakle god su krenuli – lijepo prikazani

2:25 u gornjem videu Život ćelije. Tijelo iz kojeg se stvaraju vezikule je odjeljak za donatore ‘donor ’, a odredište za koje se kasnije spajaju je odjeljak za prihvaćanje ‘ ’.

Proces pupanja zahtijeva da G proteini u membrani regrutiraju Coat proteine. Konkretno, za anterogradni transport, G protein Sar1 (gen: SAR1A) regrutira COPII (‘cop dva ’) za retrogradni transport, ARF G protein regrutira COPI (izgovara se ‘cop one ’). Ovi G proteini se aktiviraju za obavljanje ovog posla kada im GEF učita GTP, zamjenjujući BDP.

Dakle, koraci u anterogradnom transportu, na primjer, su sljedeći:

  1. Sec12-GEF (Sec označava sekretorni) učitava Sar1 sa GTP. Kada se veže za BDP, Sar1 samo pluta oko odjeljka za donatore, ali kada je vezan za GTP, prolazi kroz konformacijsku promjenu koja uzrokuje da njegov inače zatrpan hidrofobni rep sa N-završetkom strši, pa se zalijepi u membranu, gdje proteini COPII tada počinju akumuliraju jer im se taj rep jako sviđa.
  2. COPII počinju da se polimeriziraju i, zbog svoje konformacije, imaju intrinzičnu preferenciju za zakrivljenost, tako da njihova akumulacija počinje da uzrokuje pupanje. U isto vrijeme, proteini vezani za membranu koje je potrebno transportirati#8211 identificirani sekvencom aminokiselina DXE (tj. Aspartat-bilo što-glutamat) koja tvori vezivno mjesto u svom citosolnom dijelu – regrutiraju se u novonastalu vezikulu . Proteini vezani za membranu djeluju kao receptori, regrutirajući lumenalne proteine ​​koji su vezani da Golgi visi u konkavnom prostoru gdje će završiti u vezikuli kada se formira.
  3. Nakon što stigne dovoljno COPII, mjehurići se otpuštaju, u kojem trenutku Sar1 hidrolizira svoj GTP, dajući mu energiju da usisa svoj hidrofobni rep nazad u sebe, oslobađajući COPII. Mjehurić je sada odvojen od odjeljka za donatore.
  4. Zbog loše objašnjenih (ili slabo razumljivih?) Razloga, sloj COPII se samo rastavlja, izlažući receptore ispod omotača koji usmjeravaju ciljanje vezikula. Kada vezikula stigne na svoje odredište, Rab-GTP ugrađen u membranu vezikule stupa u interakciju s Rab efektorom ugrađenim u membranu akceptorskog odjeljka. Razmjenjuje se pogled u stranu, rasplamsava se interesovanje. Uskoro će se vezikula spojiti s membranom. proteini prisutni i na vezikularnoj i na argentnoj membrani (V-SNARE i T-SNARE) međusobno djeluju kako bi još više približili membrane. U ovom primjeru ćemo razmotriti VAMP (VAMP_ geni) kao V-SNARE i Sintaksin (geni STX__) i SNAP25 (gen SNAP25) kao T-SNARE. Sintaksin i SNAP25 su oba membranski proteini. Sintaksin ima 1 alfa spiralu, a SNAP25 2, sve na citosolnoj strani. Alfa spirale pokreću interakciju sa VAMP-om. Suprotne strane i#8217 alfa spirale imaju izuzetno jak afinitet jedna prema drugoj, čime se membrane približavaju dovoljno blizu da se spoje. Jednom kada se to dogodi, ponovno razdvajanje V-SNARE-a i T-SNARE-ova zahtijeva dva proteina: NSF (gen: NSF je skraćenica za NEM osjetljivi faktor) i alfa-SNAP (gen: NAPA), rastvorljivi protein vezivanja za NSF. NSF je ATPaza i sagorijeva ATP kako bi pokrenuo energetsko uzbrdo rastavljanja kompleksa.

Sada za retrogradni transport. Zašto uopće postoji retrogradni transport? Evo neiscrpne liste nekih razloga:

  • Neki membranski proteini započinju svoj život u hitnoj pomoći, potrebno ih je modificirati u Golgiju, ali se onda moraju vratiti u hitnu. Oni to rade sa sekvencom aminokiselina KKXX.
  • Postoji i KDEL aminokiselinska sekvenca na C terminusu nekih lumenalnih proteina koja bi trebala da ih zadrži u urgentnom centru, ali nije savršena – ponekad završe u Golgiju, u kom slučaju ponovo ciljano natrag u ER putem retrogradnog transporta, ovisno o tom nizu KDEL -a za prepoznavanje. Mehanizam je nekako uredan – proteini koji prepoznaju i vezuju se za KDEL to rade samo pri niskom pH, a pH Golgija je niži od ER, tako da oni vežu KDEL u Golgiju, a zatim ga oslobađaju kada&# 8217re se vratio u neutralniji pH ER.
  • Također, razmislite o tome, svi proteini koji sudjeluju u anterogradnom transportu – V-SNARES, Rab, itd. – moraju se vratiti u hitnu da bi mogli sve iznova, kao što je autobus da se na kraju dana vratite u autobusku depo.
  • Kao što ćemo uskoro vidjeti, Golgi dolazi u više faza koje zavise od dodavanja enzima dalje nizvodno.

Proces retrogradnog transporta nije toliko različit od anterogradnog. Koristi ARF umjesto Sar1, COPI umjesto COPII, ali radi isto: ARF napunjen GTP -om dopušta da se njegov hidrofobni rep zalijepi u membranu, privlačeći pažnju COPI -ja. COPI ima dvije komponente, COPIalpha i COPIbeta, obje u interakciji s tom sekcijom KKXXX kako bi regrutirale proteine ​​vezane za membranu namijenjene za retrogradni transport. Neki proteini također imaju RR sekvencu (bilo gdje u proteinu) koja ih može označiti za retrogradni transport.

Golgijev aparat nije susjedan. To je naslagani skup zasebnih pododjeljenja koji se nazivaju vrećice ili cisterne. Različiti odjeljci imaju različita svojstva i proteini ih posjećuju određenim redoslijedom. U redu od ER do ćelijske membrane, Golgi kompartmenti se nazivaju cis, medijalna, trans i trans-Golgi mreža. Svaki odjeljak ima različite enzime koji modificiraju proteine, a promjene se moraju dogoditi određenim redoslijedom, pa stoga postoji potreba za složenim odjeljkom.

