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Exotické tvary buniek

Exotické tvary buniek


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Pokiaľ viem, tvary rastlinných buniek je ťažké určiť. Rastlinné bunky však majú bunkové steny, a preto môžu byť veľmi tuhé. Avšak jediné rastlinné bunky, ktoré som videl, boli buď v tvare bloku, alebo v tvare trubice. Existujú exotickejšie príklady tvarov buniek, najmä v rastlinách? Použili niekedy rastliny napríklad plástové vzory alebo iné formy mozaikovania?


Bunky chodníka by vám mali vyhovovať:

Z http://dev.biologists.org/content/131/21/5215.full


25 najznámejších exotických kvetov

Exotické kvety: Podľa definície exotické rastliny sú rastliny, ktoré boli zavlečené do regiónu alebo ekosystému, kde sú cudzie alebo nepôvodné.

Častejšie sa tieto druhy rastlín stávajú inváznymi a dominujú na mieste. Predpokladá sa, že táto tendencia spôsobuje bezprostredné poškodenie ich susedných rastlín a zvierat, ako aj životné prostredie.

Vedeli ste, že mnohé známe pestované okrasné rastliny sú exotické?


Bunková biológia Ch.7

Táto postupná amplifikácia umožňuje bunkám rýchlo syntetizovať veľké množstvá proteínu, kedykoľvek je to potrebné. Zároveň môže byť každý gén transkribovaný a jeho RNA translatovaná rôznymi rýchlosťami, čo bunke poskytuje spôsob, ako vytvoriť obrovské množstvá niektorých proteínov a malé množstvá iných.

(B) RNA obsahuje bázu uracil, ktorá sa líši od tymínu, ekvivalentnej bázy v DNA, absenciou -CH3 skupiny.

• V eukaryotoch každá mRNA typicky nesie informáciu prepísanú len z jedného génu, ktorý kóduje jeden proteín v baktériách, súbor susedných génov sa často prepisuje ako jedna mRNA, ktorá teda nesie informáciu pre niekoľko rôznych proteínov.

Iniciácia transkripcie je obzvlášť kritický proces, pretože je to hlavný bod, v ktorom bunka vyberá, ktoré proteíny alebo RNA sa majú produkovať.
• Na začatie transkripcie musí byť RNA polymeráza schopná rozpoznať začiatok génu a pevne sa naviazať na DNA v tomto mieste.

• Spôsob, akým RNA polymerázy rozpoznávajú miesto začiatku transkripcie génu, sa medzi baktériami a eukaryotmi trochu líši.

2. RNA polymeráza sa pevne prichytí až potom, čo sa stretne s génovou oblasťou nazývanou promótor, ktorá obsahuje špecifickú sekvenciu nukleotidov, ktorá leží bezprostredne pred východiskovým bodom syntézy RNA.

3. Akonáhle je RNA polymeráza pevne naviazaná na túto sekvenciu, otvorí dvojitú špirálu bezprostredne pred promótorom, aby obnažila nukleotidy na každom vlákne krátkeho úseku DNA.

4. Jedno z dvoch odkrytých reťazcov DNA potom pôsobí ako templát pre komplementárne párovanie báz s prichádzajúcimi ribonukleozidtrifosfátmi, z ktorých dva sú spojené polymerázou, aby sa začala syntéza reťazca RNA.

5. Predlžovanie reťazca potom pokračuje, kým enzým nenarazí na druhý signál v DNA, terminátor (alebo stop miesto), kde sa polymeráza zastaví a uvoľní tak DNA templát, ako aj novo vytvorený RNA transkript. Táto terminátorová sekvencia je obsiahnutá v géne a je transkribovaná do 3. konca novovytvorenej RNA.

2. Druhým rozdielom je, že kým bakteriálna RNA polymeráza (spolu so svojou sigma podjednotkou) je schopná iniciovať transkripciu sama, eukaryotické RNA polymerázy vyžadujú asistenciu veľkého súboru doplnkových proteínov. Hlavnými z nich sú všeobecné transkripčné faktory, ktoré sa musia zhromaždiť v každom promótore spolu s polymerázou predtým, ako môže polymeráza začať transkripciu.

3. Treťou charakteristickou črtou transkripcie u eukaryotov je, že mechanizmy, ktoré riadia jej iniciáciu, sú oveľa prepracovanejšie ako u prokaryotov. V baktériách majú gény tendenciu ležať v DNA veľmi blízko seba, pričom medzi nimi sú len veľmi krátke dĺžky neprepisovanej DNA. Ale v rastlinách a zvieratách, vrátane ľudí, sú jednotlivé gény rozložené pozdĺž DNA. Táto architektúra umožňuje, aby bol jeden gén riadený veľkým množstvom regulačných sekvencií DNA rozptýlených pozdĺž DNA a umožňuje eukaryotom zapojiť sa do zložitejších foriem transkripčnej regulácie ako baktérie.

RNA polymeráza II – prepisuje všetky gény kódujúce proteín, gény miRNA plus gény pre iné nekódujúce RNA (napr. tie v spliceozómoch)

(A) Mnohé eukaryotické promótory obsahujú sekvenciu DNA nazývanú TATA box.

(B) TATA box je rozpoznávaný podjednotkou všeobecného transkripčného faktora TFIID, nazývaného TATA-binding protein (TBP).

(C) Väzba TFIID umožňuje susednú väzbu TFIIB.

(D) Zvyšok všeobecných transkripčných faktorov, ako aj samotná RNA polymeráza, sa zostavuje v promótore.

1. Polymeráza prepisuje DNA na RNA.

2. Polymeráza nesie aj proteíny na spracovanie RNA, ktoré pôsobia na novovytvorenú RNA.

3. Proteíny sa viažu na koniec RNA polymerázy, keď je fosforylovaný neskoro v procese iniciácie transkripcie.

• Vzdialenosti pozdĺž RNA medzi tromi zostrihovými sekvenciami sú veľmi variabilné, avšak vzdialenosť medzi bodom vetvenia a spojom 5 spojov je zvyčajne oveľa väčšia ako vzdialenosť medzi spojom troch spojov a bodom vetvenia.

• SnRNP rozpoznávajú sekvencie miesta zostrihu prostredníctvom komplementárneho párovania báz medzi ich zložkami RNA a sekvenciami v pre-mRNA a tiež sa úzko zúčastňujú na chémii zostrihu.

• Zostrih RNA tiež poskytuje eukaryotom ďalšiu výhodu, ktorá bola pravdepodobne veľmi dôležitá v ranej evolučnej histórii génov.

60 % ľudských génov podlieha alternatívnemu zostrihu.
o Zostrih RNA umožňuje zvýšiť potenciál kódovania ich genómov.
• Existuje päť snRNP, ktoré sa nazývajú U1, U2, U4, U5 a U6

1. U1 a U2 sa viažu na 5' miesto zostrihu (U1) a bod vetvenia lariatu (U2) prostredníctvom komplementárneho párovania báz.

2. Ďalšie snRP sú priťahované k miestu zostrihu a interakcie medzi ich proteínovými zložkami riadia zostavenie kompletného zostrihu.

• Videli sme, ako prebieha syntéza a spracovanie eukaryotickej pre-mRNA usporiadaným spôsobom v bunkovom jadre. Tieto udalosti však vytvárajú špeciálny problém pre eukaryotické bunky: z celkového počtu pre-mRNA transkriptov, ktoré sú syntetizované, bude pre bunku užitočná iba malá časť - zrelé mRNA.
o Zostávajúce fragmenty RNA – vyrezané intróny, rozbité RNA a aberantne zostrihané transkripty – sú nielen zbytočné, ale ak by sa im umožnilo opustiť jadro, mohli by byť pre bunku nebezpečné.

