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3.8.3: Među vlakna i mikrotubule - Biologija

3.8.3: Među vlakna i mikrotubule - Biologija


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Mikrotubule su dio citoskeleta stanice, pomažući stanici da se odupre kompresiji, pomakne vezikule i odvoji kromosome pri mitozi.

ciljevi učenja

  • Opišite uloge mikrotubula kao dijela citoskeleta stanice

Ključne točke

  • Mikrotubule pomažu stanici da se odupre kompresiji, pružaju trag duž kojeg se mjehurići mogu kretati po stanici, a sastavni su dio cilija i flagela.
  • Cilia i flagella su strukture nalik na kosu koje pomažu pri kretanju u nekim stanicama, kao i postavljaju različite strukture kako bi zarobile čestice.
  • Strukture cilija i flagella su "niz 9+2", što znači da je prsten od devet mikrotubula okružen s još dvije mikrotubule.
  • Mikrotubule se tijekom diobe stanica vežu za replicirane kromosome i razdvajaju ih na suprotne krajeve pola, dopuštajući stanici da se podijeli s kompletnim setom kromosoma u svakoj stanici kćeri.

Ključni uvjeti

  • mikrotubule: Male cjevčice napravljene od proteina koje se nalaze u stanicama; dio citoskeleta
  • flagellum: flagellum je dodatak poput trepavica koji viri iz staničnog tijela određenih prokariotskih i eukariotskih stanica
  • citoskelet: Stanična struktura poput kostura, sadržana u citoplazmi.

Mikrotubule

Kao što im naziv govori, mikrotubule su male šuplje cijevi. Mikrotubule, zajedno s mikrofilamentima i srednjim vlaknima, spadaju u klasu organela poznatih kao citoskelet. Citoskelet je okvir stanice koji čini strukturnu potpornu komponentu. Mikrotubule su najveći element citoskeleta. Stijenke mikrotubule izrađene su od polimeriziranih dimera α-tubulina i β-tubulina, dva globularna proteina. S promjerom od oko 25 nm, mikrotubule su najšire komponente citoskeleta. Oni pomažu stanici da se odupre kompresiji, pružaju trag duž kojeg se mjehurići kreću kroz stanicu i povlače replicirane kromosome na suprotne krajeve stanice koja se dijeli. Kao i mikrofilamenti, mikrotubule se mogu brzo otopiti i reformirati.

Mikrotubule su također strukturni elementi flagela, cilija i centriola (potonji su dva okomita tijela centrosoma). U životinjskim stanicama centrosom je centar za organiziranje mikrotubula. U eukariotskim stanicama flagele i cilije strukturno se prilično razlikuju od svojih kolega u prokariotima.

Srednje niti

Intermedijarni filamenti (IF) su citoskeletne komponente koje se nalaze u životinjskim stanicama. Sastoje se od obitelji srodnih proteina koji imaju zajedničke značajke strukture i sekvence. Srednji filamenti imaju prosječni promjer od 10 nanometara, što je između onog aktina od 7 nm (mikrofilamenti) i 25 nm mikrotubula, iako su u početku označeni kao "srednji" jer je njihov prosječni promjer između onih užih mikrofilamenata (aktin) i širi miozinski filamenti koji se nalaze u mišićnim stanicama. Srednji filamenti doprinose staničnim strukturnim elementima i često su ključni za držanje zajedno tkiva poput kože.

Flagella i Cilia

Flagele (singular = flagellum) su duge strukture nalik na kosu koje se protežu od plazma membrane i koriste se za pomicanje cijele stanice (na primjer, spermija, Euglena). Kad je prisutna, stanica ima samo jedan flagelum ili nekoliko flagella. Međutim, kada su prisutne cilije (singular = cilium), mnoge se protežu duž cijele površine plazma membrane. To su kratke strukture nalik na kosu koje se koriste za pomicanje cijelih stanica (poput paramecije) ili tvari duž vanjske površine stanice (na primjer, cilije stanica koje oblažu jajovode koje pomiču jajnu stanicu prema maternici, ili cilije koje oblažu stanice respiratornog trakta koje hvataju čestice i pomiču ih prema nosnicama).

Unatoč razlikama u duljini i broju, flagele i cilije imaju zajednički strukturni raspored mikrotubula koji se naziva „niz 9 + 2“. Ovo je prikladan naziv jer se jedan flagellum ili cilium sastoji od prstena od devet dubleta mikrotubula koji okružuju jedan doubletutule u sredini.


3.3 Eukariotske stanice

U ovom trenutku treba biti jasno da eukariotske stanice imaju složeniju strukturu od prokariotskih stanica. Organele dopuštaju da se u stanici istodobno pojavljuju različite funkcije. Prije nego raspravljamo o funkcijama organela unutar eukariotske stanice, prvo ćemo ispitati dvije važne komponente stanice: plazma membranu i citoplazmu.

Vizualna veza

Koje strukture biljna stanica ima, a životinjska nema? Koje strukture ima životinjska stanica a biljna nema?

Plazma membrana

Poput prokariota, eukariotske stanice imaju plazma membranu (slika 3.8) sačinjenu od fosfolipidnog dvosloja s ugrađenim proteinima koji odvaja unutarnji sadržaj stanice od okoline. Fosfolipid je molekula lipida sastavljena od dva lanca masnih kiselina, okosnice glicerola i fosfatne skupine. Plazma membrana regulira prolaz nekih tvari, poput organskih molekula, iona i vode, sprječavajući prolaz nekih radi održavanja unutarnjih uvjeta, dok druge aktivno unosi ili uklanja. Ostali spojevi se pasivno kreću po membrani.

Plazma membrane stanica koje su specijalizirane za apsorpciju presavijene su u izbočine slične prstima koje se nazivaju mikrovilici (singularno = mikrovili). Ovo preklapanje povećava površinu plazma membrane. Takve se stanice obično nalaze u tankom crijevu, organu koji apsorbira hranjive tvari iz probavljene hrane. Ovo je izvrstan primjer oblika koji odgovara funkciji strukture.

Ljudi s celijakijom imaju imunološki odgovor na gluten, protein koji se nalazi u pšenici, ječmu i raži. Imunološki odgovor oštećuje mikrovile, pa stoga oboljeli pojedinci ne mogu apsorbirati hranjive tvari. To dovodi do pothranjenosti, grčeva i proljeva. Pacijenti koji boluju od celijakije moraju se pridržavati dijete bez glutena.

Citoplazma

Citoplazma sadrži sadržaj stanice između plazma membrane i jezgrene ovojnice (struktura o kojoj ćemo uskoro raspravljati). Sastoji se od organela suspendiranih u citosolu poput gela, citoskeleta i raznih kemikalija (slika 3.7). Iako se citoplazma sastoji od 70 do 80 posto vode, ima polutvrdu konzistenciju, koja potječe od proteina u njoj. Međutim, proteini nisu jedine organske molekule koje se nalaze u citoplazmi. Glukoza i drugi jednostavni šećeri, polisaharidi, aminokiseline, nukleinske kiseline, masne kiseline i derivati ​​glicerola također se nalaze tamo. Ioni natrija, kalija, kalcija i mnogi drugi elementi također su otopljeni u citoplazmi. Mnoge metaboličke reakcije, uključujući sintezu proteina, odvijaju se u citoplazmi.

Citoskelet

Kad biste uklonili sve organele iz stanice, bi li plazma membrana i citoplazma ostale jedine komponente? Ne. Unutar citoplazme još bi bilo iona i organskih molekula, plus mreža proteinskih vlakana koja pomažu u održavanju oblika stanice, učvršćuju određene organele u određenim položajima, omogućuju kretanje citoplazme i vezikula unutar stanice i omogućuju jednostanični organizmi koji se samostalno kreću. Zajedno, ta mreža proteinskih vlakana poznata je kao citoskelet. Unutar citoskeleta postoje tri vrste vlakana: mikrofilamenti, poznati i kao aktinski filamenti, srednji filamenti i mikrotubule (slika 3.9).