No, kako proteini sazrijevaju u Golgiju, nije tako da izviru u mjehurićima iz jednog odjeljka i prelaze u sljedeći. Tačnije, kupe koji su već unutra pomiče prema van i ‘ sazrijeva ’ jer mu se dodaju novi enzimi (od dalje niz Golgijev lanac) putem retrogradnog transporta. Čudno, zar ne? To je nekako kao da ste umjesto da pređete iz osnovne škole u srednju školu u srednju, ostali samo u jednoj školskoj zgradi cijelo svoje djetinjstvo i adolescenciju, a oni su samo donosili nove udžbenike i nastavnike svake godine kako bi ih zadržali odgovara ocjeni koju ste vi i vaši drugovi iz razreda sada postigli. Evo kako izgledaju Golgijevi dok se kreću i razvijaju:

Dakle, nema (malo ili) nikakvog anterogradnog transporta unutar Golgijeve mreže, ali dosta retrogradnog transporta za unos svake nove runde enzima. Kada proteini konačno završe puni K-12 nastavni plan i program Golgijeve mreže, oni se podvrgavaju transportu do prelaze na svoju konačnu destinaciju. Rasturaju u vezikuli koja će otići na jedno od tri mjesta:

    – fuzija sa staničnom membranom. Tako će se lumenalni proteini izlučivati ​​izvanstanično, a membranski proteini će postati proteini stanične membrane. – oni se samo zadržavaju kao vezikule u ćeliji dok ne zatrebaju – gdje ‘potrebno ’ znači da na kraju podliježu egzocitozi. U neuronima se tu pohranjuju neurotransmiteri sve dok akcijski potencijal ne zahtijeva njihovo lučenje u sinapsu. U želucu, ćelije koje proizvode želučane enzime drže te enzime u sekretornim mjehurićima sve dok unos hrane ne pokrene njihovo otpuštanje u želudac. - gde pogrešno savijeni proteini odlaze da se razgrade.

Transport od trans-Golgijeve mreže do ovih odredišta razlikuje se od drugog prije spomenutog transporta i često uključuje klatrin (CLT__ geni). Vezikule koje pupaju imaju dvoslojni omotač, sa kompleksom dapter p roteina (AP) kao unutrašnjim slojem i klatrinom kao vanjskim slojem. Adapterski proteini imaju ciljni signal sa YXXh motivom (h = Φ = bilo koja hidrofobna aminokiselina). Klatrin tvori takozvanu formaciju ‘clathrin-triskelion ’ koja je prikazana ovdje:


(Slika zahvaljuje korisniku Wikimedia Commons Phoebus87)

Klatrin je takođe odgovoran za endocitozu – pupanje iz vezikula ekstracelularnog materijala (i proteina ćelijske membrane) koji će doći into ćelija. To se zove endocitoza posredovana klatrinom. Receptori u ćelijskoj membrani se endocitozuju vrlo često: čitava populacija hormonskih receptora se okreće otprilike svakog sata, posebno kada se hormoni primaju. Uzimanje receptora u vezikulu jedan je od načina na koji ćelija može prekinuti dolazni signal dok se ne može obraditi.
Bilješke o plazma membranama ukratko govore o cističnoj fibrozi: CFTR je ABC transporter odgovoran za ispumpavanje Cl - iz ćelije (također propušta Na + unutra). Mutanti gubitka funkcije nemaju pumpu Cl-koja uklanja pokretačku snagu osmoze, zgušnjavajući sluz i uzrokujući probleme s disanjem. Postoji najmanje 127 različitih CFTR mutanata sa gubitkom funkcije (barem je toliko Natera testova) koji (ako su oba alela onemogućena) uzrokuju cističnu fibrozu. Najčešća mutacija je ΔF508, tj

3% svih europskih CFTR alela i oko 70% mutiranih. Gubitak tog fenilalanina mijenja konformaciju CFTR-a tako da dikiselinski izlazni kod (aminokiseline D565 i D567) koji cilja CFTR za egzocitotične vezikule nije više pravilno izložen i protein nikada ne stiže do stanične membrane [Wang 2004 ].

odeljak za diskusiju

U odeljku čitamo Hu 2009, koji je pokazao da su proteini atlastina uključeni u stvaranje tubularne ER mreže. Dokazi su gotovo u potpunosti došli iz interakcija protein-protein. Iznenadio sam se da je ovaj rad bio velika stvar, jer je postojao milion radova koji pokazuju interakcije proteina i proteina za Huntingtin, i niko im ne veruje svima i nije nas nužno približio saznanju šta Huntingtin radi ili šta pođe po zlu u Huntingtonovoj bolesti. Ali očito je Hu bio u stanju napraviti prilično čist slučaj za interakcije atlastina i retikulona kao što implicira ulogu u formiranju ER. Pomaže što je Hu uspio pokazati fizičku (vezujuću) interakciju ‘genetsku interakciju ’. A ‘genetska interakcija ’ (morao sam to potražiti) znači kada “Ponekad mutacije u dva gena proizvedu fenotip koji je iznenađujući u svjetlu pojedinačnih efekata svake mutacije. Ovaj fenomen, koji definira genetsku interakciju, može otkriti funkcionalne odnose između gena i puteva. ” [Mani 2007].

Ovo je staro desetljeće, tako da su neke stvari možda zastarjele, ali smatrao sam da je Harrisov pregled biologije PrP ćelija iz 2003. (ft) bio izuzetno jasan i koristan. Kim & Hegde 2002 je također bio od pomoći. PrP je protein sekretornog puta. Njegove prve 22 aminokiseline (MANLGCWMLVLFVATWSDLGLC) su signalni peptid koji uzrokuje kotranslacijsku translokaciju u ER. Normalno, PrP se samo poveže GPI-om na svom C kraju i usidruje se na egzoplazmatsku stranu membrane. Ali aminokiseline 111-134 (HMAGAAAAGAVVGGLGGYMLGSAM) su neka vrsta slabe sekvence sidrenja signala (tip II, sa +++ aminokiselinama koje dolaze prije sidra signala) koje ponekad, ali ne uvijek postaje transmembranski domen, invertujući C-terminus u lumen. Što je još zbunjujuće, ta sekvenca ponekad može jednostavno završiti kao transmembranska domena bez inverziju, tako da je N kraj u lumenu. Dakle, postoje tri membranske topologije PrP-a: regularna stara GPI usidrena i dvije transmembranske orijentacije, kako je prikazano na Harrisu 2003. Slika 3:

Zapazite koliko je čudan Ctm PrP. On je transmembranski, ali i GPI-usidren, a signalni peptid N-terminala nikada se ne odcjepljuje. Normalno, transmembranski oblici čine <10% ukupnog PrP. U nekim laboratorijskim uvjetima postotak je veći, a dvije mutacije koje uzrokuju GSS (A117V i P105L) također povećavaju udio Ctm PrP na 20-30% svih PrP. Od ova tri oblika, postoji dobra količina dokaza da je Ctm PrP toksičan i da bi mogao igrati ulogu u formiranju priona, iako se čini da većina mutacija genetske prionske bolesti (uključujući FFI D178N) ne utječe na topologiju membrane PrP ili dio Ctm PrP.