• Transport mRNA z jadra do cytosolu, kde sa mRNA prekladajú na proteín, je vysoko selektívny: exportujú sa iba správne spracované mRNA. Tento selektívny transport je sprostredkovaný komplexmi jadrových pórov, ktoré spájajú nukleoplazmu s cytozolom a pôsobia ako brány, ktoré kontrolujú, ktoré makromolekuly môžu vstúpiť do jadra alebo z neho vystúpiť.

o Aby bola molekula mRNA pripravená na "export", musí byť naviazaná na vhodný súbor proteínov, z ktorých každý rozpoznáva rôzne časti zrelej molekuly mRNA. Tieto proteíny zahŕňajú poly-A-viažuce proteíny, komplex viažuci čiapočku a proteíny, ktoré sa viažu na mRNA, ktoré boli vhodne zostrihané.

• O tom, či molekula mRNA opustí jadro, rozhoduje skôr celý súbor naviazaných proteínov než akýkoľvek jednotlivý proteín. "Odpadové RNA", ktoré zostanú v jadre, sa tam degradujú a ich nukleotidové stavebné bloky sa znova použijú na transkripciu.

2. V kroku 2 sa karboxylový koniec polypeptidového reťazca (aminokyselina 3 v kroku 1) odpojí od tRNA v mieste P a pripojí sa peptidovou väzbou k voľnej aminoskupine aminokyseliny pripojenej k tRNA v mieste P. stránka A. Táto reakcia je katalyzovaná enzymatickým miestom vo veľkej podjednotke.

3. V kroku 3 posun veľkej podjednotky vzhľadom na malú podjednotku presunie dve tRNA do miest E a P veľkej podjednotky.

4. V kroku 4 sa malá podjednotka presunie presne o tri nukleotidy pozdĺž molekuly mRNA, čím ju vráti späť do pôvodnej polohy vzhľadom na veľkú podjednotku. Tento pohyb vysunie vyčerpanú tRNA a resetuje ribozóm s prázdnym miestom A, aby sa mohla viazať ďalšia nabitá molekula tRNA. Ako je uvedené, mRNA sa translatuje v smere 5 až 3 a najskôr sa vytvorí N-terminálny koniec proteínu, pričom v každom cykle sa pridá jedna aminokyselina na C-koniec polypeptidového reťazca. Ak chcete sledovať cyklus prekladu v atómových detailoch, viď.
• V danom čase sú obsadené iba dve lokality.

• rRNA sú poskladané do vysoko kompaktných, presných trojrozmerných štruktúr, ktoré tvoria jadro ribozómu (obrázok 7-35). Na rozdiel od centrálneho umiestnenia rRNA sú ribozomálne proteíny vo všeobecnosti umiestnené na povrchu, kde vyplňujú medzery a štrbiny poskladanej RNA. Zdá sa, že hlavnou úlohou ribozomálnych proteínov je pomáhať skladať a stabilizovať jadro RNA, pričom umožňujú zmeny v konformácii rRNA, ktoré sú potrebné na to, aby táto RNA katalyzovala účinnú syntézu proteínov.

• Nielenže sú tri miesta viazania tRNA (miesta A, P a E) na ribozóme tvorené primárne rRNA, ale katalytické miesto pre tvorbu peptidovej väzby je tvorené 23S rRNA veľkej podjednotky blízkej ribozomálny proteín je umiestnený príliš ďaleko na to, aby sa dostal do kontaktu s prichádzajúcou nabitou tRNA alebo s rastúcim polypeptidovým reťazcom. Katalytické miesto v tejto rRNA – peptidyltransferáze – je v mnohých ohľadoch podobné tomu, ktoré sa nachádza v niektorých proteínových enzýmoch: je to vysoko štruktúrovaná kapsa, ktorá presne orientuje dva reaktanty – predlžujúci sa polypeptid a nabitú tRNA – čím sa výrazne zvyšuje. pravdepodobnosť produktívnej reakcie.

• Iniciátor tRNA spojený s metionínom je naložený na malom
ribozomálna podjednotka + proteíny = faktory iniciácie translácie.
• Len nabitý iniciátor tRNA je schopný tesne sa viazať
P-miesto malej ribozomálnej podjednotky.
• Potom sa viaže na 5' koniec mRNA, čo signalizuje prítomný uzáver
mRNA.
• Pohybuje sa dopredu (5' až 3') pozdĺž mRNA a hľadá prvý AUG.
• Niekoľko iniciačných faktorov disociuje z malej ribozomálnej podjednotky
aby sa uvoľnilo miesto na zostavenie veľkej ribozomálnej podjednotky.
• Syntéza bielkovín začína pridaním ďalšej nabitej tRNA
na A-stránku.

• Translácia mRNA začína kodónom AUG a na spustenie translácie je potrebná špeciálne nabitá tRNA. Tento iniciátor tRNA vždy nesie aminokyselinu metionín (alebo modifikovanú formu metionínu, formyl-metionín, v baktériách). Všetky novo vyrobené proteíny teda majú metionín ako prvú aminokyselinu na svojom N-konci, konci proteínu, ktorý sa syntetizuje ako prvý. Tento metionín je zvyčajne neskôr odstránený špecifickou proteázou.

• V eukaryotoch sa iniciačná tRNA nabitá metionínom najskôr vloží do P miesta malej ribozomálnej podjednotky spolu s ďalšími proteínmi nazývanými translačné iniciačné faktory, 5. koniec molekuly mRNA, ktorý je označený 5 čiapočkou, ktorá je prítomný na všetkých eukaryotických mRNA.

• Malá ribozomálna podjednotka sa potom pohybuje dopredu (5 až 3) pozdĺž mRNA a hľadá prvý AUG. Keď sa s týmto AUG stretne a rozpozná ho iniciačná tRNA, niekoľko iniciačných faktorov sa oddelí od malej ribozomálnej podjednotky, aby uvoľnilo cestu veľkej ribozomálnej podjednotke, aby sa naviazala a dokončila ribozomálne zostavenie.

• Pretože iniciačná tRNA je naviazaná na miesto P, syntéza proteínov je pripravená začať pridaním ďalšej nabitej tRNA do miesta A.

• Mechanizmus výberu štartovacieho kodónu je u baktérií odlišný. Bakteriálne mRNA nemajú 5 uzáverov, ktoré by ribozómu povedali, kde má začať hľadať začiatok translácie. Namiesto toho obsahujú špecifické sekvencie viažuce ribozómy, dlhé až šesť nukleotidov, ktoré sa nachádzajú niekoľko nukleotidov pred AUG, v ktorých má translácia začať. Na rozdiel od eukaryotického ribozómu sa prokaryotický ribozóm môže ľahko viazať priamo na štartovací kodón, ktorý leží vo vnútri mRNA, pokiaľ ho väzbové miesto pre ribozóm predchádza o niekoľko nukleotidov. Takéto sekvencie viažuce ribozómy sú nevyhnutné v baktériách, pretože prokaryotické mRNA sú často polycistronické – to znamená, že kódujú niekoľko rôznych proteínov, z ktorých každý je preložený z rovnakej molekuly mRNA. Na rozdiel od toho eukaryotická mRNA zvyčajne nesie informáciu pre jeden proteín.
o Baktérie používajú sekvenciu Shine-Delgarno (