Mikrofilamenti su najtanja od citoskeletnih vlakana i funkcioniraju u kretanju staničnih komponenti, na primjer, tijekom diobe stanica. Oni također održavaju strukturu mikrovila, opsežno nabiranje plazma membrane pronađeno u stanicama posvećenim apsorpciji. Ove komponente su također česte u mišićnim stanicama i odgovorne su za kontrakciju mišićnih stanica. Srednji filamenti su srednjeg promjera i imaju strukturne funkcije, poput održavanja oblika ćelije i sidrenja organela. Keratin, spoj koji jača kosu i nokte, tvori jednu vrstu srednjih niti. Mikrotubule su najdeblje od citoskeletnih vlakana. To su šuplje cijevi koje se mogu brzo otopiti i reformirati. Mikrotubule vode kretanje organela i strukture su koje vuku kromosome na svoje polove tijekom diobe stanica. Oni su također strukturne komponente flagela i cilija. U cilijama i flagelama mikrotubule su organizirane kao krug od devet dvostrukih mikrotubula izvana i dvije mikrotubule u sredini.

Centrosom je regija u blizini jezgre životinjskih stanica koja funkcionira kao središte za organiziranje mikrotubula. Sadrži par centriola, dvije strukture koje leže okomito jedna na drugu. Svaki centriol je cilindar od devet tripleta mikrotubula.

Centrosom se replicira prije nego što se stanica podijeli, a centrioli igraju ulogu u povlačenju dupliciranih kromosoma na suprotne krajeve stanice koja se dijeli. Međutim, točna funkcija centriola u staničnoj diobi nije jasna, budući da se stanice koje imaju uklonjene centriole još uvijek mogu podijeliti, a biljne stanice kojima nedostaje centriola sposobne su za diobu stanica.

Flagella i Cilia

Flagele (singular = flagellum) su duge strukture nalik na kosu koje se protežu od plazma membrane i koriste se za pomicanje cijele stanice (na primjer, spermija, Euglena). Kad je prisutna, stanica ima samo jedan flagelum ili nekoliko flagella. Kad su prisutne trepetljike (singular = cilium), one su brojne i protežu se duž cijele površine plazma membrane. Oni su kratke strukture nalik na kosu koje se koriste za pomicanje cijelih stanica (poput paramecija) ili za pomicanje tvari duž vanjske površine stanice (na primjer, cilije stanica koje oblažu jajovode koje pomiču jajnu stanicu prema maternici, ili cilije koje oblažu stanice respiratornog trakta i koje pomiču čestice prema grlu koje je sluz zarobila).

Endomembranski sustav

Endomembranski sustav (endo = unutar) skupina je membrana i organela (slika 3.13) u eukariotskim stanicama koje zajedno rade na izmjeni, pakiranju i transportu lipida i proteina. Uključuje nuklearnu ovojnicu, lizosome i vezikule, endoplazmatski retikulum i Golgijev aparat, koje ćemo uskoro pokriti. Iako tehnički ne unutar stanica, plazma membrana je uključena u endomembranski sustav jer, kao što ćete vidjeti, stupa u interakciju s ostalim endomembranoznim organelama.

Nukleus

Tipično, jezgra je najistaknutija organela u stanici (slika 3.7). U jezgri (množina = jezgre) nalazi se DNK stanice u obliku kromatina i usmjerava sintezu ribosoma i proteina. Pogledajmo to detaljnije (slika 3.10).

Nuklearna ovojnica je struktura s dvostrukom membranom koja čini najudaljeniji dio jezgre (slika 3.10). I unutarnja i vanjska membrana jezgre omotača su fosfolipidni dvoslojevi.

Nuklearni omotač pun je pora koje kontroliraju prolaz iona, molekula i RNA između nukleoplazme i citoplazme.

Da bismo razumjeli kromatin, korisno je prvo razmotriti kromosome. Kromosomi su strukture unutar jezgre koje se sastoje od DNK, nasljednog materijala i bjelančevina. Ova kombinacija DNK i proteina naziva se kromatin. U eukariota kromosomi su linearne strukture. Svaka vrsta ima određeni broj kromosoma u jezgri svojih tjelesnih stanica. Na primjer, kod ljudi je broj kromosoma 46, dok je kod voćnih mušica broj kromosoma osam.

Kromosomi su vidljivi i međusobno se razlikuju samo kada se stanica sprema za diobu. Kad je stanica u fazi rasta i održavanja svog životnog ciklusa, kromosomi nalikuju odmotanoj, zbrkanoj hrpi niti.

Već znamo da jezgra usmjerava sintezu ribosoma, ali kako to čini? Neki kromosomi imaju dijelove DNK koji kodiraju ribosomsku RNK. Tamno obojeno područje unutar jezgre, nazvano nukleolus (množina = nukleoli), agregira ribosomsku RNA s povezanim proteinima kako bi sastavilo ribosomske podjedinice koje se zatim transportiraju kroz nuklearne pore u citoplazmu.

Endoplazmatski retikulum

Endoplazmatski retikulum (ER) (slika 3.13) je niz međusobno povezanih membranskih cjevčica koje zajedno mijenjaju proteine ​​i sintetiziraju lipide. Međutim, ove dvije funkcije obavljaju se u zasebnim područjima endoplazmatskog retikuluma: grubi endoplazmatski retikulum i glatki endoplazmatski retikulum.

Šuplji dio ER tubula naziva se lumen ili cisternalni prostor. Membrana ER -a, koji je fosfolipidni dvosloj ugrađen s proteinima, kontinuirana je s jezgrom.

Grubi endoplazmatski retikulum (RER) nazvan je tako jer mu ribosomi pričvršćeni na citoplazmatsku površinu daju šiljast izgled kada se gledaju pod elektronskim mikroskopom.

Ribosomi sintetiziraju proteine ​​dok su vezani za ER, što rezultira prijenosom njihovih novosintetiziranih proteina u lumen RER -a gdje se podvrgavaju modifikacijama kao što su nabiranje ili dodavanje šećera. RER također proizvodi fosfolipide za stanične membrane.

Ako fosfolipidi ili modificirani proteini nisu predodređeni da ostanu u RER -u, bit će zapakirani u vezikule i transportirani iz RER -a pupanjem iz membrane (slika 3.13). Budući da je RER modificiran proteinima koji će se izlučivati ​​iz stanice, obiluje stanicama koje luče proteine, poput jetre.

Glatki endoplazmatski retikulum (SER) kontinuiran je s RER -om, ali ima malo ili nimalo ribosoma na svojoj citoplazmatskoj površini (vidi sliku 3.7). Funkcije SER -a uključuju sintezu ugljikohidrata, lipida (uključujući fosfolipide) i steroidne hormone za detoksikaciju lijekova i otrovaju metabolizam alkohola i skladištenje kalcijevih iona.

Golgijev aparat

Već smo spomenuli da vezikule mogu pupati iz ER -a, ali kamo mjehurići idu? Prije nego stignu na svoje konačno odredište, lipide ili proteine ​​unutar transportnih vezikula potrebno je sortirati, pakirati i označiti tako da završe na pravom mjestu. Razvrstavanje, označavanje, pakiranje i distribucija lipida i proteina odvija se u Golgijevom aparatu (koji se naziva i Golgijevo tijelo), nizu spljoštenih opnastih vrećica (slika 3.11).

Golgijev aparat ima prijemno lice u blizini endoplazmatskog retikuluma i otpuštajuće lice sa strane udaljene od ER, prema staničnoj membrani. Transportni mjehurići koji nastaju iz ER -a putuju do prijemnog lica, stapaju se s njim i prazne svoj sadržaj u lumen Golgijevog aparata. Kako proteini i lipidi putuju kroz Golgi, podvrgavaju se daljnjim izmjenama. Najčešća modifikacija je dodavanje kratkih lanaca molekula šećera. Novo modificirani proteini i lipidi tada se označavaju malim molekularnim skupinama kako bi im se omogućilo usmjeravanje na njihova odgovarajuća odredišta.

Konačno, modificirani i označeni proteini pakirani su u vezikule koje pupaju sa suprotne strane Golgija. Dok neki od ovih mjehurića, transportni mjehurići, odlažu svoj sadržaj u druge dijelove stanice gdje će se koristiti, drugi, sekrecijski mjehurići, spajaju se s plazma membranom i oslobađaju njihov sadržaj izvan stanice.

Količina Golgija u različitim tipovima stanica ponovno pokazuje da oblik slijedi funkciju unutar stanica. Stanice koje se bave velikom sekretornom aktivnošću (kao što su stanice žlijezda slinovnica koje luče probavne enzime ili stanice imunološkog sustava koje luče antitijela) imaju veliki broj Golgija.