Nakon što PrP prođe kroz Golgi, cilja se na staničnu membranu. Ali prema Harrisu, on ne samo sjedi tamo već često kroz endocitozu posredovanu klatrinom i kruži kroz ćeliju svaki

60 minuta, pri čemu se neki molekuli cijepaju u svakom ciklusu. Bakar stimulira ovu endocitozu PrP. Većina genetskih mutacija prionske bolesti mijenja lokalizaciju PrP – obično kada je prisutna mutacija, manje PrP se nalazi na površini ćelije, a više se akumulira u ER.

O Ericu Vallabhu Minikelu

Eric Vallabh Minikel je u doživotnoj potrazi za sprečavanjem prionske bolesti. On je naučnik sa sjedištem na Broad institutu MIT-a i Harvarda.


PREGLED članka

  • Ključna laboratorija za biologiju algi, Institut za hidrobiologiju, Kineska akademija nauka, Wuhan, Kina

Cilije i flagele su visoko očuvani organeli u eukariotskim stanicama koji pokreću ćelijsko kretanje i djeluju kao stanične antene koje primaju i prenose signale. Osim što primaju i prenose vanjske signale koji aktiviraju kaskade signala, cilije izlučuju i cilijarne ektosome koji šalju signale ćelijama primateljima i na taj način posreduju ćelijskoj komunikaciji. Abnormalna cilijarna funkcija dovodi do različitih ciliopatija, a precizan transport i lokalizacija proteina cilijarne membrane bitni su za funkciju ciliuma. Ovaj pregled sažima trenutno znanje o transportnim procesima proteina cilijarne membrane nakon njihove sinteze u endoplazmatskom retikulumu: modifikacija i sortiranje u Golgijevom aparatu, transport kroz vezikule do cilijarne baze, ulazak u cilije kroz difuzijsku barijeru i promet lučenjem ektosoma . Također se raspravlja o molekularnim mehanizmima i regulaciji uključenim u svaki korak. Transport proteina cilijarne membrane je složen, precizan stanični proces koordiniran između više organela. Sistematskom analizom postojećih istraživanja identificiramo teme koje treba dodatno istražiti kako bi se promovirao napredak u ovoj oblasti istraživanja.


Rezultati

Snimanje pojedinačnih sinaptičkih mikrovezikula

Ovdje smo snimili pojedinačne sinaptičke mikrovezikule u živim stanicama s mikroskopijom potpune unutrašnje refleksije fluorescencije (TIRF) 5. Konkretno, koristili smo mikrovezikularno ciljanu pH osjetljivu fluorescentnu sondu (VAChT-pH) zasnovanu na VAChT (slika 1a) 6. Pojedinačne vezikule koje sadrže ovu sondu svijetle kada se fuzione pore vezikula otvore nakon egzocitoze i kiseli lumen vezikule se neutralizira ekstracelularnim puferom 6 . Slika 1b prikazuje dvije ćelije koje izražavaju VAChT-pH. Fluorescencija je bila raspršena po donjoj površini ćelije, gdje je bila ograničena na male punkte. Da bismo testirali da li su ove punkte bile na vanjskoj strani ćelije, superfuzirali smo stanice s niskim pH otopinom (pH 5,5 dopunska slika S1). Tokom ovog tretmana izmjereno je dramatično zatamnjenje ćelija (dopunska slika S1a-c). Pojedinačna VAChT-pH punkta je zamračena, a zatim ponovno osvijetljena, što ukazuje na to da su mnogi punktati bili na izvanćelijskoj strani plazma membrane. Neke točke nisu zamračene, što ukazuje na to da se nalaze u unutarćelijskim odjeljcima. Da bismo testirali da li je VAChT-pH sadržan u kiselim odjeljcima, superfuzirali smo ćelije amonijum hloridom (dopunska slika S1d-f). Ova hemikalija razbija intracelularne pH gradijente. Ćelije i neke fluorescentne tačke izložene ovoj otopini su posvijetlile, što ukazuje na to da se neki VAChT-pH nalazi u unutarćelijskim kiselinskim odjeljcima (dopunska slika S1d-f). Zajedno, ovi rezultati pokazuju da je VAChT-pH bio prisutan i u klasterima na plazma membrani i u kiselim odjeljcima unutar ćelije.

(a) Crtani film mikrovezikularne sonde VAChT-pH. (b) Slika dvije PC12 ćelije koje eksprimiraju VAChT-pH snimljena pomoću TIRF-a. Skala je 5 μm. (c) Okviri iz filma u kojem jedna vezikula koja sadrži VAChT-pH prolazi kroz egzocitozu izazvanu depolarizacijom, i (d) odgovarajuća fluorescencija iz centralnog kruga radijusa od 750 nm te regije. Skala je jednaka 2 μm. (e) Srednja VAChT-pH fluorescencija iz aktiviranih egzocitnih vezikula (83 događaja, 13 ćelija). (f) Crtani film o proporcionalnoj pH sondi VAChT-pH-mCherry. (g) Prosječna fluorescencija VAChT-pH-mCherry iz aktiviranih egzocitnih vezikula u pHluorinskom i mCherry kanalu (36 događaja, 3 ćelije). Odnos ova dva intenziteta prikazan je u h. Trake grešaka su s.e.m. Norm. fluor., normalizirana fluorescencija.

Da bismo izazvali egzocitozu, depolarizirali smo stanice s visokim kalijem. Ovo rješenje izazvalo je brze i brojne egzocitne događaje. Svijetli bljeskovi su se mogli vidjeti preko donje površine ćelije. Ovi događaji bili su rijetki u nestimuliranim stanicama. Slika 1c prikazuje primjer događaja (Dopunski film 1). Deset sekundi prije egzocitoze mjehurić nije vidljiv, ali kad se fuzijske pore otvore, u jednom kadru (500 ms) dolazi do jakog bljeska i stvara cvjetanje fluorescencije koje zrači prema van u svim smjerovima i zatamnjuje (slika 1c). Nakon egzocitoze, dio ove fluorescencije je uhvaćen na prethodno formiranim VAChT-pH klasterima u blizini egzocitnog mjesta (slika 1c). Fluorescencija iz područja lokalne membrane (krug od 750 radijusa) koja okružuje ovu vezikulu prikazana je na slici 1d. Ovaj trag pokazuje da se VAChT-pH povećava pri egzocitozi, raspada se u roku od nekoliko sekundi, a zatim visoravni do intenziteta blizu polovice vršne vrijednosti. The corresponding fluorescence from many events (83 events, 13 cells) is plotted in Fig. 1e. On average, VAChT-pH brightens rapidly and then decays. There is, however, a substantial amount of fluorescence (61±0.03%) that remains near the site of fusion (Fig. 1e).