Časť 3: Myozín a aktín Steer Plant Cell Division

00:00:06.00 Ahoj. Volám sa Magdaléna Bezanilla a som
00:00:09 z University of Massachusetts at
00:00:11:00 Amherst. A dnes vám to poviem
00:00:13.00 príbeh o priesečníku medzi
00:00:16.00 dva cytoskelety a ako fungujú
00:00:20,00 spoločne, aby pomohli umiestniť bunkové delenie
00:00:22.00 lietadlo v rastlinách. Takže umiestnenie bunky
00:00:26.00 rovina divízie je veľmi dôležitá, pretože
00:00:28.00 nakoniec určuje tvar
00:00:30,00 bunka. A tak toto je v podstate an
00:00:33.00 záujem o laboratórium, pretože moje laboratórium
00:00:35.00 študuje molekulárny základ bunky
00:00:37,00 tvar. Takže tento film, pre ktorý hrám
00:00:40, tu je film cytoskeletu
00:00:45.00 pod plazmatickou membránou a
00:00:48.00 vláknitá bunka v machu
00:00:51.00 Physcomitrella patens, čo je organizmus
00:00:53.00 na ktorom pracuje moje laboratórium. A v červenom vidíš
00:00:56.00 mikrotubuly a v zelenej farbe vidíte aktín
00:00:58.00 vlákna a čo chcem, aby ste si vzali
00:00:59.00 ďaleko od tohto filmu je len ako
00:01:01.00 krásne komplexné a integrálne
00:01:04.00 cytoskelet je a aký je dynamický,
00:01:06 a že sa neustále prerába.
00:01:10.00 Takže najprv vám musím trochu povedať
00:01:11.00 o tom, ako sa delia rastlinné bunky. Oni
00:01:14.00 deliť pomocou cytoskeletu a
00:01:18 ich rozdelenie je trochu iné
00:01:19 zo zvieracích buniek. Takže v mnohých rastlinných bunkách
00:01:23.00 existuje mikrotubulová štruktúra nazývaná
00:01:25.00 predprofázové pásmo, ktorým je a
00:01:27.00 štruktúra mikrotubulov vpravo
00:01:29.00 pod plazmatickou membránou. A čo
00:01:32.00 to znamená, že to v podstate znamená
00:01:35.00 bunková kôra a hovorí, toto je miesto
00:01:38.00 nová rovina bunkového delenia by mala
00:01:40,00 prísť. To je predprofázová kapela.
00:01:44.00 A potom, ako mitóza postupuje, a jadro
00:01:47.00 obálka sa rozpadne, mikrotubuly sú v nej
00:01:51.00 predprofázový pás rozobrať a
00:01:53.00 sa stávajú súčasťou mitotického vretienka.
00:01:55.00 Ako tu môžete vidieť, toto je vreteno
00:01:58.00 metafáza, kde sú všetky chromozómy
00:02:00,00 zarovnané pozdĺž metafázovej platne. Potom
00:02:03.00 nastáva anafáza a chromozómy sa pohybujú
00:02:06 na dve žrde a potom na tie
00:02:08.00 mikrotubuly sa znova použijú a
00:02:10.00 prerobia sa na štruktúru
00:02:12.00 nazývaný fragmoplast. Takže, fragmoplast
00:02:14 je založená na tejto krásnej štruktúre.
00:02:17.00 z mikrotubulov a čo to robí
00:02:20.00 pomáha presunúť vezikuly do
00:02:23.00 poloha v strede fragmoplastu,
00:02:25 a tie vezikuly sú plné
00:02:27.00 materiál bunkovej steny. Takže sa spájajú
00:02:29 a tvoria túto lipidovú dvojvrstvu a
00:02:34.00 potom vo vnútri je nová bunková stena
00:02:37,00 materiál. Takže to, čo v podstate robí, je
00:02:39.00 je to šablóna novej bunkovej dosky, ktorá je
00:02:42.00 potom oddelíme dve dcéry
00:02:44,00 bunky. Tento fragmoplast sa usadzuje v
00:02:48.00 uprostred medzi dvoma dcérskymi jadrami,
00:02:50, ale potom musí expandovať od polovice
00:02:52.00 bunka von do bunkovej kôry, k tomu
00:02:55.00 miesto, ktoré bolo definované
00:02:57.00 predprofázové pásmo. Tu, tá nová membrána
00:03:00 a materiál bunkovej steny sa spája s
00:03:02.00 materská bunková stena a cytokinéza je
00:03:05,00 dokončené. Takže mikrotubuly sú absolútne
00:03:08.00 nevyhnutné pre tento proces, ak by ste boli
00:03:10,00 aby ste sa zbavili mikrotubulov
00:03:11.00 cytoskelet, zastavili by ste mitózu, vy
00:03:14.00 by pomohlo cytokinéze. Ale ten druhý
00:03:17.00 cytoskelet v rastlinných bunkách,
00:03:19.00 aktínový cytoskelet. takže,
00:03:22.00 je prítomný aktínový cytoskelet
00:03:24.00 predprofázové pásmo. Tiež sa nájde
00:03:27 vo fragmoplaste. teda aktín
00:03:29.00 Ukážem vám tu je v modrej farbe a
00:03:32.00 mikrotubuly boli ružové a
00:03:36.00 aktínový cytoskelet je prítomný všade
00:03:37.00 bunkovú kôru, takže priamo pod
00:03:40 plazmatická membrána v celom tomto celku
00:03:42.00 proces. A už to bolo trochu
00:03:44.00 náročné zistiť, aká je úloha
00:03:47.00 aktín je počas bunkového delenia. Čo je
00:03:49.00 to robí v predprofázovom pásme, čo
00:03:51.00 robí to vo fragmoplaste, čo
00:03:53.00 robí to v bunkovej kôre. A
00:03:55.00 hlavným dôvodom, prečo to bolo náročné, je
00:03:57.00 pretože skutočne môžete depolymerizovať
00:03:58.00 celý aktínový cytoskelet a bunku
00:04:01.00 divízia akosi pokračuje a stále
00:04:04.00 sa stane. A tak to nie je podstatné pre
00:04:07.00 bunkové delenie, ale v každej jednej rastline
00:04:09.00 druh, ktorý bol skúmaný na aktín
00:04:12.00 prítomný, takže to musí niečo robiť.
00:04:13.00 Len sme neprišli na to, čo
00:04:16.00 to robí. Takže moje laboratórium pracuje na tomto mechu
00:04:21.00 Physcomitrella patens a sme
00:04:23 naozaj ma zaujímajú tieto krásne
00:04:25.00 vláknité bunky a jedna z vecí
00:04:28.00 o machu, ktorý v skutočnosti je
00:04:30.00 atraktívne je, že v týchto vláknitých
00:04:32.00 bunky sa v skutočnosti delia
00:04:34 bez predprofázového pásma. takže,
00:04:37.00 apikálne bunky, subapikálne bunky, oni
00:04:40 00:04:40 bez predprofázového pásma.
00:04:42.00 Takže toto je obrázok mikrotubulov v a
00:04:47.00 živá bunka a ukázali by sa modré bodky
00:04:50 na tomto konkrétnom obrázku, ktorý
00:04:53.00 je stredná časť jadra
00:04:55.00 pred rozpadom jadrového obalu, to je
00:04:58.00 kde by ste videli mikrotubuly
00:04:59.00 fluorescenciu, ak by tam bola. ale je
00:05:02.00 tam nie je, dobre? Takže neexistuje
00:05:05 predprofázové pásmo. Teraz sa idem hrať
00:05:07.00 tento film a vy ho uvidíte
00:05:09.00 rozpad jadrového obalu, vznik
00:05:10.00 mitotické vreteno a metafáza a potom
00:05:13.00 bude sa to opakovať znova a znova
00:05:14,00 znova. Takže neexistuje žiadna predprofázová kapela
00:05:17 v týchto celách. Takže, čo nám to umožňuje
00:05:20 je konkrétne opýtať sa, aká je úloha
00:05:23 aktínu vo fragmoplaste, však?
00:05:26.00 Môžeme sa zbaviť komplikácií
00:05:28.00 predprofázová kapela, pretože neexistuje
00:05:29.00 predprofázová kapela a môžeme rozobrať,
00:05:31.00 akú funkciu má aktín
00:05:33 fragmoplast? A dostali sme okno
00:05:37.00 tento problém analýzou molekulárnej
00:05:40.00 motor, ktorý chodí po aktínových vláknach.
00:05:43.00 Takže myozíny sú molekulárne motory, na ktoré sa viažu
00:05:45 a kráčajte po aktínových vláknach a
Je to veľmi veľká, rôznorodá rodina
00:05:50,00 molekúl. Toto je len fylogenetika
00:05:54.00 strom myozínov na základe ich motora
00:05:56.00 domén. v eukaryotoch a vy
00:06:00 môžete vidieť, že je tam aspoň 35 myozínu
00:06:02.00 tried, keď bol tento konkrétny papier
00:06:04.00 zverejnené a čo je naozaj zaujímavé
00:06:07 a bol vždy veľmi zaujímavý
00:06:09.00 mne je fakt, že rastliny majú len dve
00:06:12.00 triedy mojich myozínov. Majú triedu
00:06:14.00 VIII a potom myozín triedy XI a oni
00:06:18.00 nemajú takú obrovskú rozmanitosť
00:06:20 môžete nájsť v iných organizmoch. Ľudia
00:06:22.00 majú mnoho rôznych tried myozínov,
00:06:24.00 napríklad. Takže, čo sú tieto myozíny?
00:06:28.00 v rastlinách a aká je ich úloha?
00:06:30.00 A tak pomocou obrátenej genetiky
00:06:33.00 ktorý nám poskytuje výkonný systém
00:06:36.00 Physcomitrella patens, vlastne sme