U biljnim stanicama, Golgi ima dodatnu ulogu sinteze polisaharida, od kojih su neki ugrađeni u staničnu stjenku, a neki se koriste u drugim dijelovima stanice.

Lizosomi

U životinjskim stanicama lizosomi su "odlaganje smeća" stanice. Probavni enzimi unutar lizosoma pomažu razgradnji proteina, polisaharida, lipida, nukleinskih kiselina, pa čak i istrošenih organela. U jednostaničnih eukariota lizosomi su važni za probavu hrane koju unose i recikliranje organela. Ti su enzimi aktivni pri mnogo nižem pH (kiseliji) od onih koji se nalaze u citoplazmi. Mnoge reakcije koje se odvijaju u citoplazmi ne mogu se dogoditi pri niskom pH, pa je očita prednost razdvajanja eukariotske stanice u organele.

Lizosomi također koriste svoje hidrolitičke enzime za uništavanje organizama koji uzrokuju bolesti i koji mogu ući u stanicu. Dobar primjer za to javlja se u skupini bijelih krvnih stanica zvanih makrofagi, koje su dio imunološkog sustava vašeg tijela. U procesu poznatom kao fagocitoza, dio plazma membrane makrofaga invaginira (savija se) i zahvaća patogen. Invaginirani dio, s patogenom unutra, odvaja se od plazma membrane i postaje mjehurić. Mjehurić se spaja s lizosomom. Hidrolitički enzimi lizosoma tada uništavaju patogen (slika 3.12).

Vezikule i vakuole

Vezikule i vakuole su membranski vezane vrećice koje funkcioniraju u skladištu i transportu. Vakuole su nešto veće od vezikula, a membrana vakuole se ne stapa s membranama drugih staničnih komponenti. Vezikule se mogu stopiti s drugim membranama unutar staničnog sustava. Osim toga, enzimi unutar biljnih vakuola mogu razgraditi makromolekule.

Vizualna veza

Zašto se cis lice Golgija nije lice prema plazma membrani?

Ribosomi

Ribosomi su stanične strukture odgovorne za sintezu proteina. Kada se promatraju elektronskim mikroskopom, slobodni ribosomi pojavljuju se kao nakupine ili pojedinačne sitne točkice koje slobodno plutaju u citoplazmi. Ribosomi se mogu pričvrstiti ili na citoplazmatsku stranu plazma membrane ili na citoplazmatsku stranu endoplazmatskog retikuluma (slika 3.7). Elektronska mikroskopija pokazala je da se ribosomi sastoje od velikih i malih podjedinica. Ribosomi su enzimski kompleksi koji su odgovorni za sintezu proteina.

Budući da je sinteza proteina bitna za sve stanice, ribosomi se nalaze u praktički svakoj stanici, iako su manji u prokariotskim stanicama. Posebno su bogati nezrelim crvenim krvnim zrncima za sintezu hemoglobina, koji djeluje u transportu kisika po cijelom tijelu.

Mitohondrije

Mitohondrije (jednina = mitohondrije) često nazivaju „elektranama“ ili „tvornicama energije“ stanice jer su odgovorne za stvaranje adenozin trifosfata (ATP), glavne molekule stanice koja prenosi energiju. Stvaranje ATP -a razgradnjom glukoze poznato je kao stanično disanje. Mitohondriji su organele dvostruke membrane ovalnog oblika (slika 3.14) koje imaju svoje ribosome i DNK. Svaka membrana je fosfolipidni dvosloj ugrađen proteinima. Unutarnji sloj ima nabore nazvane cristae, koji povećavaju površinu unutarnje membrane. Područje okruženo naborima naziva se mitohondrijski matriks. Krista i matrica imaju različite uloge u staničnom disanju.

U skladu s našom temom funkcije slijeđenja forme, važno je naglasiti da mišićne stanice imaju vrlo visoku koncentraciju mitohondrija jer mišićnim stanicama treba mnogo energije za kontrakciju.

Peroksisomi

Peroksisomi su mali, okrugli organeli zatvoreni pojedinačnim membranama. Provode oksidacijske reakcije koje razgrađuju masne kiseline i aminokiseline. Također detoksiciraju mnoge otrove koji mogu ući u tijelo. Alkohol se detoksicira peroksisomima u stanicama jetre. Nusprodukt ovih oksidacijskih reakcija je vodikov peroksid, H2O.2, koji se nalazi u peroksisomima kako bi se spriječilo da kemikalija ošteti stanične komponente izvan organele. Vodikov peroksid se sigurno razgrađuje peroksisomskim enzimima u vodu i kisik.

Životinjske stanice nasuprot biljnim stanicama

Unatoč temeljnim sličnostima, postoje neke zapanjujuće razlike između životinjskih i biljnih stanica (vidi tablicu 3.1). Životinjske stanice imaju centriole, centrosome (o kojima se govori pod citoskeletom) i lizosome, dok stanice biljaka nemaju. Biljne stanice imaju staničnu stijenku, kloroplaste, plazmodezmate i plastide koji se koriste za skladištenje, te veliku središnju vakuolu, dok životinjske stanice nemaju.

Stanični zid

Na slici 3.7b, dijagram biljne stanice, vidite strukturu izvan plazma membrane koja se naziva stanična stijenka. Stanična stijenka je kruti pokrov koji štiti stanicu, pruža strukturnu potporu i daje stanici oblik. Stanice gljivica i protista također imaju stanične stijenke.

Dok je glavna komponenta staničnih stijenki prokariota peptidoglikan, glavna organska molekula u staničnoj stijenci biljaka je celuloza, polisaharid sastavljen od dugih, ravnih lanaca jedinica glukoze. Kad se nutritivne informacije odnose na dijetalna vlakna, to se odnosi na sadržaj celuloze u hrani.

Kloroplasti

Kao i mitohondriji, kloroplasti također imaju vlastitu DNK i ribosome. Kloroplasti funkcioniraju u fotosintezi i mogu se naći u eukariotskim stanicama poput biljaka i algi. U fotosintezi se ugljični dioksid, voda i svjetlosna energija koriste za stvaranje glukoze i kisika. Ovo je glavna razlika između biljaka i životinja: biljke (autotrofi) mogu sami stvarati hranu, poput glukoze, dok se životinje (heterotrofi) moraju oslanjati na druge organizme za svoje organske spojeve ili izvor hrane.

Poput mitohondrija, kloroplasti imaju vanjsku i unutarnju membranu, ali unutar prostora zatvorenog unutarnjom membranom kloroplasta nalazi se niz međusobno povezanih i naslaganih membranskih vrećica ispunjenih tekućinom zvanih tilakoidi (slika 3.15). Svaki hrpetina tilakoida naziva se granum (množina = grana). Tekućina zatvorena unutarnjom opnom i okružujući granu naziva se stroma.

Kloroplasti sadrže zeleni pigment nazvan klorofil, koji hvata energiju sunčeve svjetlosti za fotosintezu. Poput biljnih stanica, fotosintetski protisti također imaju kloroplaste. Neke bakterije također vrše fotosintezu, ali nemaju kloroplaste. Njihovi fotosintetski pigmenti nalaze se u tilakoidnoj membrani unutar same stanice.

Evolucijska veza

Endosimbioza

Spomenuli smo da i mitohondriji i kloroplasti sadrže DNA i ribosome. Jeste li se pitali zašto? Čvrsti dokazi ukazuju na endosimbiozu kao objašnjenje.

Simbioza je odnos u kojem organizmi iz dvije odvojene vrste žive u bliskoj vezi i tipično pokazuju specifične prilagodbe jedan prema drugom. Endosimbioza (endo-= unutar) je odnos u kojem jedan organizam živi unutar drugog. Endosimbiotski odnosi obiluju prirodom. Mikrobi koji proizvode vitamin K žive unutar crijeva čovjeka. Taj je odnos koristan za nas jer ne možemo sintetizirati vitamin K. Također je koristan za mikrobe jer su zaštićeni od drugih organizama i pružaju im stabilno stanište i obilnu hranu živeći u debelom crijevu.