To determine if the VAChT-pH decay was due to loss of the protein into the plasma membrane (full-fusion) or was due to the direct recapture and re-acidification of the vesicle (kiss-and-run), we tagged VAChT-pH with mCherry. VAChT-pH-mCherry, positions the pH-sensitive pHluorin inside the vesicle lumen and the pH-insensitive mCherry into the cytoplasm (Fig. 1f). This sensor acts as a ratio-metric probe for pH changes in vesicles. The green fluorescence from VAChT-pH-mCherry rapidly brightens and then decays to 58±0.07% (Fig. 1g). Similarly, the mCherry fluorescence increases and then decays at the same rate, and to the same extent as pHluorin (62±0.07% Fig. 1g). The ratio of the red-to-green is shown in Fig. 1h, and shows that after exocytosis there is no change. Thus, the decay in VAChT-pH after exocytosis is not due to direct recapture and acidification (kiss-and-run), but instead is due to the loss of the probe into the membrane. These results support a full-fusion mechanism of exocytosis for VAChT.

To analyse the spread of VAChT-pH, we measured four radial line scans centred over vesicles (Fig. 2a). These scans generated an average one-dimensional kymograph of the two-dimensional membrane surrounding exocytic events. Figure 2a shows results of averaging the kymographs of many VAChT-pH fusion events together. Upon exocytosis, fluorescence brightens and spreads rapidly. Fluorescence, however, progressed only a short distance. As seen in the example in Fig. 1c, some of the diffusing fluorescence remains near the site of fusion (Fig. 2a). The fluorescence from these kymographs is plotted in Fig. 2b. Analysis of these data is shown in Fig. 2c, where two-dimensional Gaussian functions were fit to vesicles. This plot shows that VAChT initially spreads rapidly, but remains relatively constant in size 10 s following exocytosis. We conclude that microvesicles release VAChT into the plasma membrane. Most of this material, however, does not spread more than a few hundred nanometres (σ-values at 30 s, 1.1±0.1 μm) from the site of exocytosis. These results are consistent with several models of release for VAChT, which we consider below.

(a) Mean normalized radial linescans of microvesicles during exocytosis (46 events, 13 cells). Scale bar equals 1.5 μm. (b) Intensity plot of the same radial linescans. (c) Mean σ-value of a Gaussian function fit to each of the events used for a i b. Error bars are s.e.m.

Vesicle material is trapped in clathrin-coated structures

The cytosolic tail of VAChT binds to the adaptor protein AP2 (refs 7, 8). To determine if endocytic proteins were responsible for clustering vesicle material on the cell surface, and are involved in the retention of VAChT near sites of exocytosis, we imaged VAChT-pH together with two endocytic markers. Figure 3a shows an image of VAChT-pH and a marker for clathrin-coated structures (clathrin light chain tagged with dsRED, mLC-dsRED) 9 . These images show that the bottom surface of PC12 cells have a large number of clathrin-coated structures (0.95 clathrin spots per μm 2 ±0.02 spots per μm 2 , n=11 cells, 79 regions (72 μm 2 area)). A similar density for clathrin was found with antibody staining (Supplementary Fig. S2). To test if MLC-dsRED labelled all the clathrin structures, we immunostained cells expressing MLC-dsRED with antibodies against the heavy chain of clathrin. Almost all the antibody-stained clathrin spots were labelled with MLC-dsRED (Supplementary Fig. S3). Furthermore, the number of antibody-stained clathrin structures was not increased compared with untransfected controls (X22 density of MLC-dsRED-transfected cells: 1.05±0.02 clathrin spots per μm 2 , X22 density of control cells: 1.06 clathrin spots per μm 2 ±0.03 Supplementary Figs S2 and S3). These results show that the density of clathrin was not changed by the expression of clathrin light chain (Supplementary Figs S2 and S3).

(a) TIRF image of a cell co-expressing VAChT-pH and MLC-dsRED along with the overlay image. Scale bar equals 5 μm. (b) Mean extracted and normalized square regions centred on VAChT-pH spots and the corresponding regions from the MLC-dsRED channel (n=11 cells, 1,374 regions). Scale bar equals 2 μm. (c) The two-dimensional correlation values between the images used in b. (d) TIRF image of a cell co-expressing VAChT-pH and AP2-mCherry along with the overlay image. Scale bar equals 5 μm. (e) Mean extracted and normalized square regions centred on VAChT-pH spots and the corresponding region from the AP2-mCherry channel (n=6 cells, 964 regions). Scale bar equals 2 μm. (f) The two-dimensional correlation values between the images used in e. Avg, average.

When green images (Fig. 3a) were compared with red, a large fraction of VAChT-pH and clathrin colocalized (Fig. 3a). To analyse colocalization, we extracted 1,374 small (8.5 × 8.5 μm) images from 11 cells. In these regions, the centre of VAChT-pH structures were aligned to the middle pixel of the image. The corresponding regions from the MLC-dsRED image were also extracted. The fluorescence from these regions were normalized and averaged. This analysis showed a small distinct spot in the VAChT-pH image that co-localized with clathrin (Fig. 3b). The correlation between these regions is plotted in Fig. 3c and shows a strong and positive correlation. Visual inspection of 215 randomly chosen regions indicated that 85% of the central VAChT-pH spots had a corresponding signal in the MLC-dsRED channel. Similar analysis was done with VAChT-pH and AP2-mCherry (Fig. 3d-f). These images and their analysis show that, similar to clathrin VAChT-pH and AP2-mCherry strongly colocalize. Average VAChT-pH and AP2-mCherry image pairs have similar correlation values, sizes and distributions (Fig. 3d-f). Again, visual inspection of 215 randomly chosen regions showed that 82% of all the central VAChT-pH spots colocalized with AP2-mCherry. To test for the association of clathrin and AP2, we measured the colocalization of MLC-dsRED and AP2-green fluorescent protein (GFP Supplementary Fig. S4). Visual inspection indicated that 98% of the MLC-dsRED had a corresponding signal in the AP2-GFP channel. Thus, almost all of the MLC-dsRED structures contained AP2-GFP. We conclude that there is a resting pool of VAChT-pH on the surface of the cell that is clustered on endocytic structures composed of clathrin and AP2.

After exocytosis, vesicle material is trapped over clathrin

We next asked if the corralling of VAChT-pH observed after exocytosis is related to clathrin. To answer this question, we triggered exocytosis in cells expressing VAChT-pH and MLC-dsRED. We then imaged the release of VAChT-pH from single exocytic vesicles and compared these images with clathrin. Figure 4a shows a single exocytic vesicle arriving at the surface and rapidly brightening. VAChT-pH left the site of exocytosis and diffused radially into the plasma membrane where a fraction of the fluorescence was rapidly (<1 s) captured on a preformed clathrin structure (Fig. 4a Supplementary Fig. S5 and Supplementary Movie 2). This trapped VAChT-pH remained associated with clathrin for over 20 s.