00:06:37.00 analyzovali funkciu oboch závodov
00:06:39.00 triedy rastlinného myozínu. Takže myozín XI sú
00:06:44.00 veľmi podobné myozínom triedy VIII z
00:06:47.00 zvierat a húb a myslíme si to
00:06:50.00 sú dôležité pri transporte vezikúl
00:06:51 a transport organel. A ukázali sme
00:06:54.00 pomocou RNA interferencie, ktorou sú
00:06:56.00 nevyhnutné pre polarizovaný rast machu.
00:06:59.00 A v iných rastlinách a semenných rastlinách
00:07:02.00 sú dôležité pre cytoplazmu
00:07:04.00 streamovanie a pohyb organel. myozín
00:07:07.00 8s, ktoré sú hviezdami dnešnej šou,
00:07:11.00 sú menej dobre študovaní a menej dobre
00:07:14,00 pochopili. Takže v semenných rastlinách majú ľudia
00:07:19,00 zatiaľ som neprišiel na žiadnu funkciu. Tam
00:07:22.00 boli obmedzené lokalizačné štúdie.
00:07:24.00 Pomocou protilátok ukázali, že áno
00:07:28.00 môže lokalizovať do steny po
00:07:31.00 cytokinézy a tiež kanálov, ktoré
00:07:34.00 sa nachádzajú medzi rastlinnými bunkami tzv
00:07:36 plazmodesmaty. Pustili sme sa teda do analýzy
00:07:41.00 funkciu myozínov triedy VIII v
00:07:42,00 rastlín. v machu a použili sme homologické
00:07:46.00 rekombinácia na generovanie knockoutov. takže,
00:07:50.00 najprv vám musím povedať, že existujú
00:07:51.00 päť génov, ktoré tvoria myozín VIII
00:07:54.00 rodina v machu a môžu byť zoskupené
00:07:56.00 do dvoch skupín na základe ich
00:07:58.00 podobnosť sekvencií. Takže fialová doména
00:08:01.00 je motorická doména a potom červená
00:08:04.00 doména je miesto, kde sa viažu ľahké reťazce a
00:08:06.00 podľa predpovedí budú modré domény
00:08:07.00 vinuté cievky, takže pravdepodobne budú
00:08:09.00 dimérne myozíny. Takže v podstate my.
00:08:15 sa pozrel aj na výraz týchto
00:08:17.00 gény v rôznych typoch tkanív a toto
00:08:20.00 sa zobrazuje tu. Takže zelené pruhy sú
00:08:23.00 vláknité tkanivá, protonemata,
00:08:26 a fialové pruhy sú
00:08:28.00 výraz v listových výhonkoch alebo v
00:08:32.00 gametofory. A to, čo môžete vidieť, je
00:08:34, že s výnimkou myozínu VIII-D
00:08:38.00 väčšina z nich je vyjadrená práve týmto
00:08:40.00 štádium vláknitého tkaniva,
00:08:43.00 protonemata a nie je to dramatické
00:08:48.00 rozdiely v úrovni ich prejavu.
00:08:50.00 Takže sa zameriame na delenie buniek
00:08:53.00 udalosti v týchto protonemálnych vláknach
00:08:55.00 pretože v nich ide o bunkové delenie
00:08:57.00 protonemálne vlákna, ktoré sú
00:08:58.00 deje v neprítomnosti a
00:08:59.00 predprofázové pásmo.
00:09:01.00 Dobre. Takže sme vytvorili panel mutantov.
00:09:06.00 Pomocou homológnej rekombinácie sme
00:09:07.00 vyradili gény myozínu VIII.
00:09:10.00 Na hornom paneli máme knockouty,
00:09:13.00 jednotlivé knockouty. Niektoré sme vygenerovali
00:09:16.00 dvojité knockouty, jeden trojitý knockout, dva
00:09:18.00 štvornásobné knockouty a potom, na
00:09:20.00 tam dole, vidíte regenerujúce sa rastliny
00:09:23.00 päťnásobného knockoutu. Takže vlastne my
00:09:25,00 vyradil všetkých päť myozínov triedy VIII.
00:09:28.00 Takže prvé prekvapenie je, že môžete
00:09:31.00 žiť bez myozínov triedy VIII. Ale prečo držať
00:09:34.00 na päť génov, ak bez nich dokážete žiť
00:09:35,00 ich? Tak to je niečo, čo stále
00:09:37.00 ma v noci obťažuje, ale je, vieš.
00:09:40 je fakt, že tieto myozíny sú
00:09:43 nie je nevyhnutný pre životaschopnosť. však
00:09:45.00 existujú určité rozdiely. Takže tieto
00:09:47.00 rastliny sú rastliny, ktoré boli regenerované
00:09:49.00 z jednej bunky a je ich približne
00:09:51.00 päť až šesť dní staré a my snímame
00:09:54.00 pomocou fluorescenčného mikroskopu a
00:09:56.00 pozeráme sa na fluorescenciu v
00:09:58.00 ich stenu, pretože sme pridali farbivo
00:10:00, ktorý sa viaže na stenu. A tak to
00:10:02.00 nám umožňuje zmerať plochu
00:10:04.00 rastliny, pretože meriame plochu
00:10:06.00 fluorescencia a tak divoký typ, na ktorý sme sa rozhodli
00:10:09.00 jeden a všetky naše údaje normalizujeme na
00:10:11.00 divoký typ. Takže, čo môžete vidieť, je to
00:10:13.00 jediní mutanti sú všetci tak trochu
00:10:16,00 menšie a potom následné knockouty
00:10:19,00 nakoniec vyústi do päťnásobného knockoutu,
00:10:22.00 kde ste rastlina, ktorá je o
00:10:24.00 58 % veľkosti
00:10:26.00 divoký typ. Existuje teda porucha rastu. A
00:10:30 ak sa na ne pozriete naozaj pozorne
00:10:32.00 mladé rastliny, všimli sme si, že existuje
00:10:34.00 tiež chyba v umiestnení
00:10:36.00 bunková doska. Takže rastliny divokého typu, ktoré sú
00:10:40.00 päť dní starý, čo volám
00:10:43.00 priečne doštičky buniek alebo uhol nula.
00:10:46.00 A mutant myozínu VIII, ako môžete
00:10:50 z tohto histogramu má veľký
00:10:53.00 populácia buniek, ktoré majú šikmé
00:10:55,00 bunkové platne. Takže namiesto polohovania
00:10:58.00 doska bunky dokonale priečne k
00:11:00 dĺžka. k dlhej osi bunky
00:11:02.00 je tu prešľap -- je tu nejaká chyba.
00:11:05.00 Aby sme získali predstavu o tom, aký je tento myozín
00:11:10.00 práce, chceli sme vytvoriť fúziu
00:11:12.00 tejto molekuly so zeleným fluorescenčným proteínom,
00:11:14.00 takže by sme mohli analyzovať jeho lokalizáciu.
00:11:16.00 A tak sme sa rozhodli pozrieť na myozín 8A
00:11:22.00 pretože. takže oranžové molekuly sú
00:11:25.00 z ktorých sú všetky veľmi podobné
00:11:26.00 ďalšie a tie modré sú tie
00:11:27.00 ktoré sú si navzájom podobné, teda tie
00:11:29.00 sú dve skupiny, skupina A a skupina
00:11:30.00 skupina B. A zo skupiny A je myozín 8A
00:11:34.00 najviac vyjadrené. Tak sme si mysleli, dobre,
00:11:36.00 vyskúšame to a spojíme to
00:11:38.00 tri tandemové molekuly GFP a my to urobíme
00:11:41.00 premeniť ho späť na päťku
00:11:43,00 knockout. Jeho premenou späť na
00:11:45.00 päťnásobný knockout, ktorý nám umožnil
00:11:47.00 príležitosť vidieť, či táto molekula
00:11:49.00 bola funkčná, pretože sme videli, či
00:11:51.00 zachránilo to fenotypy, ktoré sme mali
00:11:53,00 pozorovaný v päťnásobnom knokaute. takže,
00:11:56.00 tento transgén z veľkej časti zachraňuje väčšinu
00:12:00 fenotypy, ktoré pozorujeme, že I
00:12:01.00 nemám čas s tebou hovoriť, ale
00:12:03.00 jeden fenotyp, ktorý je dôležitý
00:12:05.00 dnes je umiestnenie dosky bunky. A
00:12:07.00 ako môžete vidieť na tomto zelenom histograme
00:12:10.00 tu, kde sme vzali myozín 8
00:12:12.00 zlúčený s GFP a vložili sme ho do
00:12:14.00 päťnásobný knockout, vidíme posun
00:12:17.00 táto distribúcia späť k divočejšiemu typu.
00:12:19.00 Takže to nám hovorí, že táto fúzia GFP
00:12:22.00 proteín je funkčný proteín a my
00:12:24.00 potom analyzujte jeho lokalizáciu. Takže najprv ja
00:12:28.00 musím vám povedať, že toto je to, čo
00:12:30.00 bunky divokého typu vyzerajú, ako keď vložíme
00:12:32.00 na našom konfokálnom mikroskope. Takže tie
00:12:34 sú nádherné chloroplasty, ktoré
00:12:37.00 autofluorescovať v tomto konkrétnom kanáli a
00:12:39, takže vždy, keď v ňom uvidíte chloroplasty
00:12:42.00 veľa obrázkov, na ktoré sa chystám
Ukážte vám, že to je len autofluorescencia.
00:12:46.00 Rastliny Myosin 8-GFP vyzerajú takto.
00:12:48.00 Takže môžete vidieť, že je tam veľmi veľký
00:12:50 rozdiel medzi divokým typom a
00:12:51.00 myozín 8-GFP v tom cytozole teraz
00:12:54 sa rozsvieti fluorescenciou, však? takže,
00:12:57.00 tie chloroplasty nie
00:12:59.00 majú v sebe akýkoľvek myozín 8 okrem