Znanstvenici su odavno primijetili da su bakterije, mitohondriji i kloroplasti slične veličine. Također znamo da mitohondriji i kloroplasti imaju DNK i ribosome, baš kao i bakterije. Znanstvenici vjeruju da su stanice domaćini i bakterije stvorile obostrano koristan endosimbiotski odnos kada su stanice domaćini unijele aerobne bakterije i cijanobakterije, ali ih nisu uništile. Kroz evoluciju su se ove unesene bakterije sve više specijalizirale u svojim funkcijama, pri čemu su aerobne bakterije postale mitohondrije, a fotosintetske bakterije kloroplasti.

Središnji vakuol

Prije smo spomenuli vakuole kao bitne komponente biljnih stanica. Ako pogledate sliku 3.7, vidjet ćete da svaka biljna stanica ima veliku središnju vakuolu koja zauzima veći dio stanice. Središnja vakuola igra ključnu ulogu u regulaciji stanične koncentracije vode u promjenjivim uvjetima okoline. U biljnim stanicama tekućina unutar središnje vakuole osigurava turgor tlak, koji je vanjski tlak uzrokovan tekućinom unutar stanice. Jeste li ikada primijetili da ako zaboravite zalijevati biljku na nekoliko dana, ona uvene? To je zato što kako koncentracija vode u tlu postaje niža od koncentracije vode u biljci, voda se iseljava iz središnjih vakuola i citoplazme u tlo. Kako se središnja vakuola smanjuje, ostavlja staničnu stijenku nepodržanom. Gubitak potpore staničnim stijenkama biljke dovodi do uvenulog izgleda. Osim toga, ova tekućina ima vrlo gorak okus, što obeshrabruje konzumaciju insekata i životinja. Središnja vakuola također funkcionira za pohranu proteina u stanicama sjemena u razvoju.

Izvanstanični matriks životinjskih stanica

Većina životinjskih stanica oslobađa materijale u izvanstanični prostor. Primarne komponente ovih materijala su glikoproteini i protein kolagen. Zajedno se ti materijali nazivaju izvanstanični matriks (slika 3.16). Ne samo da izvanstanični matriks drži stanice zajedno da tvore tkivo, već i omogućuje stanicama unutar tkiva da međusobno komuniciraju.

Zgrušavanje krvi primjer je uloge izvanstaničnog matriksa u staničnoj komunikaciji. Kad su stanice koje oblažu krvnu žilu oštećene, one pokazuju receptor proteina koji se naziva tkivni faktor. Kada se faktor tkiva veže s drugim čimbenikom u izvanstaničnom matriksu, uzrokuje prianjanje trombocita na stijenku oštećene krvne žile, potiče susjedne stanice glatkih mišića u krvnom sudu da se stežu (čime se sužava krvna žila) i započinje niz koraci koji potiču trombocite na stvaranje faktora zgrušavanja.

Međustanični spojevi

Stanice također mogu međusobno komunicirati izravnim kontaktom, što se naziva međustaničnim spojevima. Postoje neke razlike u načinima na koje to čine biljne i životinjske stanice. Plasmodesmata (singular = plazmodesma) su spojevi između biljnih stanica, dok kontakti životinjskih stanica uključuju tijesne i razmaknute spojeve i desmosome.

Općenito, duga protezanja plazma membrana susjednih biljnih stanica ne mogu se međusobno dodirivati ​​jer su odvojene staničnim stjenkama koje okružuju svaku stanicu. Plazmodezmati su brojni kanali koji prolaze između staničnih stijenki susjednih biljnih stanica, povezujući njihovu citoplazmu i omogućujući prijenos signalnih molekula i hranjivih tvari iz stanice u stanicu (slika 3.17.a).

Čvrsto spajanje je vodonepropusna brtva između dvije susjedne životinjske ćelije (slika 3.17b). Proteini drže stanice čvrsto jedne uz druge. Ovo čvrsto prianjanje sprječava curenje materijala između ćelija. Čvrsti spojevi obično se nalaze u epitelnom tkivu koje oblaže unutarnje organe i šupljine i čini većinu kože. Na primjer, čvrsti spojevi epitelnih stanica koje oblažu mokraćni mjehur sprječavaju curenje urina u izvanstanični prostor.

Samo u životinjskim stanicama nalaze se i desmosomi, koji djeluju poput točkastih zavara između susjednih epitelnih stanica (slika 3.17.c). Oni drže stanice zajedno u obliku lista u organima i tkivima koji se protežu, poput kože, srca i mišića.

Pukotine u životinjskim stanicama slične su plazmodesmatama u biljnim stanicama jer su kanali između susjednih stanica koje omogućuju transport iona, hranjivih tvari i drugih tvari koje omogućuju stanicama komunikaciju (slika 3.17.d). Strukturno, međutim, raspori i plazmodesmati se razlikuju.

Stanična komponenta Funkcija Prisutni u Prokariotima? Prisutan u životinjskim stanicama? Prisutni u biljnim stanicama?
Plazma membrana Odvaja stanicu od vanjskog okruženja kontrolira prolaz organskih molekula, iona, vode, kisika i otpada u i iz stanice Da Da Da
Citoplazma Pruža strukturu staničnom mjestu mnogih medija za metaboličke reakcije u kojima se nalaze organele Da Da Da
Nukleoid Položaj DNK Da Ne Ne
Jezgra Stanične organele u kojima se nalazi DNK i usmjerava sintezu ribosoma i proteina Ne Da Da
Ribosomi Sinteza proteina Da Da Da
Mitohondrije Proizvodnja ATP -a/stanično disanje Ne Da Da
Peroksisomi Oksidira i razgrađuje masne kiseline i aminokiseline, te detoksicira otrove Ne Da Da
Vezikule i vakuole Skladištenje i transport probavne funkcije u biljnim stanicama Ne Da Da
Centrosom Nespecificirana uloga u diobi stanica u životinjskim stanicama koje organiziraju središte mikrotubula u životinjskim stanicama Ne Da Ne
Lizosomi Probava makromolekula recikliranjem dotrajalih organela Ne Da Ne
Stanične stijenke Zaštita, strukturna potpora i održavanje oblika stanice Da, prvenstveno peptidoglikan u bakterijama, ali ne i u arhejama Ne Da, prvenstveno celuloza
Kloroplasti Fotosinteza Ne Ne Da
Endoplazmatski retikulum Modificira proteine ​​i sintetizira lipide Ne Da Da
Golgijev aparat Modificira, sortira, označava, pakira i distribuira lipide i proteine Ne Da Da
Citoskelet Održava oblik stanice, učvršćuje organele u određenim položajima, dopušta kretanje citoplazme i vezikula unutar stanice i omogućuje jednostaničnim organizmima da se samostalno kreću Da Da Da
Flagella Stanično kretanje Neki Neki Ne, osim neke biljne sperme.
Cilija Stanično kretanje, kretanje čestica duž izvanstanične površine plazma membrane i filtriranje Ne Neki Ne

Ova tablica daje komponente prokariotskih i eukariotskih stanica i njihove odgovarajuće funkcije.


Pozadina

Do prijelaza epitela u mezenhim (EMT) dolazi kada usko povezane epitelne stanice stječu migracijski mezenhimalni fenotip tijekom embrionalnog razvoja, zacjeljivanja rana i bolesti [1, 2]. Povijesno gledano, EMT je bio povezan s dubokom reorganizacijom citoskeleta kako bi se oslabilo vezivanje stanica -stanica i ojačale adhezije staničnog matriksa [3], a prvi ju je primijetila Elizabeth Hay kao odgovor na poučne znakove izvanstaničnog matriksa [4] . Doista, invazija tumora i rezistencija na lijekove povezani su s nereguliranim izvanstaničnim matriksom, s aberantnim taloženjem matriksa i remodeliranjem što rezultira povećanom ukočenošću [5]. Citoskeletni elementi dobro su uspostavljeni kao biomarkeri EMT -a, osobito posredni filamenti poput keratina (u epitelnim stanicama) i vimentina (u mezenhimskim stanicama) [6]. Ipak, funkcionalna važnost EMT -a i povezanih citoskeletnih promjena ostaje slabo shvaćena, osobito za progresiju raka kod ljudi [7]. Nedavne inovacije u bioinženjeringu omogućile su biomimetička ispitivanja i alate za mjerenje veće razlučivosti da razjasne EMT na razini jedne stanice tijekom vremena i prostora.