(a) Sequential two-colour TIRF images of a single vesicle fusing with the plasma membrane. The location of fusion is indicated by the blue arrow. After fusion, VAChT-pH is captured on a preformed clathrin-coated pit (yellow arrow) located close to the site of exocytosis. Scale bar equals 2 μm. (b) Mean radial linescans through images centred on clathrin structures located within 1 μm of an exocytic event (n=7 cells, 35 regions). Fusion occurs at zero seconds. Scale bar equals 2 μm. (c) The measured fluorescence from VAChT-pH and MLC-dsRED from a circle with a radius of 375 nm centred on the clathrin structures shown in b. (d) Mean radial linescans through images centred on clathrin-free areas located within 1 μm of an exocytic event (n=7 cells, 43 regions). (e) The measured fluorescence from VAChT-pH and MLC-dsRED from a circle with a radius of 375 nm centred on the centre of the clathrin-free areas shown in d. Spatial analysis comparing sites of exocytosis (f, yellow cross) to clathrin (f, red circles). Scale bar equals 2 μm. The blue circle indicates an area 1 μm from the centre of exocytosis. (g) Plot of the number of clathrin structures located within 1 μm of fusion events (n=11 cells, 79 regions, 4,383 clathrin structures). (h) Histogram showing nearest-neighbour distances between fusion sites and clathrin spots. (i) Histogram showing the nearest-neighbour distances between random sites and clathrin spots. Error bars are s.e.m. Prosj. fluor., average fluorescence Norm. fluor., normalized fluorescence.

To analyse the capture of VAChT-pH on clathrin, we measured fluorescence around clathrin structures located within1 μm of fusion events. The average kymographs centred over clathrin is shown in Fig. 4b. From this analysis, it is evident that clathrin is stable at the membrane before exocytosis. At the moment of fusion, VAChT-pH brightens in the membrane area surrounding clathrin, steadily increases and is then focused onto the central clathrin-containing spot (Fig. 4b). We observe that the width of the VAChT-pH spot decrease after exocytosis and aligns with the central spot of clathrin in the kymograph (Fig. 4b). A plot of the fluorescence intensity during exocytosis measured in a small membrane area (375 nm radius circle) over clathrin is shown in Fig. 4c. In these clathrin-containing regions, VAChT-pH fluorescence steadily increases after exocytosis and then plateaus (Fig. 4c). The small and slow decline of fluorescence over clathrin during the plateau phase could be due to several experimental issues, including photobleaching, vesicle movement, re-acidification and endocytosis. These data demonstrate that the trapping of VAChT-pH occurs over resident clathrin structures located within a micron of exocytosis.

To show that VAChT-pH is specifically trapped over clathrin after exocytosis, we measured VAChT-pH in regions that did not contain clathrin and were located within 1 μm of fusion events. The average kymograph centred over clathrin-free areas is shown in Fig. 4d. In these regions, VAChT-pH brightens at exocytosis and is then rapidly lost. Specifically, VAChT-pH diffuses from the central, clathrin-free area, and is then captured on the surrounding clathrin. Unlike Fig. 4c, the fluorescence from clathrin-free areas (375 nm radius circle) increases at exocytosis, does not continue to increase and then rapidly decays (Fig. 4e). These data demonstrate that after exocytosis, VAChT-pH freely diffuses in membranes that do not contain clathrin, and is caught on membranes that do contain clathrin.

To test for a spatial coupling between sites of exocytosis and endocytosis, we mapped the location of 79 exocytic vesicles in relation to all clathrin spots. Figure 4f shows an example image of a field of clathrin where an exocytic event occurs in the centre (Fig. 4f, yellow cross). For this analysis, we determined the location of all the clathrin structures in each image surrounding exocytic sites (11 cells, 79 regions, 4,383 spots). The same analysis was done for control images from the same cells (7 cells, 67 images, 3,941 spots). This analysis shows that there is a dense network of clathrin across the cell surface. Interestingly, there was no increase in the number of clathrin structures near sites of exocytosis (2.9 spots±0.2 spots) compared with the control images (2.7 spots±0.2 spots Fig. 4g). A histogram showing the distance between exocytic sites and the closest clathrin structure is plotted in Fig. 4h. The closest clathrin structure to each fusion site was 482±27 nm. Figure 4i shows that the nearest-neighbour spacing of clathrin structures is 706±11 nm (4,373 structures, 79 regions, 11 cells). For controls, the clathrin structure nearest to the centre of the image was 435±23 nm and the nearest-neighbour spacing of clathrin was 711±11 nm. Thus, material exiting a vesicle needs to go no further than a few hundred nanometres before it encounters a preformed clathrin structure. This distribution of clathrin, however, is not linked to sites of exocytosis. Instead, it is randomly generated by the spacing and high density of resident clathrin structures.

To support the hypothesis that classical clathrin-mediated endocytosis was linked to the surface distribution of VAChT, we treated cells with the dynamin inhibitor dynasore 10 . Like MLC-dsRED and AP2-mCherry, Dynamin1-mCherry (Dyn1-mCherry) co-localized with VAChT-pH (Supplementary Fig. S6). Visual inspection indicated that 64% of VAChT-pH spots co-localized with Dyn1-mCherry spots (Supplementary Fig. S6). When cells were exposed to 80 μM dynasore for 30 min, a dramatic increase in surface fluorescence over time was observed (Supplementary Fig. S6). This increase in fluorescence occurred in both the stable fluorescent puncta and plasma membrane regions not associated with puncta. Thus, the surface distribution and recycling of VAChT-pH is dependent on the action of components of clathrin-coated pits, namely dynamin.

Architecture of clathrin determined by iPALM

From the above data, we conclude that a large fraction of vesicle material is rapidly and locally captured over pre-formed clathrin structures after triggered exocytosis. Standard optical microscopy (for example, TIRF) is restricted by the diffraction limit of light. This fact limits the resolution of cellular structures to

200 nm with TIRF. Thus, we used a super-resolution optical technique known as interferometric photo-activation localization microscopy (iPALM) to image the three-dimensional sub-diffraction size and distribution of clathrin in PC12 cells 11 . To accomplish this, we tagged clathrin light chains with the photo-switchable fluorescent protein mEOS2 (mLC-mEOS2 Fig. 5a) 12 . iPALM was used to reconstruct the size, shape and distribution of clathrin at the bottom surface of the plasma membrane. iPALM also allows one to analyse the distribution of clathrin molecules in the plane perpendicular to the coverslip 11 . Figure 5b shows a single cell imaged with iPALM. The images to the left shows the cell imaged with a conventional TIRF, and to the right shows the same cell imaged with iPALM. Clearly, individual spots of clathrin are difficult to distinguish with conventional imaging. However, distinct clathrin structures are clearly visible with iPALM.

(a) Cartoon of a single clathrin triskeleon with a light chain labelled with a fluorescent protein. The triskeleons assemble together to form clathrin-coated structures. (b) Conventional TIRF optical image and (c) iPALM measurement of MLC-mEOS2 in a PC12 cell. Scale bar equals 5 μm. U b, the blue arrows point to gold particles used for alignment. In both cases, a magnified view of the white box is shown below. Scale bar equals 1 μm. (d) Histogram of clathrin spacing in PC12 cells measured with iPALM. (e) Histogram showing the width of the same structures. (f) Plot of width versus depth of the clathrin objects measured with iPALM. Error bars are s.e.m.