00:13:01.00 cytoplazma má veľa myozínu 8-GFP,
00:13:03 a dúfam, že to tam vidíte
00:13:06 sú malé častice, málo intenzívne
00:13:08 častice a niektoré z týchto častíc
00:13:10 sa zdajú byť viac obohatené na hrote
00:13:12.00 celu. Takže, toto je aký druh
00:13:15.00 globálny pohľad vyzerá ako napr.
00:13:17.00 konfokálny mikroskop s rotujúcim diskom, ale
00:13:20 chceli sme sa pozrieť priamo pod
00:13:21.00 plazmatickú membránu a chceli sme ju analyzovať
00:13:23.00 lokalizácia týchto molekúl v
00:13:26 plazmatická membrána.
00:13:27.00 Takže používame úplný vnútorný odraz
00:13:29.00 fluorescenčná mikroskopia na to a
00:13:31.00 čo vám tu ukazujem, je obrázok
00:13:33.00 z mikroskopu TIRF, kde môžete vidieť
00:13:36.00 myozín 8-GFP tvorí tieto krásne
00:13:38.00 malé dynamické častice, ktoré sa pohybujú
00:13:41.00 pozdĺž povrchu plazmatickej membrány.
00:13:42 A ak sa teda chcete opýtať, čo s tým
00:13:45.00 aktín? Potom sa môžete pozrieť na aktín pomocou a
00:13:48.00 sonda s názvom Lifeact, ktorá sa viaže na
00:13:51.00 aktínové vlákna a to, čo môžete vidieť, je
00:13:53.00 že mnohé z týchto častíc sa pohybujú
00:13:55,00 pozdĺž aktínových vlákien. A toto je čo
00:13:58 by sme očakávali. Toto je myozínový motor
00:14:00 má chodiť po aktínových vláknach.
00:14:02.00 Tak toto je veľmi potešujúce vidieť.
00:14:04.00 Vlastne sme merali pohyblivosť
00:14:07.00 pozdĺž týchto vlákien a boli sme schopní
00:14:09.00 zistiť, ako rýchlo sa pohybovali, ako dlho
00:14:12.00 ich trajektórie boli tiež. Takže jeden
00:14:14 deň, keď môj postgraduálny študent prišiel do laboratória
00:14:16 a povedal: Tento obrázok som dostal ako posledný
00:14:18.00 noc a toto vyzerá ako naozaj
00:14:20 zaujímavá štruktúra a pomyslel som si, oh
00:14:22.00 môj bože, toto vyzerá ako mitotický
00:14:23.00 vreteno. A pomyslel som si, ako je to s myozínom 8
00:14:26.00 sa lokalizuje do mitotického vretienka.
00:14:28.00 Vieme, že aktín je vo fragmoplaste,
00:14:30 ale v skutočnosti sme aktín nikdy nevideli
00:14:33.00 mitotické vreteno -- ktoré je vyrobené z mikrotubulov.
00:14:34.00 Aby sme to naozaj potvrdili
00:14:37.00 že táto štruktúra bola mitotická
00:14:39.00 vreteno, potrebovali sme vygenerovať čiaru
00:14:41.00 ktorý mal fluorescenčný myozín
00:14:44 fluorescenčné mikrotubuly. Takže sme vygenerovali a
00:14:47.00 linka, ktorá mala obe označené
00:14:49 dve rôzne farby. A toto ide
00:14:53.00 aby to bol film, v ktorom budem hrať
00:14:56.00 myozín 8 v zelenej farbe a mCherry tubulín v červenej farbe
00:14:59.00 v zlúčení. A to
00:15:03 je tesne pred jadrovou obálkou
00:15:04.00 rozpis, takže môžete vidieť tie krásne
00:15:06.00 mikrotubuly, ktoré idú presne okolo
00:15:08.00 jadro, potom jadro
00:15:10.00 sa rozložíte a vytvoríte
00:15:12.00 mitotické vreteno. Takže, keď hráme tento film
00:15:15.00 môžete vidieť, že môj myozín 8 je jasne
00:15:16.00 lokalizácia do mikrotubulov v celom rozsahu
00:15:19.00 celý proces rozdelenia a ako
00:15:23.00 jadrová obálka sa pokazí
00:15:25.00 hneď na začiatku filmu
00:15:26.00 sa viaže na vreteno a potom, ako je
00:15:29.00 sa zmení na fragmoplast, ktorý
00:15:30.00 happens very quickly after that, you can
00:15:32.00 see a very fine band of myosin 8
00:15:35.00 that localizes to the leading edge of
00:15:37.00 the phragmoplast. So, that was very
00:15:39.00 exciting. So, we
00:15:41.00 to ensure that actin really isn't in the
00:15:43.00 mitotic spindle because maybe it is in
00:15:46.00 these cells and we hadn't looked
00:15:47.00 carefully enough. So, we looked at a line
00:15:49.00 that had myosin 8 and actin. And so, what you can
00:15:53.00 see here is that myosin 8 localizes to the
00:15:55.00 spindle, but it. when it turns into that
00:15:58.00 very, very narrow band, that's when
00:16:00.00 actin appears. Before then, really, actin
00:16:04.00 is not accumulating. So, actin is not in
00:16:07.00 the mitotic spindle but it seems to be
00:16:09.00 appearing right when that spindle
00:16:10.00 transitions into this phragmoplast
00:16:12.00 that's so important for generating the
00:16:14.00 new cell plate. So, then we thought, okay,
00:16:17.00 well, we have these tools, we have these
00:16:19.00 drugs that we can get rid of the actin
00:16:20.00 cytoskeleton, so what happens if we get
00:16:22.00 rid of the actin cytoskeleton? So, we
00:16:24.00 treated cells with Latrunculin,
00:16:25.00 which is a drug that depolymerizes
00:16:27.00 actin filaments, and then we looked at
00:16:29.00 myosin, and we had mCherry tubulin so
00:16:32.00 we could look at mitotic spindles and
00:16:34.00 phragmoplasts. And what you can see is
00:16:36.00 that myosin localizes just fine to the
00:16:38.00 mitotic spindle and it localizes just
00:16:40.00 fine to the mid-zone of the phragmoplast
00:16:42.00 during phragmoplast expansion.
00:16:45.00 So, this was very puzzling, we were a little
00:16:47.00 concerned, so we did a little recap. Okay.
00:16:50.00 Myosins are actin-based motors. We know
00:16:52.00 this and they're walking along actin
00:16:54.00 filaments in the cell cortex. But during cell
00:16:57.00 division they seem to be binding the
00:16:59.00 microtubules, not actin necessarily, and
00:17:02.00 they show up at the site of cell
00:17:04.00 division before actin ever shows up, and
00:17:07.00 it doesn't seem like our localization
00:17:10.00 depends on actin during cell division. takže,
00:17:12.00 this was a little bit puzzling, we were a
00:17:14.00 little bit concerned, and so we asked the
00:17:16.00 question, does myosin 8 actually work
00:17:18.00 with actin during cell division or is it
00:17:20.00 doing something entirely novel? Did we
00:17:23.00 actually find a kinesin or something?
00:17:26.00 So, we looked more carefully. So, again,
00:17:29.00 this is this movie that I've already
00:17:30.00 shown you where myosin 8 is localizing
00:17:33.00 to the mitotic spindle, there, and then as
00:17:37.00 it turns into a phragmoplast,
00:17:38.00 actin accumulates in the phragmoplast zone. So, we
00:17:42.00 thought, maybe what's going on is that
00:17:44.00 myosin 8 needs to get the right area and
00:17:48.00 it gets to the right area by interacting
00:17:50.00 with microtubules but really its
00:17:52.00 function is during cytokinesis,
00:17:54.00 and that function works with actin. takže,
00:17:58.00 to really address that what we did was
00:18:00.00 look very carefully at expanding
00:18:02.00 phragmoplasts. So, this is a wild-type
00:18:05.00 cell labeled with GFP-tubulin and the
00:18:08.00 membranes that are getting incorporated
00:18:10.00 into that new cell plate are labeled
00:18:12.00 with FM4-64 which is a lipophilic
00:18:14.00 membrane dye. And so what you can see is
00:18:17.00 this beautiful phragmoplast structure makes
00:18:20.00 a very linear sort of line that's very
00:18:24.00 uniform, and you form a new cell plate.
00:18:28.00 And then these are just examples, still
00:18:31.00 images, of a beautiful, nicely formed phragmoplast
00:18:35.00 with membrane in the center, in
00:18:37.00 wild-type cells. So, then we thought, okay,
00:18:40.00 well, let's look at what happens in the
00:18:42.00 myosin 8 mutant. So, in the myosin 8
00:18:45.00 mutant, again, there is your phragmoplast.