Klasični testovi migracije stanica temelje se na eksperimentalnim uvjetima koji mogu umjetno utjecati na epitelna ili mezenhimalna stanja [8]. Na primjer, testovi "zacjeljivanja rana" događaju se na plastici krute kulture tkiva, na temelju kolektivne migracije čvrsto povezanih epitelnih jednoslojnih ploča [9]. Alternativno, Transwell (Boydenovi) testovi temelje se na plastičnoj mikroporoznoj membrani, koju stanice moraju prelaziti kao pojedinačne stanice, ometajući kolektivnu migraciju [10]. Trodimenzionalni (3D) uvjeti kulture, temeljeni na ugrađivanju stanica u usklađeni biomaterijal, predstavljaju obećavajuću alternativu za istraživanje invazije i EMT [11, 12]. Konkretno, epitelne stanice mliječne žlijezde kultivirane u rekonstituiranoj bazalnoj membrani (tj. Matrigel) rekapituliraju diferenciranu strukturu nalik tkivu s lumenima i stanično-staničnim spojevima, koje se postupno dezorganiziraju i šire kao odgovor na aberantne signale mikro okoliša [13]. U novije vrijeme projektirani biomaterijali [14] i mikroproizvodni uređaji [15] osigurali su povećanu kontrolu nad krutošću materijala, razgradljivošću i arhitekturom. Ti kontrolirani uvjeti mikro okruženja mogu pružiti nove uvide u to kako interakcije stanica i matriksa posreduju u kolektivnim i individualnim fenotipima migracije.

Fenotipska heterogenost i plastičnost definiraju značajke stanica raka i ostaju izazovni za istraživanje pomoću skupnih analiza na krajnjim točkama [16]. Vjeruje se da se EMT rijetko javlja u maloj podpopulaciji stanica, što se može zanemariti bez opsežnih mjerenja pojedinačnih stanica. Snimanje živih stanica s visokom prostornom i vremenskom rezolucijom može omogućiti nove uvide u molekularnu i staničnu dinamiku tijekom invazije i EMT-a [17]. Na primjer, izbočine citoskeleta posebno su važne za usmjerenu migraciju, a stanice mogu znatno deformirati okolni ECM [18]. Zauzvrat, stanice mogu doživjeti značajne deformacije kako bi prešle ECM, što može biti olakšano sukladnijim citoskeletom. Nadalje se pretpostavljalo da su stanice raka znatno mekše od svojih netransformiranih kolega, osobito u kontekstu stanja sličnih stabljikama koje izražavaju vimentin [19]. Općenito, postoji povećan interes za podstaničnu razlučivost adhezija stanica i matriksa [20], kao i za kolektivno ponašanje posredovano spojevima stanica i stanica [21].

Ovdje pregledavamo najnovija dostignuća u EMT -u i citoskeletu u raku (osobito srednjim vlaknima) omogućenim biomimetičkim materijalima i tehnologijama mjerenja veće rezolucije. Usredotočujemo se na publikacije u posljednjih 5 godina i naglašavamo moguće veze između mehanobiologije unutarstaničnog citoskeleta i izvanstaničnog matriksa. Za opsežnije liječenje EMT -a u raku, upućujemo čitatelje na druge novije recenzije o ovoj temi [1,2,3, 7, 22]. Prvo dajemo kratki uvod u biokemiju i mehaniku citoskeleta, kao i operativne definicije za EMT. Zatim razmatramo ponašanje poput EMT-a tijekom kolektivne i individualne migracije na ravnu podlogu. Dalje razmatramo dinamiku vimentina i EMT u 2D jednoslojnoj i 3D matričnoj kulturi. We then address the effect of submicron topographies (“2.5D”) and compliant 3D matrix on migration and EMT. Finally, we provide our perspective on EMT and cancer metastasis, as well as future directions for the field.


The Cytoplasm

The cytoplasm comprises the contents of a cell between the plasma membrane and the nuclear envelope (a structure to be discussed shortly). It is made up of organelles suspended in the gel-like cytosol, the cytoskeleton, and various chemicals. Even though the cytoplasm consists of 70 to 80 percent water, it has a semi-solid consistency, which comes from the proteins within it. However, proteins are not the only organic molecules found in the cytoplasm. Glucose and other simple sugars, polysaccharides, amino acids, nucleic acids, fatty acids, and derivatives of glycerol are found there too. Ions of sodium, potassium, calcium, and many other elements are also dissolved in the cytoplasm. Many metabolic reactions, including protein synthesis, take place in the cytoplasm.


Pozadina

Mechanoreceptors contain compliant elements, termed “gating springs,” that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments they have not been identified molecularly or ultrastructurally.

Rezultati

We show that the filamentous structures that connect the plasma membrane to the microtubules are compliant structural elements in the mechanoreceptive organelle of fly campaniform receptors. These filaments colocalize with the ankyrin-repeat domain of the transient receptor potential (TRP) channel NOMPC. In addition, they resemble the purified ankyrin-repeat domain in size and shape. Most importantly, these filaments are nearly absent in nompC mutants and can be rescued by the nompC gen. Finally, mechanical modeling suggests that the filaments provide most of the compliance in the distal tip of the cell, thought to be the site of mechanotransduction.

Zaključci

Our results provide strong evidence that the ankyrin-repeat domains of NOMPC structurally contribute to the membrane-microtubule connecting filaments. These filaments, as the most compliant element in the distal tip, are therefore good candidates for the gating springs.


Rasprava

The flattened nucleus is a common feature of cultured cells, but the mechanisms by which it is flattened have remained obscure. There is mounting evidence that the cytoskeleton exerts forces on the nucleus to position it (20,46�). In this study, however, we show that as long as the cell was able to spread, inhibiting actomyosin forces, microtubule-based forces and intermediate filaments, as well as the LINC complex, did not prevent nuclear flattening. Remarkably, nuclear height correlated tightly with the degree of cell spreading. Independent of the type of cytoskeletal force perturbed, the nucleus is flat unless the perturbation prevents initial cell spreading, or rounds a spread cell.

This robust feature of nuclear shaping suggests that it is the dynamic deformation of the cell shape itself that causes nuclear flattening consistent with our previous results reporting reversible nuclear deformation caused by proximal cell protrusions in migrating cells (49). The fact that the nuclear apex collapses during the nuclear flattening, opening up a significant distance between the cell apex and the nuclear apex (on the order of a few microns), argues against the cell cortex directly compressing the nucleus downward. The near complete absence of apical actomyosin bundles argues against any explanation for flattening that requires a downward compressive force on the nuclear apex by large actomyosin bundles (see, for example, (50)). That apical fibers do not participate in the flattening process does not argue against later distortion of the nucleus by fully developed actomyosin bundles as reported by others (51).

Neither intermediate filaments nor microtubules are required for flattening. Finally, disruption of the LINC complex via KASH4 overexpression failed to prevent nuclear flattening. It slowed the rate of cell spreading, suggesting perhaps that a coupled nuclear-cytoskeleton is required for rapid F-actin polymerization, but it did not prevent flattening over longer times. Given that myosin activity, microtubules and vimentin intermediate filaments are not required, that the LINC complex is dispensable is perhaps not surprising. We have shown before that KASH4 overexpression results in rounded nuclear shapes in cells on polyacrylamide gels (52). This difference may be because of possibly different cell spreading dynamics on gels versus glass. We note that the cell spreading area in KASH4 cells was lower on gels, suggesting that the relationship between nuclear flattening and cell spreading is conserved on other types of surfaces.

Our computational model demonstrates that expansive/compressive stresses arising from movement of the cell boundaries and centripetal flow of cytoskeletal network from the cell membrane is sufficient to explain translation of the nucleus toward the substratum and subsequent flattening against the substratum. The fact that the experimentally observed flattening dynamics could be closely reproduced using one constitutive equation (Eq. 1), a simple model for cell mechanics, and one fitted parameter (va) provides strong support for the validity of the model assumptions. Moreover, the model successfully predicts a number of experimental findings: the approach to a steady-state flattened nuclear shape despite continued cell spreading, nuclear flattening in the absence of actomyosin tension, increased nuclear flattening in the absence of lamin A/C, and the cessation of nuclear flattening upon the cessation of cell spreading.

Consistent with the assumption that the nucleus is under tension, we found that the volume of the nucleus decreased in myosin-inhibited cells. However, our experiments show that flattening is not a consequence of tension. As explained by the computational model, flattening can instead arise from the motion of the cell boundary transmitting stresses to the nuclear surface because the intervening cytoskeletal network resists expansion or compression. As a result, the nuclear shape changes tend to mimic changes in cell shape during cell spreading.