From images of entire cells, we were able to calculate the sub-diffraction distribution of clathrin. The total density of resolvable spots was 2.00±0.09 spots per μm 2 (3 cells). Nearest-neighbour analysis showed that the average spacing was 560±1 nm (n=3 cells, 305 spots Fig. 5d). This was 200 nm closer than we observed with conventional TIRF. To systematically analyse the shape of all the structures on the surface, we measured the width and depth of each structure from three cells in the x, y and z dimensions relative to the glass coverslip. Figure 5e shows that the size of clathrin-coated structures ranged from 50 to 450 nm in diameter (mean: 224±5 nm, n=3 cells, 401 structures). These structures varied in shape with 42% less than 75 nm in depth (Fig. 5f). From these measurements, there was no strong correlation between the width and depth of individual pits. These data support the finding that the density of clathrin structures in these cells is very high. Furthermore, individual structures were either flat or domed. The functional importance of these two types of structures is yet to be determined. Finally, the two-fold increase in density observed with iPALM can be explained by the inability of traditional optical imaging (TIRF) to delineate between two closely spaced structures and one large structure.

Architecture of clathrin determined by electron microscopy

To further map the architecture of clathrin, we performed electron microscopy. We first prepared platinum-shadowed membrane sheets from PC12 cells 13 . We then used whole-cell transmission electron microscopy to visualize the entire inner surface of the plasma membrane (Supplementary Fig. S7). We observed two types of clathrin structures: (1) distinctive flat patches and (2) domes, or pit-like structures (Supplementary Movie 3). Figure 6a shows four regions where both flat patches and pits of clathrin can be seen. To determine the shape of these structures, we measured the minimum and maximum dimension (1,045 clathrin structures, 96 regions, 3 cells). The structures were further classified by eye as flat or domed. Figure 6b shows a scatter plot of these measurements. The structures have a maximum diameter of 129±1 nm (1,045 clathrin structures, 96 regions, 3 cells). Figure 6c show this nearest-neighbour analysis. Clathrin structures were distributed at a nearest-neighbour density of 463±8 nm (3 cells). The total density was 1.6 clathrin structures per μm 2 ±0.11 (96 regions, 3 cells). To further confirm these densities, we performed super-resolution imaging (Supplementary Fig. S8). The density observed with ground-state depletion (GSD) imaging showed 2.3 structures per μm 2 ±0.08 (92 regions, 6 cells). Again, these data support the finding that the density of clathrin on the surface is very high, between 2 and 11 times greater than previously reported from other cell types 14,15,16,17,18,19,20 . Furthermore, these data demonstrate that the density of clathrin structures measured in iPALM was not a consequence of overexpression. The differences between our EM, iPALM and GSD-measured clathrin densities likely resulted from the systematic measurement errors of each method. For example, some clathrin structures are hidden by other cellular structures in our EM images. Furthermore, background fluorescence could result in a slight overcounting of clathrin structures in iPALM and GSD imaging. These measurements support the model in which fusion occurs randomly among sites of endocytosis, and diffusing material can be rapidly captured on preformed clathrin-coated structures waiting on the cell surface to trap material destined for endocytosis.

(a) Four representative transmission electron microscopic images of the inner surface of the plasma membrane of PC12 cells. A representative flat (yellow arrow) and domed (pink arrow) structure are indicated. Scale bar equals 200 nm. (b) Plot of minimum and maximum caliper measurement for all visible clathrin structures over the entire cell (n=3 cells, 64 regions, 1,045 clathrin structures). Pink circles are structures classified as domed and purple circles are structures classified as flat. (c) Nearest-neighbour analysis of all the clathrin structures shown in b.

Modelling

To test the hypothesis that a dense field of traps can limit the spread of VAChT, we developed a simple physical model (Fig. 7a). First, particles in the centre of a small field were allowed to undergo random two-dimensional Brownian diffusion. These simulations were observed to lose all the particles rapidly (Fig. 7b and Supplementary Movie 4). When we introduced a population of traps into the simulation (1, 2 and 5 traps per μm 2 ), we observe a rapid capture of particles near sites of release (Fig. 7b and Supplementary Movie 5). The amount of capture was directly related to the density of traps. This is observed in Fig. 7b and Supplementary Movie 5. A plot measuring the fluorescence from simulations shows that without traps fluorescence is rapidly lost from the centre (750 nm radius region) into the surrounding membrane (Fig. 7c). The plot, however, of average simulations that included traps shows a rapid loss and then a plateau of fluorescence (Fig. 7c). The size of the plateau is proportional to the number of traps. These simulations support the hypothesis that a dense network of traps can account for the limited spread and rapid capture of VAChT. Furthermore, the density of traps can modulate the amount of material lost or remaining near any individual site of release.

(a) Cartoon of the model for the release and capture of VAChT. (b) Radial kymograph of fluorescence from the mean of five diffusion simulations. The simulations contained 0, 1, 2 and 5 traps per μm 2 . The material rapidly exits the centre, but a large amount of the particles are caught and remain in the centre in simulations that contain traps. Scale bar equals 2 μm. (c) Plot of the mean intensity contained in circular regions with a radius of 750 nm from the above simulations. Error bars are s.e.m.


Some of the integral membrane proteins that a cell displays at its surface are receptors for particular components of the ECF. For example, iron is transported in the blood complexed to a protein called transferin. Cells have receptors for transferrin on their surface. When these receptors encounter a molecule of transferrin, they bind tightly to it. The complex of transferrin and its receptor is then engulfed by endocytosis. Ultimately, the iron is released into the cytosol. The strong afinitet of the transferrin receptor for transferrin (its ligand) ensures that the cell will get all the iron it needs even if transferrin represents only a small fraction of the protein molecules present in the ECF. Receptor-mediated endocytosis is many thousand times more efficient than simple pinocytosis in enabling the cell to acquire the macromolecules it needs.

Another Example: the Low-Density Lipoprotein (LDL) Receptor

Cells take up cholesterol by receptor-mediated endocytosis. Cholesterol is an essential component of all cell membranes. Most cells can, as needed, either synthesize cholesterol or acquire it from the ECF. Human cells get much of their cholesterol from the liver and, if your diet is not strictly "100% cholesterol-free", by absorption from the intestine.

Cholesterol is a hydrophobic molecule and quite insoluble in water. Thus it cannot pass from the liver and/or the intestine to the cells simply dissolved in blood and ECF. Instead it is carried in tiny droplets of lipoprotein. The most abundant cholesterol carriers in humans are the low-density lipoproteins ili LDLs.