00:18:47.00 spindle and then it turns into a
00:18:50.00 phragmoplast about now. and you can
00:18:52.00 start to see membranes getting
00:18:54.00 incorporated into that phragmoplast. A
00:18:57.00 you can see that the phragmoplast is
00:18:59.00 messier, it's not as uniform, and you can
00:19:03.00 see that the membranes are not getting
00:19:05.00 incorporated into a nice linear line. A
00:19:07.00 over here we have these stills, alright?
00:19:11.00 And in the stills you can see that the
00:19:12.00 membrane is waves and then you can see
00:19:16.00 that you can form these sort of not very
00:19:19.00 normal-looking cell plates. So, it does
00:19:23.00 look like in the myosin 8 mutant
00:19:25.00 that phragmoplast expansion and the
00:19:28.00 building of the new cell plate is
00:19:30.00 aberrant. So, if myosin 8 requires
00:19:34.00 actin for its function during phragmoplast
00:19:36.00 expansion, then we should be able
00:19:39.00 to see a copy this mutant phenotype by
00:19:42.00 getting rid of actin. And so that's what
00:19:44.00 we did. We looked at cells treated with
00:19:47.00 Latrunculin during this stage of
00:19:49.00 phragmoplast expansion and what we saw was
00:19:52.00 that we got very similar results to
00:19:57.00 knocking out the five myosins. We see a
00:20:01.00 disorganized phragmoplast and we see
00:20:04.00 buckling of the membranes as the membranes
00:20:06.00 incorporate. And in these still
00:20:09.00 images, which are just other examples of
00:20:11.00 this happening, you can see that the
00:20:14.00 phragmoplasts are aberrant and you can see
00:20:16.00 that the planes of the membranes are not
00:20:19.00 uniform. So, that led us to think, okay, so
00:20:22.00 this is probably a myosin and it
00:20:24.00 probably walks on actin filaments, and
00:20:26.00 it's probably doing a job very
00:20:28.00 specifically during cytokinesis and
00:20:31.00 during cell expansion. during phragmoplast
00:20:34.00 expansion. So, this is a movie, now,
00:20:36.00 where we're looking at myosin 8
00:20:39.00 and microtubules, and we're going to play
00:20:42.00 it through once as the phragmoplast
00:20:44.00 expands, and then I want you to focus where
00:20:48.00 that circle is because there are a lot
00:20:51.00 of peripheral cytoplasmic. of peripheral
00:20:55.00 microtubules at the edge of this
00:20:56.00 structure. And those peripheral
00:20:59.00 microtubules, interestingly, all have
00:21:02.00 myosin 8s on their ends. So, it looks like
00:21:04.00 these peripheral microtubules interact.
00:21:07.00 have myosin 8 on the ends and we know
00:21:09.00 that in this structure the ends of these
00:21:11.00 microtubules are the plus ends of the
00:21:13.00 microtubules. So, it looks like myosin 8
00:21:15.00 is binding to the plus ends of these
00:21:17.00 peripheral microtubules, and they very,
00:21:20.00 very rapidly incorporate into the
00:21:22.00 expanding phragmoplast as the
00:21:24.00 phragmoplast expands. They also search the
00:21:27.00 cell cortex, where you can also see an
00:21:29.00 accumulation of myosin 8 at the cell
00:21:31.00 cortex. So, it looks like myosin 8 is
00:21:35.00 helping to direct the expansion of this
00:21:37.00 structure to the right place on the cell
00:21:39.00 cortex. If we look at a very similar cell,
00:21:43.00 but now a cell that is treated with
00:21:47.00 Latrunculin, so there are no actin
00:21:49.00 filaments, what we see is that now myosin 8
00:21:53.00 highly decorates those peripheral
00:21:56.00 microtubules. They stay associated with
00:21:59.00 the cell cortex for a very long period
00:22:01.00 of time, as those search and search
00:22:04.00 and search, but there are no actin
00:22:05.00 filaments around, so those myosin 8s
00:22:07.00 just stay bound to the plus ends of the
00:22:09.00 microtubules, and the whole structure
00:22:12.00 actually can slip. So, instead of being
00:22:16.00 stuck in one place in the middle of the
00:22:18.00 cell, the structure actually slips
00:22:20.00 up and down, and sometimes skews. So, our
00:22:24.00 hypothesis based on these images in
00:22:26.00 these movies was that we think that
00:22:28.00 actin is actually forming in the mid-zone,
00:22:30.00 the edge of the phragmoplast. So, we
00:22:34.00 work on molecules that polymerize actin
00:22:37.00 filaments and we thought, well, let's look
00:22:39.00 at them during cell division and see
00:22:40.00 where they are. And so we have a line
00:22:44.00 that has a functional fusion of formin 2A,
00:22:47.00 which polymerizes actin filaments
00:22:50.00 and. fused to GFP. and a line that has.
00:22:53.00 mCherry tubulin. And as this. toto. toto je
00:22:56.00 a spindle that's in metaphase and it's
00:22:58.00 going to go into anaphase when I play
00:23:00.00 the movie. So, you can see anaphase from
00:23:02.00 those shadows moving apart from each
00:23:04.00 other and then you can see formin 2A
00:23:06.00 accumulate beautifully in the mid-zone
00:23:09.00 of the phragmoplast as the phragmoplast
00:23:11.00 expands out to the mother cell
00:23:13.00 cortex. So, formin is exactly where it
00:23:16.00 needs to be if actin filaments are being
00:23:18.00 polymerized off of this expanding
00:23:20.00 structure. So, then we needed to just bite
00:23:23.00 the bullet and look at actin and
00:23:24.00 microtubules at the same time. Actin is
00:23:27.00 very challenging to image because it's
00:23:28.00 very, very dynamic, and so we were
00:23:31.00 fortunate enough to be able to get
00:23:33.00 good images of this using a fairly fast
00:23:36.00 microscope. And you can see here. actin
00:23:39.00 is in green, microtubules are in red in
00:23:42.00 this merge image, and the circle is just
00:23:44.00 pointing to that area where those
00:23:45.00 peripheral microtubules are and they're
00:23:48.00 embedded in a meshwork of actin
00:23:50.00 filaments, and that meshwork of actin
00:23:52.00 filaments is connected to the cell
00:23:53.00 cortex. So, the microtubules are being
00:23:57.00 templated by this meshwork to
00:23:59.00 incorporate in along a specific plane in
00:24:02.00 the middle of the cell, allowing for the
00:24:04.00 expansion to occur and so that that
00:24:06.00 structure doesn't slip up or down, and
00:24:08.00 doesn't tweak. And so it's a. and, remember,
00:24:11.00 those peripheral microtubules have
00:24:12.00 myosin 8 on their plus ends, so they
00:24:14.00 are then incorporating and interacting
00:24:17.00 with the actin filaments to then make
00:24:18.00 this structure incorporate the right way.
00:24:20.00 So, this led us to a model where we think
00:24:25.00 myosin 8 is binding to the mitotic
00:24:27.00 spindle essentially as a way to get to
00:24:29.00 the right place and be there at the
00:24:31.00 right time. And then umm. in anaphase,
00:24:34.00 myosin 8 concentrates at
00:24:36.00 the cell cortex, it also concentrates in
00:24:38.00 the middle of the spindle, and also in
00:24:40.00 the poles. We don't understand the polar
00:24:42.00 concentration, we haven't figured that
00:24:43.00 out yet, but we understand what's going
00:24:46.00 on with this in the middle, here. Ako toto
00:24:49.00 turns into a phragmoplast, then actin
00:24:53.00 gets polymerized off of the middle there,