Interestingly, the presence of actomyosin contraction in normal cells does not alter the dynamics of nuclear shape changes during cell spreading. In the presence of contraction, the net stress on the nuclear surface in the absence of any F-actin assembly from the membrane (such as in serum-starved cells) is likely tensile. However, even if this stress in the network is net compressive (such as when myosin is inhibited), the differential stresses between apex and sides of the nucleus that drive nuclear shape dynamics during cell spreading are predicted to be similar ( Fig.ꀐ C).

In summary, our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes. Our computational model suggests that nuclear shape changes result from transmission of stress from the moving cell boundary to the nuclear surface because of frictional resistance to expansion/compression of the intervening cytoskeletal network. Nuclear shaping are thus driven by cell shape changes.


Rasprava

The present results demonstrated that both CD and CB prevented fusion of nucleoli in activated mouse oocytes, suggesting that the microfilaments are essential for nucleolar fusion. Brangwynne et al. [ 28] showed that nucleoli in germinal vesicles (GVs) of Xenopus laevis oocytes were mostly isotropic in shape, much like liquid droplets, and that two or more of the spherical nucleoli often fused with each other. In CD-treated oocytes, however, nucleoli showed irregular shapes and did not relax to spheres, and fusion of these nucleoli was frequently incomplete. According to these authors, actin can act as a scaffold, giving both structure and stability to the nucleoli, and the disruption of actin leads to loss of liquid-like behavior. Further observations made by Feric and Brangwynne [ 29] showed that GVs contained an elastic F-actin scaffold that mechanically stabilized these large nuclei against gravitational forces, and upon actin disruption, nucleoli and histone locus bodies underwent gravitational sedimentation and fusion. Together, these data suggest that actin forms not only an intranucleolus scaffold that maintains a spherical shape of nucleoli but also an internucleoli scaffold that supports an active movement of the nucleoli both the spherical shape and the active movement of nucleoli are essential for their normal fusion. Furthermore, a number of reports have revealed nuclear localization of myosins as well as actin and actin-binding proteins [ 30], although their concentration is found lower than that in the cytoplasm [ 31]. The discovery of actin as a component of the transcription apparatus, chromatin-remodeling complexes, as well as RNA processing machines, implies important intranuclear roles for actin in the readout of genetic information [ 18].

This study demonstrated that whereas acrylamide inhibits nucleolar fusion efficiently, IDPN shows no effect when used at various concentrations (data not shown). IDPN is a neurotoxin that segregates neurofilaments from microtubules and retards neurofilament transport [ 32]. It is known that most types of intermediate filaments are cytoplasmic, but one type, the lamins, is nuclear [ 33]. A review of published papers indicated that whereas IDPN had been used mostly to destroy vimentin [ 34– 36], acrylamide was often used to disrupt not only vimentin [ 37] but also keratin [ 38]. Immunofluorescence microscopy revealed keratin but not vimentin in mouse oocytes and early embryos [ 39– 42]. Lamins were found on all pronuclei during mouse fertilization [ 43]. Furthermore, ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was associated with a significant decrease in the thickness of the nuclear lamina [ 13]. Taken together, we proposed that nuclear lamins might be the intermediate filaments that supported the fusion of nucleoli.

Newly ovulated mouse oocytes are arrested at the metaphase II stage and they resume meiosis, extrude the Pb2, and form pronuclei upon fertilization or activation stimulus. Verlhac et al. [ 44] reported that Pb2 began to extrude at about 45 min, pronuclei were clearly visible 4 h later, and 70% of the eggs reached the first mitotic metaphase by 14 h after ethanol activation of mouse oocytes. Our previous study [ 9] showed that small nucleoli began to take shape at about 4 h after activation stimulus, and it took about 1.5 h for them to fuse with each other into a single large nucleoli. The fused nucleoli (NPB) lasted for about 10 h before disappearance. The pronuclei appeared and disappeared at the same time as did the nucleoli. Furthermore, no difference in the fusion of nucleoli was observed between male and female pronuclei in fertilized zygotes. In the present study, when mouse oocytes were treated with CD or acrylamide for different times at different intervals after the onset of Sr 2+ activation, nucleolar fusion was successfully prevented only when the drugs were present from the second or third to fifth hour after the onset of Sr 2+ treatment. This drug-sensitive window fell well within the temporal window for nucleolar fusion in activated mouse oocytes reported by Li et al. [ 9]. Thus, the results further confirmed that both microfilaments and intermediate filaments were involved in the fusion of nucleoli.

The present results showed that although treatment of activated mouse oocytes with microtubule inhibitors demecolcine or nocodazole prevented pronuclear formation, it did not affect fusion of nucleoli, suggesting that microtubules are not involved in the regulation of nucleolar fusion. Despite the best efforts, we found no report on the presence of microtubules in the nucleus. According to a recent review by Simon and Wilson [ 45], the metazoan nucleoskeleton includes nuclear pore-linked filaments, A-type and B-type lamin intermediate filaments, nuclear mitotic apparatus networks, spectrins, titin, “unconventional” polymers of actin, and at least 10 different myosin and kinesin motors. It is known that microtubules function mainly in the cytoplasm, including the movement of secretory vesicles, organelles, metaphase chromosomes, and intracellular substances [ 46]. Although microtubules were found to influence gene expression, the effect was caused by their action within the cytoplasm. For example, although cold-induced depolymerization of microtubules led to I kappa B degradation and activation of NF-kappa B, the activated factor remained in the cytoplasm and translocated to the nucleus only upon warming to 37°C [ 47].

In the present study, whereas continuous incubation with demecolcine prevented pronuclear formation, normal pronuclei formed when demecolcine was excluded during the first hour of activation treatment. Xue et al. [ 48] also observed that the influence of microtubules on nuclear assembly was restricted to a limited time window. Thus, if nocodazole treatment was delayed by 30 min, microtubule depolymerization no longer rescued the effects of dppa2 depletion on nuclear shape. Although microtubule assembly was found essential for the formation of the male and female pronuclei during mouse but not sea urchin fertilization [ 22], the mechanisms are not known. Our Western analysis at 1 h of activation treatment showed that whereas the MPF activity dropped dramatically in the absence of demecolcine, it remained high in the presence of demecolcine. Previous studies had also observed a dramatic drop of MPF activity within 1 h after regular activation of mouse oocytes [ 9, 24, 25]. Taken together, the present results suggested that microtubule disruption prevented pronuclear formation in activated oocytes by maintaining their MPF activity. Many studies have demonstrated that defects in spindle assembly or spindle kinetochore attachment, or artificial depolymerization of microtubules, activate the SAC proteins such as MAD2 and BUB1, which arrest cells prior to the metaphase-anaphase transition with stable cyclin B and elevated MPF activities [ 49– 51]. Further studies have confirmed that a complex between the anaphase-promoting complexes/cyclosome (APC), Cdc20, and SAC proteins renders APC inactive and thus activates MPF by preventing cyclin B proteolysis [ 52– 55].

Although it is known that protein synthesis inhibition does not preclude pronuclear formation (CHX is often used in some protocols for chemical activation of oocytes), whether it affects nucleolar fusion is not known. In this study, neither rate of pronuclear formation nor rate of nucleolar fusion in activated oocytes was affected by the presence of CHX, although oocyte maturation was efficiently blocked after the same CHX treatment. This suggested that proteins required for nucleolar fusion including the microfilaments and intermediate filaments were made during oocyte maturation but not de novo synthesized after activation. It is known that following fertilization the early embryo is essentially transcriptionally silent and the early development is directed by the complement of maternally inherited mRNAs and proteins [ 56]. Because CHX inhibits protein biosynthesis by blocking translational elongation, our results suggest that the microfilaments and intermediate filaments essential for nucleolar fusion in activated oocytes were ready-made proteins that had been translated and stored before ovulation. Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice [ 57]. Arnault et al. [ 58] reported the presence of lamin during oogenesis and in isolated mouse oocytes.