LDL particles are spheres covered with a single layer of phospholipid molecules with their hydrophilic heads exposed to the watery fluid (e.g., blood) and their hydrophobic tails directed into the interior. Some 1,500 molecules of cholesterol (each bound to a fatty acid) occupy the hydrophobic interior of LDL particles. One molecule of a protein called apolipoprotein B (apoB) is exposed at the surface of each LDL particle.

The first step in acquiring LDL particles is for them to bind to LDL receptors exposed at the cell surface. These transmembrane proteins have a site that recognizes and binds to the apolipoprotein B on the surface of the LDL. The portion of the plasma membrane with bound LDL is internalized by endocytosis. A drop in the pH (from

5) causes the LDL to separate from its receptor. The vesicle then pinches apart into two smaller vesicles: one containing free LDLs the other containing now-empty receptors. The vesicle with the LDLs fuses with a lysosome to form a secondary lysosome. The enzymes of the lysosome then release free cholesterol into the cytosol. The vesicle with unoccupied receptors returns to and fuses with the plasma membrane, turning inside out as it does so (exocytosis). In this way the LDL receptors are returned to the cell surface for reuse.

People who inherit two defective (mutant) genes for the LDL receptor have receptors that function poorly or not at all. This creates excessively high levels of LDL in their blood and predisposes them to atherosclerosis and heart attacks. The ailment is called familial (because it is inherited) hiperholesterolemija.

Mutations in APOB, the apoB gene, cause another form of inherited hypercholesterolemia.

  • the retinoid vitamin A (retinol) bound to the retinol-binding protein
  • the steroids
      bound to the vitamin D binding protein bound to the corticosteroid binding globulin and estrogens bound to the sex hormone binding globulin
  • and there is growing evidence that, like cholesterol, they are taken into the cell by receptor-mediated endocytosis.


    Egzocitoza

    Exocytosis is the process by which cells release particles from within the cell into the extracellular space.

    Ciljevi učenja

    Describe exocytosis and the processes used to release materials from the cell.

    Key Takeaways

    Key Points

    • Exocytosis is the opposite of endocytosis as it involves releasing materials from the cell.
    • Exocytosis has five stages, each leading up to the vesicle binding with the cell membrane.
    • Many bodily functions include the use of exocytosis, such as the release of neurotransmitters into the synaptic cleft and the release of enzymes into the blood.

    Ključni uslovi

    • sekrecija: The act of secreting (producing and discharging) a substance, especially from a gland.
    • vezikula: A membrane-bound compartment found in a cell.

    Egzocitoza

    Exocytosis’ main purpose is to expel material from the cell into the extracellular fluid this is the opposite of what occurs in endocytosis. In exocytosis, waste material is enveloped in a membrane and fuses with the interior of the plasma membrane. This fusion opens the membranous envelope on the exterior of the cell and the waste material is expelled into the extracellular space. Exocytosis is used continuously by plant and animal cells to excrete waste from the cells.

    Egzocitoza: In exocytosis, vesicles containing substances fuse with the plasma membrane. The contents are then released to the exterior of the cell.

    Exocytosis is composed of five main stages. The first stage is called vesicle trafficking. This involves the steps required to move, over a significant distance, the vesicle containing the material that is to be disposed. The next stage that occurs is vesicle tethering, which links the vesicle to the cell membrane by biological material at half the diameter of a vesicle. Next, the vesicle’s membrane and the cell membrane connect and are held together in the vesicle docking step. This stage of exocytosis is then followed by vesicle priming, which includes all of the molecular rearrangements and protein and lipid modifications that take place after initial docking. In some cells, there is no priming. The final stage, vesicle fusion, involves the merging of the vesicle membrane with the target membrane. This results in the release of the unwanted materials into the space outside the cell.

    Some examples of cells releasing molecules via exocytosis include the secretion of proteins of the extracellular matrix and secretion of neurotransmitters into the synaptic cleft by synaptic vesicles. Some examples of cells using exocytosis include: the secretion of proteins like enzymes, peptide hormones and antibodies from different cells, the flipping of the plasma membrane, the placement of integral membrane proteins(IMPs) or proteins that are attached biologically to the cell, and the recycling of plasma membrane bound receptors(molecules on the cell membrane that intercept signals).


    The Back Story

    by Eleonora Aquilini

    What work led to this paper?

    Apicomplexan parasites are single-celled, obligate intracellular parasites defined by the presence of an “apical complex” - a group of cytoskeletal structures and organelles located at the anterior end of the cell. The apical complex is a critical compartment for parasites, because it regulates the events leading to host cell invasion. Out of all the remarkable structures of the apical complex, rhoptries are pear-shaped organelles that inject proteins into the host-cell (crossing not only the plasma membrane of the parasite but also that of the host), to support invasion and subversion of host immune function. But, how!?

    The molecular mechanism by which they are discharged and their effectors delivered into the host cytoplasm was unclear, and no orthologues of secretion genes from bacteria, yeasts, animals or plants were found associated to rhoptry secretion in Apicomplexa (with only one exception). Rhoptry secretion could thus represent an unconventional, parasite specific, secretory mechanism…

    Luckily, pioneering ultrastructural work done at the turn of the '80s pointed out some resemblance between secretory organelles in apicomplexan parasites and ciliates - their free-living relatives. Moreover, a rosette of 8 intramembranous particles, essential for fusion and secretion in free-living ciliates, was found to be present also in several apicomplexan parasites at the site of exocytosis, where the fusion takes place.

    A) Free-living Paramecium , Dubremetz et al. 1976 b) Eimeria parasite, Dubremetz et al. 1977 c) Plasmodium parasite merozoite, Dubremetz et al. 1979 d) Roof of Casa Batlo, by A. Gaudí in Barcelona – we were so obsessed with the rosette that we could spot it literally everywhere , Lebrun 2017 e) free-living Paramecium, Froissard et al. 2002 f) Plasmodium parasite sporozoite, Dubremetz et al. 1979 g) Toxoplasma parasite tachizoite, Aquilini et al. 2021

    These resemblances were all we needed to start.

    What did we do?

    Back and forth between free-living Ciliata and apicomplexan parasites.

    Building on the ultrastructural work done previously, we explored the similarities between organelle secretion system in ciliates (Tetrahimena thermophila i Paramecijum tetraurelija) and apicomplexan parasites (Toxoplasma gondii i Plasmodium falciparum). We used cutting edge imaging techniques to study the structural elements and their architecture, and state-of-the-art molecular biology methods to explore the mechanism and molecular composition of these secretion systems.

    We cultivated tons of parasites and free-living ciliates, performed uncountables experiments, established the most amazing collaborations (Turkewitz Lab, specialists in secretory organelle biogenesis of Ciliata and for hobby in monarca butterfly metamorphosis and release and Chang Lab, masters of Cryo-electron tomography of the parasite apex in unfixed condition), I lived in Chicago for a while, we shared and discussed results in conferences (won one of the prizes at the Molecular Parasitology Meeting with preliminary results in 2018!), passed hours and hours and MORE hours looking at parasites under different types of microscopes . Dare I say we looked at them for hundreds of hours? Da.