00:24:55.00 towards the cell cortex where myosin 8
00:24:58.00 can hold on to it, and that essentially
00:25:00.00 makes a network that then the
00:25:02.00 microtubules will connect to and then
00:25:05.00 incorporate along this plane, allowing
00:25:08.00 for cell division to occur along it in
00:25:10.00 the correct plane and divide the cell
00:25:12.00 properly. So, that's what's happening in
00:25:15.00 division in an apical cell, in these
00:25:18.00 protonemal filaments. So, we were
00:25:21.00 wondering about sub-apical cells, because
00:25:23.00 these sub-apical cells, as you can see
00:25:25.00 here in these division events, are
00:25:27.00 asymmetric events, where you actually
00:25:30.00 have to move the nucleus to the division
00:25:33.00 site and then division occurs at the
00:25:35.00 division site. So, we know that there are
00:25:38.00 no pre-prophase bands there, but what does
00:25:40.00 myosin 8 look like when you have this
00:25:42.00 branching event where you have to have
00:25:44.00 this large nuclear migration. Jeden z
00:25:48.00 things that we noticed right away in our
00:25:50.00 mutant analysis is that the myosin 8
00:25:52.00 knockout, so this is the quintuple
00:25:54.00 knockout, has dramatic defects in cell
00:25:58.00 plate positioning right at branch sites.
00:26:01.00 So, this is an image with Calcofluor
00:26:03.00 fluorescence, where Calcofluor binds very
00:26:06.00 specifically to new cell plates. And so
00:26:08.00 you can see that some of these cell
00:26:09.00 plates are incomplete, and some of them
00:26:12.00 are very aberrant in their positioning. takže,
00:26:16.00 it seems as if the phenotype actually is
00:26:19.00 enhanced at branch sites. So, myosin 8
00:26:23.00 at branch site localizes to the emerging
00:26:26.00 branch neck, and it localizes to the
00:26:29.00 emerging branch neck before mitosis even
00:26:32.00 begins. And once this movie will loop
00:26:34.00 you'll see that as the cell goes into
00:26:38.00 mitosis and then there's a phragmoplast,
00:26:39.00 you actually have two rings of myosin 8
00:26:41.00 that are concentric: one that was
00:26:43.00 very static on the cell cortex and then
00:26:46.00 one that's on the phragmoplast,
00:26:47.00 that expands out and reaches that
00:26:49.00 cortical population. So, this actually
00:26:52.00 looks like a pre-prophase band. except it
00:26:56.00 doesn't have any microtubules -- it just
00:26:58.00 has myosin 8. So, myosin 8 is defining
00:27:01.00 the cortical region that the phragmoplast
00:27:04.00 needs to expand to, okay? And this requires
00:27:08.00 actin, because if we treat cells with
00:27:12.00 Latrunculin, you'll see that the myosin 8
00:27:15.00 doesn't have any actin filaments
00:27:18.00 to interact with and you see buckling of
00:27:21.00 the cell. of the cell plate as it's
00:27:23.00 expanding. And this is the phenotype that
00:27:26.00 we see so clearly in the myosin 8
00:27:28.00 knockout. Okay. So, that's what's happening
00:27:33.00 in moss, where there is no pre-prophase
00:27:35.00 band. But we wanted to ask the question,
00:27:38.00 well, what happens in a plant cell that
00:27:40.00 does have a pre-prophase band?
00:27:43.00 Where would my myosin 8 localize in that cell?
00:27:45.00 And the other thing is that we needed to
00:27:47.00 redraw what the actin looks like in an
00:27:50.00 expanding phragmoplast. So, the actin
00:27:53.00 actually is coming out of the center of
00:27:55.00 the phragmoplast and it's not
00:27:56.00 necessarily collinear with the phragmoplast,
00:27:59.00 which is what's in the textbooks. Okay.
00:28:02.00 So, in order to analyze myosin 8
00:28:05.00 localization in a cell that has a
00:28:07.00 pre-prophase band, we decided to turn to
00:28:10.00 tobacco BY2 suspension culture cells,
00:28:12.00 because this is a plant cell culture
00:28:14.00 line that has been used to analyze cell
00:28:17.00 division for years and years, and
00:28:18.00 cytokinesis as well. And it's a beautiful cell
00:28:21.00 type that is very large and very easy to
00:28:23.00 image. So, we actually decided to put the
00:28:27.00 moss myosin 8 into these cells and see
00:28:29.00 how the moss myosin 8 localizes. And, to our
00:28:32.00 delight, we got images that look like
00:28:34.00 this. So, this is a cell that's about to
00:28:39.00 undergo mitosis -- you can actually see a
00:28:41.00 halo of the nuclear envelope -- and you can
00:28:44.00 see a band of myosin 8 right
00:28:48.00 underneath the cell cortex. Toto je
00:28:49.00 exactly what the pre-prophase ban looks
00:28:52.00 like. So, myosin 8 from moss is binding
00:28:56.00 to the pre-prophase band of tobacco
00:28:58.00 cells. And what happens
00:29:00.00 in cytokinesis? So, in the cortical
00:29:05.00 division site. you can see it at the
00:29:06.00 cortical division site in a maximum
00:29:08.00 projection image, but if you look at the
00:29:10.00 midplane of that same cell what you see
00:29:14.00 is the phragmoplast. I'm going to point
00:29:16.00 out here in circles where the cortical
00:29:18.00 division site is right at the cell
00:29:20.00 cortex, but then you also see myosin 8
00:29:23.00 accumulating in the mid-zone of
00:29:24.00 the phragmoplast. So, myosin 8 is
00:29:27.00 both at cortical division site and in
00:29:29.00 the phragmoplast mid-zone. So, if we look
00:29:32.00 at a cell that's undergoing division and
00:29:34.00 we label myosin 8 and membranes, we
00:29:37.00 can see here that the myosin 8 is in
00:29:40.00 green and the membranes labeled by FM4-64
00:29:43.00 are in red. what you can see is that
00:29:47.00 the phragmoplast expands out to a site,
00:29:50.00 and that arrow is pointing to the myosin 8
00:29:53.00 accumulation at the cortical
00:29:54.00 division site. So, you have myosin 8 both
00:29:57.00 in the phragmoplast mid-zone and at the
00:29:59.00 cortical division site, providing a
00:30:01.00 mechanistic link between the expansion
00:30:04.00 of the phragmoplast and the site where
00:30:06.00 it needs to join the mother cell wall.
00:30:09.00 So, what I've told you today is that during
00:30:11.00 cell division myosin 8 marks the
00:30:14.00 future site of division in these
00:30:15.00 branching cells. In the apical cells that
00:30:18.00 divide symmetrically, that mark happens
00:30:20.00 around anaphase, but in these cells where
00:30:23.00 you have to actually move the nucleus to
00:30:25.00 the site of division, it happens before
00:30:27.00 mitosis begins. And then myosin 8
00:30:31.00 localizes to the mitotic spindle and to
00:30:34.00 the phragmoplast and you have this
00:30:36.00 internal ring of myosin 8 on the
00:30:39.00 expanding phragmoplast that grows out
00:30:42.00 and reaches the cortical population. takže,
00:30:46.00 myosin 8, together with actin, provide
00:30:49.00 this mechanistic and spatial link
00:30:51.00 between the phragmoplast and the
00:30:53.00 cortical division site. So, with that, I
00:30:56.00 want to thank you for watching and I
00:30:58.00 want to thank you Shu-Zon Wu, a very talented
00:31:00.00 graduate student and postdoc, who has been
00:31:02.00 working on this project and has been just a
00:31:04.00 wonderful imager. Thank you.