Our observation on embryo development showed that whereas activated oocytes developed into blastocysts following CD treatment, the acrylamide-treated oocytes could not cleave, although both drugs prevent nucleolar fusion efficiently. It has been reported that slow freezing destroys intermediate filaments in mouse oocytes, leading to failure of embryo cleavage [ 59]. In addition, the result that oocytes developed into blastocysts following CD inhibition of nucleolar fusion seems contradictory with reports that the number, position, and distribution of nucleoli in human pronuclei can be used as an indicator of embryonic developmental potential [ 5, 10– 12]. However, this discrepancy may be caused by species difference, because mouse embryos are well known to be much more tolerant of in vitro culture than embryos from other species. Furthermore, whether inhibition of nucleolar fusion would affect mouse postimplantation development remains to be determined.

In summary, we have studied the role of microfilaments, microtubules, and intermediate filaments in regulating the formation and fusion of nucleoli during the M/G1 transition using the activated mouse oocyte model. Results (summarized in Table 9) suggest that 1) microfilaments and intermediate filaments but not microtubules are essential for nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized in activated oocytes, and 3) microtubule disruption prevents pronuclear formation by maintaining high MPF activities. To our knowledge, this is the first report on the relationship between cytoskeleton and nucleolar events, which may evoke further work and provide a new avenue of investigation on nucleoskeleton and nucleologenesis. Understanding the assembly behavior of nucleoli, and what regulates their size and shape, is key to understanding the underlying causes of some human diseases [ 60] and to providing a marker for embryo developmental potential.


UVOD

Microtubule (MT) filaments are dynamically self-organized, semiflexible polymers that permeate the cell interior, supporting numerous cellular functions and constituting major determinants of cell morphological, signaling, and mechanical properties ( Alberts etਊl., 2002 Dumont and Mitchison, 2009 Pollard and Cooper, 2009 Fletcher and Mullins, 2010 Robison etਊl., 2016.). However, although the molecular aspects of MT functions have been intensively studied, how the global MT networks collectively contribute to the physical and biochemical attributes of the cells has not been fully understood ( Karsenti etਊl., 2006 Ando etਊl., 2015 ). Nonetheless, the potential importance of the latter is supported by emerging examples in which the physical properties of MTs directly participate in cellular physiology in a highly regulated manner ( Schaedel etਊl., 2015 Robison etਊl., 2016 ).

The architecture of the MT network is subject to complex regulatory mechanisms, interdependent with cell shape and responsive to several biochemical factors, including the classical MT-associated proteins, motor and severing proteins, MT end𠄻inding proteins ( Desai and Mitchison, 1997 Akhmanova and Steinmetz, 2015 Alfaro-Aco and Petry, 2015 ), and posttranslational modifications ( Song and Brady, 2015 ). With a subdiffraction-limit (�-nm) diameter but multimicrometer cell-spanning lengths, the complexity of the MT networks imposes major challenges for their experimental characterization and quantitative analysis ( Shariff etਊl., 2010 ). In particular, except for sparse regions near the periphery, traces of the MTs are difficult to resolve by conventional diffraction-limited fluorescence microscopy. Fortunately, the recent development of superresolution microscopy methods has enabled optical imaging with precision comparable to the MT dimension ( Kanchanawong and Waterman, 2012 ). Single-molecule localization microscopy (SMLM) methods, such as fluorescence/photoactivated localization microscopy and stochastic optical reconstruction microscopy (STORM Betzig etਊl., 2006 Hess etਊl., 2006 Rust etਊl., 2006 Fölling etਊl., 2008 Heilemann etਊl., 2008 ), have been widely used to image MTs either on their own or in conjunction with other cellular organelles, yielding highly resolved and information-rich images ( Huang etਊl., 2008a Dempsey etਊl., 2011 ). However, quantitative analysis of such superresolved images is difficult. The large-scale nature of single-molecule localization data makes it challenging for individual researchers to perform analysis and annotation in a timely manner, and thus there is a need for a tool to enable comprehensive extraction and reconstruction of the complete filament networks.

Here we present an open-source software package called SIFNE (for “SMLM image filament network extractor”), which provides a highly customizable computational tool for the global analysis of whole-cell MT networks imaged by SMLM. Our computational strategy involves two major stages: first, the iterative extraction of the global filament networks from the empirical data sets, and subsequently, the identification and assignment of every detected filament. Postextraction analysis tools for quantification of the MT network properties are also provided. Owing to the SMLM-resolvable dimension of the MTs and an unbranched filament topology, we also rigorously assessed the performance of our method by manual validation. We used the examples of the network architecture phenotypes promoted by the small GTPase Rac1 to demonstrate how SIFNE analysis can be used to quantitatively distinguish between different network architectures in fibroblasts.


1. Uvod

Photodynamic therapy (PDT) has emerged as a minimally invasive regimen for the treatment of cancers, offers an attractive alternative or complement to conventional therapies [1 2 3]. The therapeutic action of PDT is based on the generation of reactive oxygen species (ROS) that are formed upon specific wavelength-light activation of a photosensitizer (PS) such as a porphyrinic pigment. For clinical PDT applications, light in the red and infrared range is generally employed for enhanced tissue penetration. The resulting excited PS transfers its energy to molecular oxygen in tissue to generate ROS. Singlet oxygen is assumed to be the key cytotoxic ROS responsible for localized oxidative cell damage and initiation of cell death [1 3 4].

PDT for prostate cancer has not yet advanced beyond clinical trials and consequently is not yet an integral part of clinical practice [5]. The successful practice of PDT requires both the delivery of a sufficient light flux to the entire prostate and the adequate accumulation of photosensiter in tumors. While Hahn's group has developed procedures to measure and optimize light source parameters for prostate PDT [6 7 8], it is expected that improved targeting delivery and accumulation of photosensitizer molecules in prostate tumor may be additionally advantageous. Our group recently has developed a method for the selective delivery of PSs by targeting the enzyme-biomarker prostatespecific membrane antigen (PSMA) [9 10]. PSMA is a type II cell-surface glycoprotein predominantly restricted to prostatic tissue and is strongly expressed on prostate tumor cells [11]. Expression levels increase with disease progression, being highest in metastatic disease, hormone refractory cancers, and higher-grade lesions [12 13]. Endothelial-expression of PSMA in the neovasculature of a variety of non-prostatic solid malignancies has also been detected. Therefore, PSMA has attracted considerable attention as a biomarker and target for the delivery of imaging and therapeutic agents.

Microfilaments, microtubules, and intermediate filaments form the cytoskeleton systems of vertebrate cells. These fibrillar networks are composed of distinctly different proteins and exhibit unique structural and functional characteristics. The dynamic cytoskeletal networks that they form as well as their crosstalk play a critical role in the maintenance of cellular morphology and membrane integrity. They are also involved in cellular processes such as cell division, adhesion and migration, intracellular transport, and apoptosis [14 15]. Cytokeratin filaments have been found to aggregate at an early stage of the apoptotic sequence, while at later stages cytokeratin is degraded [16 17]. Reorganization of the microfilament and microtubule cytoskeleton has also been observed during the execution phase of apoptosis [18]. Cleavage of α-tubulin [19], cytokeratin 18 [20 21], and actin [22] by caspases during apoptosis has also been reported. With respect to PDT, there are characteristic changes in the cytoskeletal networks induced when phthalocyanines or 5-aminolevulinic acid are used as the PS [23 24 25 26].

We previously reported that phosphoramidate peptidomimetic PSMA inhibitors were capable of both cell-surface labeling of prostate cancer cells and intracellular delivery for targeted PDT applications [9 10]. Apoptosis of prostate cancer cells following PSMA-targeted PDT with Ppa-CTT-54 ( Fig. 1 ) was confirmed by the appearance of early chromatin condensation, poly(ADP-ribose) polymerase (PARP) p85 fragment, and DNA fragmentation [10]. In this present study, we attempted to delineate the molecular mechanism by which PSMA-targeted PDT induces apoptosis and concluded that disruption of three kinds of filamentous networks and degeneration of cytoskeletal components occur at the initiation sequence of cell death or apoptosis.

Structures of Ppa and Ppa-CTT-54.


Metode

Purification of Tubulin and Tau

Tubulin was purified from MAP-rich microtubules extracted from bovine brains. MAP-rich microtubules were obtained from crude brain extract by three polymerization/depolymerization cycles, after which tubulin was separated from MAPs with a phosphocellulose anionic exchange column. Tubulin was suspended in PEM50 (50 mM PIPES (pH 6.8), 1 mM MgSO4 and 1 mM EGTA) with protein concentration between 7 and 12 mg ml 𢄡 , as measured by bovine serum albumin concentration standard. Solution was drop-frozen in liquid nitrogen and stored in a � ଌ freezer until use.