    What did we find?

    That rhoptry exocytosis depends on a fusion rosette of intramembrane particles visible on the plasma membrane at the site of secretion. Rosette formation requires the “non discharge” complex (Nd6, Nd9, NdP1, NdP2). This critical structure (the fusion rosette) and the genetic elements necessary for its assembly are akin to the exocytic machinery of ciliates, their free-living relatives. Our data suggests a common ancestry for this fusion machinery that adapted, in two groups of protists that diverged hundreds of millions of years ago, to radically different environments . This same machinery is involved in self- defence in the free-living ciliates and supports host-cell invasion in intracellular parasites.

    We also noticed something that caught our attention: one cardinal protein responsible for exocytosis was present on an enigmatic apical vesicle visible in at the very tip of several apicomplexan parasites, but a bsent in free-living ciliates. Our results indicate that this parasite-exclusive vesicle is sandwiched between the tip of the rhoptry and the rosette, and may reflect the additional complexity required for parasitism and cell invasion, in which exocytosis must be coupled with injection of rhoptry content through the host cell membrane – in contrast with free-living organisms, where secretory organelles are more simply discharged into the environment to thwart predators. In support of this hypothesis, a similar vesicle is present at the apex of Perkinsus marinus, an oyster’s parasite phylogenetically considered an ancestor both of free-living ciliates and apicomplexan parasites, and a key taxon for understanding unique adaptations to parasitism.

    CRYO-ET view of the fusion rosette of intramembranous particles visible on the plasma mambrane and sagittal view and 3D reconstruction of the interactions between the tip of the rhoptry (orange), the apical vesicle (pink) and the fusion rosette (purple).

    Next challenge? Understand precisely the role and constitution of the apical vesicle, and further clarify how the molecular elements interact with each other to promote membrane fusion and rhoptry exocytosis.

    ZOOM-party paper accepted! From left to right, top line: Maryse Lebrun, Eleonora Aquilini, Marta Cova in the middle: Yi-Wei Chang, Nicolas Dos Santos Pacheco, Aaron Turkewitz bottom line: Laurence Berry and Daniela Sparvoli

    Summary and Future Perspectives

    We have summarized the above-mentioned mechanisms regarding exocytosis, endocytosis and possible coupling factors in Figure 2. From a macroscopic view, exocytosis may be matched with endocytosis: full fusion with clathrin-mediated endocytosis, KR and KS with clathrin-independent endocytosis, and sequential fusion and multivesicular exocytosis with bulk endocytosis. In this sense, the fate of the components of the fusing vesicle may be pre-determined at the moment of its choice of fusion modes. Therefore, understanding the early fusion intermediates of a vesicle, such as the hemifusion state, pore opening, dilation, and shape retention, will be instrumental for the understanding of the whole coupled process.

    Figure 2. Different types of exocytosis, endocytosis and coupling factors in secretory cells. Coupling factors and their roles in different steps are also listed on the scheme.

    The listed classification of different exocytosis and endocytosis subtypes is not based on molecular mechanism but rather hinges on studies that involve different experiments conducted on different cell types. The terminologies defined by different methods may not be mutually inclusive or exclusive. For example, bulk endocytosis is usually regarded as a subcategory of clathrin-independent endocytosis. However, the bulk membrane invaginations observed in secretory cells under EM, which are often taken as evidence supporting bulk endocytosis, may support the internalization of small or large chunks of membrane in a clathrin-dependent manner in live cell studies. KR and KS may be one uniform process at different stages but could also be two distinct processes with non-overlapping mechanisms. To differentiate these controversies, it is important to sort out molecules that are exclusively used for some specific processes, in addition to actin for clathrin-independent endocytosis (He et al., 2008 Delvendahl et al., 2016). Alternatively, we shall examine the same process in the same cells using multiple techniques. For example, combining cell-attached membrane capacitance measurements with imaging vesicular lipids in endocrine cells will help clarify whether lipid exchange occurs between the vesicle and the plasma membrane during the flickering of a small fusion pore. Simultaneous imaging of vesicular components and extracellularly applied fluorescent dextran of different sizes will help monitor the dilation of a fusion pore from ߡ nm to a much larger in diameter (Takahashi et al., 2002). This will differentiate KR and KS and ultimately determine the size of fusion pores accompanying KS exocytosis. Monitoring the shape of the membrane may reveal clues of hemifusion in live cells (Zhao et al., 2016) and will also confirm or disapprove the compound fusion/multivesicular exocytosis theories and their physiological significance. Finally, operating at a nanometer scale with lifetimes of milliseconds, most of the fusion intermediate structures described here can hardly be directly discerned even with state-of-the-art super-resolution microscopy methodologies (Huang et al., 2009 Schermelleh et al., 2010). Despite differences in exocytosis kinetics and the organization of fusion sites between synapses and endocrine cells, we believe that the core exo-endocytosis coupling mechanism is conserved. Therefore, if we can improve the temporal and spatial resolution and duration of current super-resolution imaging technologies, direct visualization of fusion pore intermediates in endocrine cells may invoke new insights that would render much of the discussed theories here obsolete.


    Understand Cell Secretion in 4 Q&As

    In secretory cells, such as the secretory cells of endocrine glands, organelles related to the production, processing and “export” of substances are widely present and well-developed. These organelles are the rough endoplasmic reticulum and the Golgi apparatus.

    The nuclear membrane of secretory cells generally has more pores to allow the intense traffic of molecules related to protein synthesis between the cytoplasm and the nucleus.

    Secretory Organelles

    3. What is the role of the rough endoplasmic reticulum and the Golgi apparatus in the production and release of proteins?

    In its outer membrane, the rough endoplasmic reticulum contains numerous ribosomes, structures where the translation of messenger RNA and protein synthesis occur. These proteins are stored in the rough endoplasmic reticulum and are later moved to the Golgi apparatus. Within the Golgi apparatus, proteins are chemically transformed and, when ready, they are put inside vesicles that detach from the organelle. These vesicles fuse with the plasma membrane (exocytosis) in the right place and its content is released outside the cell.

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    Protein Degradation

    When a protein has outlived its usefulness or become damaged, it is degraded by the cell. In eukaryotes, a protein that is to be degraded has a number of copies of the small protein ubiquitin attached to it by a series of ubiquitin-adding enzymes. Ubiquitin serves as a tag that marks the protein for degradation. A tagged protein is then sucked into a large cellular machine called the proteasome, which itself is made up of a number of protein components and looks something like a trash can. Inside the proteasome, the tagged protein is digested into small peptide fragments that are released into the cytoplasm where they can be further digested into free amino acids by other proteases. The life of a protein begins in one cellular machine called the ribosome and ends in another called the proteasome.


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