Tau was expressed in BL21(DE3) competent cells (Life Technologies, Carlsbad, CA) that were transfected with the pRK172 expression vector, coded for the appropriate WT isoform or truncated Tau. After incubation in auto-induction media (10 g of tryptone (CAS#: 91079�𠄲), 5 g of yeast extract (CAS#: 8013�𠄲), 0.5 g of dextrose (CAS#: 50�𠄷), 2 g of α- D -lactose (CAS#: 5989�𠄱) and 5 ml of glycerol (CAS#: 56�𠄵) per litre of 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl and 5 mM Na2TAKO4 in deionized (DI) water) for 24 h, cells were collected, lysed and resuspended in BRB80 buffer (80 mM PIPES at pH 6.8, 1 mM EGTA and 1 mM MgSO4). The solution was then bound to a phosphocellulose anionic exchange column, eluted with increasing concentration of (NH4)2TAKO4 in BRB80. Tau was further purified by a HiTrap hydrophobic interaction chromatography column (GE Healthcare Life Sciences, Pittsburgh, PA), eluted with decreasing concentration of (NH4)2TAKO4 in BRB80. Tau was then concentrated and buffer exchanged through successive centrifugation cycles using Amicon Ultra-15 Centrifugal Units with MWCO=10,000 (EMD Millipore, Darmstadt, Germany). The concentration of each Tau stock was determined by SDS–polyacrylamide gel electrophoresis comparison with a Tau mass standard (originally measured via amino-acid analysis).

Truncated Tau mutants were designed via the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with appropriate introduction/deletion of start and stop codons: 3RS㥌 (truncation of the entire CTT, deleting residues 280� of 3RS), 3RΔ(N-) (truncation of the anionic component of the NTT, deleting residues 2� of 3RL) and 3RΔN (truncation of the entire NTT, deleting residues 2� of 3RL). Truncated Tau mutants were then expressed/purified, as above.

Priprema uzorka

After thawing frozen tubulin and Tau stocks, samples were prepared on ice, mixing tubulin, GTP and Tau such that final concentrations were 5 mg ml 𢄡 , 2 mM and appropriate molar ratio of Tau to tubulin, respectively, in a final volume of 50 μl of PEM50 buffer. Samples were then polymerized in a 37 ଌ for 40 min. If necessary, sample was brought to appropriate KCl concentration.

Osmotic pressure samples

A previous study 34 measured the osmotic pressure (in Pa), P, of an aqueous solution of varying concentrations (cg ml 𢄡 ), wt%, of poly(ethylene oxide) (MW=105,000 g mol 𢄡 ) at 35 ଌ, which was taken as an reasonable approximation of the behaviour of PEO-100k at 37 ଌ, absent further data. Data were fit to a second-order polynomial (following the mathematical form of a virial expansion) to determine a formula to relate an arbitrary PEO-100k concentration to a corresponding osmotic pressure (P, in Pa):

Alternatively, following a derivation in Rau et al. 35 , the osmotic pressure (P) on cylinders in a hexagonal lattice can be converted to a force per unit length between nearest cylinder pairs f as a function of the hexagonal lattice parameter aH:

PEO-100k was used as the osmotic depletant of choice compared with better-characterized depletants to parameters unique to our system: as stable inter-microtubule distances of up to 41 nm were observed, the size of the depletant had to be equal or greater than that distance to create a concentration differential inside/outside the microtubule bundle. Prior work 36 measured the radius of gyration (RG) of a function of PEO molecular weight (MW):

Thus, the effective depletant radius 21 , a=2RGπ 𢄡/2 =19.95 nm, or an effective depletant diameter, d� nm, satisfies our experimental conditions that polymer not penetrate the space between microtubules in microtubule bundles.

Small-angle X-ray scattering

After polymerization, samples are loaded into 1.5-mm diameter quartz mark tubes (Hilgenberg GmbH, Malsfeld, Germany) and subsequently spun in a capillary rotor in a Universal 320R centrifuge (Hettich, Kirchlengern, Germany) at 9,500g, 37 ଌ for 30 min to protein density suitable for synchrotron SAXS. To ensure that structures were not induced by centrifugation, samples were observed over a period of 36 h, with no major changes to scattering or extracted parameters (Supplementary Fig. 1).

After centrifugation, varying concentration of PEO-100k in PEM50 was added for SAXS samples under osmotic pressure. Samples are subsequently sealed with epoxy and placed in a custom-made sample oven (maintained at 37 ଌ) with X-ray-transparent Kapton windows for scattering measurements.

SAXS measurements are carried out at the Stanford Synchrotron Radiation Laboratory (Palo Alto, CA) beamline 4𠄲 at 9 KeV (λ=1.3776 Å) with a Si(111) monochromator. Scattering data are taken with a 2D area detector (MarUSA, Evanston, Illinois) with a sample to detector distance of 𢒃.5 m (calibrated with a silver behenate control). X-ray beam size on the sample was 150 μm in the vertical and 200 μm in the horizontal directions. To ensure reproducibility, scattering data were retaken for most samples at similar sample conditions using tubulin and Tau from different purifications and expressions/purifications, respectively.

SAXS analysis

Scattering data were azimuthally averaged and small-angle scattering was subsequently background subtracted by fitting the minima of scattering intensities to a polynomial equation. Data were then fit to the appropriate model using a custom MATLAB fitting routine using the Levenberg–Marquadt non-linear fitting routine. Microtubules were modelled as homogenous, hollow cylinders (with no expected scattering from Tau/PEO due to low electron density relative to water) with ensemble-averaged inner radius <ru> (a fit parameter), wall thickness δ (49 Å, an input parameter 18 ) and microtubule length L (20 μm, an input parameter for Tau-stabilized microtubules 16 ), averaged all orientations in q-space:

Gdje q˙ i qz are wavevectors perpendicular and parallel to the tubular axis, and J1 is the Bessel function of order 1. The structure-factor peaks (at reciprocal lattice vector for a hexagonal array, |Ghk|=q10(h 2 +k 2 +hk) 1/2 ) were modelled as squared lorentzians with peak amplitude Ahk (a fit parameter) and peak width κhk (a fit parameter, with κ10 corresponding to the average bundle width L𢒂(πln4) 1/2 /κ10) 13 :

Plastic-embedded TEM sample preparation and TEM

Samples for thin sections were centrifuged to a pellet at 9,500g in 37 ଌ for 30 min. Supernatant was removed and pellet fixed with 2% glutaraldehyde and 4% tannic acid overnight. The pellet was stained with 0.8% OsO4 in PEM50 buffer for 1 h and subsequently rinsed four times with PEM50. Another stain of 1% uranyl acetate stain was applied for 1 h and rinsed with DI water.

Fixed and stained pellets were subsequently dehydrated with 25/50/75/100% solutions of acetone in DI water for 15 min apiece. Samples were embedded in resin, then embedded in spur plastic and incubated overnight, with resin poured into flat embedding moulds and held at 65 ଌ for 48 h and cooled overnight.

Plastic-embedded samples were then cut to �-nm slices with a microtome (Ted Pella, Redding, CA) and transferred to highly stable Formvar carbon-coated copper EM grids (Ted Pella, Redding, CA). Data were taken using the JEOL 1230 Transmission Electron Microscope.

DIC Samples and DIC

A SensiCam CCD camera (Cooke, Auburn Hills, MI) mounted on a Nikon Diaphot 300 with Xenon lamp (Sutter Instrument, Novato, CA) was used for optical microscopy measurements. Samples were centrifuged to a pellet at 9,500g for 30 min in 37 ଌ and placed between two microscopic slides sealed by wax. Images were taken while slides were kept at 37 ଌ by heat stage.

Calculation of R G

Previously, the radius of gyration (RG) of WT Tau and truncated Tau domains in solution were found 20 ,37 to scale as an unstructured protein with random-coil behaviour, with RG=0.1927N 0.588  nm, which was subsequently used to calculate the RG of the PD and truncated Tau used in our experiments.

Dostupnost podataka

The authors declare that the data supporting the findings of this study are available within the article, and its Supplementary Information files, or from the authors on reasonable request.


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