  • Part 1: Understanding Cell Shape: Big Insights From Little Plants

Types of Epithelial Cells

Not all the layers of cells have to be of one particular type for the tissue to be considered a stratified epithelium. Some of the deeper layers may contain cells of different shapes. For example, a stratified squamous epithelium could have columnar or cuboidal cells at the deeper layers of the tissue.

Squamous Epithelial Cells

Squamous epithelial cells are characterized by their flat appearance, like tiles on a bathroom floor. They can either form a simple squamous epithelium or a stratified squamous epithelium.

A simple squamous epithelium consists of a single layer of these flat cells. This type of epithelium is leaky and therefore enables materials to pass through it quite easily. As a result, it is most often found in regions of the body where fluid and gas exchange is critical. For example, simple squamous epithelium lines the blood vessels, some cells of the lungs, and the heart.

A stratified squamous epithelium is made of two or more layers of squamous epithelial cells. It is associated with rapid regeneration by cell division the outer layers can be ‘sloughed’ off and replaced by new cells. As a result, it is particularly suitable for regions that are subject to abrasion, such as the outer layers of the skin, mouth, esophagus, vagina, and anus.

Columnar Epithelial Cells

Columnar epithelial cells are long, vertically arranged cells, with an appearance like bricks standing upright. They can either form a simple columnar epithelium (which can be further subclassified as ciliated or non-ciliated), a stratified columnar epithelium, or a pseudostratified columnar epithelium.

A simple columnar epithelium consists of a single layer of non-ciliated columnar epithelial cells. These cells are most often associated with producing secretions and are therefore found lining the cells of the gastrointestinal tract. There is also a sub-type of columnar epithelial cells that are ciliated. These cells feature an arrangement of hair-like structures on their surface that act to move mucus down a tract. Simple ciliated columnar epithelial tissues are found in the cells of the respiratory system and the female reproductive system.

Stratified columnar epithelia are quite rare, but can be found in part of the eye, and some parts of both the male and female reproductive systems.

There is an additional type of columnar epithelial tissue, a pseudostratified columnar epithelium. This tissue is comprised of only a single layer of cells, but the polar arrangement of these cells (i.e., the positioning of the nuclei) makes the cells look more like those in a stratified epithelium. These cells are involved in secretion and absorption and can be ciliated or non-ciliated. Ciliated pseudostratified columnar epithelium line the respiratory tract. Non-ciliated pseudostratified columnar epithelia line portions of the male reproductive system.

Cuboidal Epithelial Cells

Cuboidal epithelial cells are cuboid in shape, and typically have a central nucleus, distinct from columnar and squamous epithelial cells. They are specialized for secretion and absorption and are frequently found in the cells of glands.

Simple cuboidal epithelia are important for secretion and absorption. The tissue also acts as a protective barrier. Simple cuboidal epithelia are found in regions such as the kidney tubules, the ovaries, and the thyroid gland. Stratified cuboidal epithelial tissues are made up of multiple layers of cuboidal cells. These tissues are most often found in glands. For example, sweat glands and salivary glands.


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Pravdepodobne ste už počuli výraz „olej a voda sa nemiešajú“ a všimli ste si, ako sa šalátový dresing zložený z octu (ktorý je vodný, t. j. z veľkej časti z vody) a oleja oddelí, keď ho necháte odstáť. Táto nekompatibilita je spôsobená skutočnosťou, že molekuly vody sú polárne, ale olej je nepolárny. Voda je polárna molekula, pretože negatívne nabité elektróny, ktoré sa točia okolo jadier atómov, nie sú rovnomerne rozložené. Atóm kyslíka má oveľa väčšiu hmotnosť ako dva atómy vodíka, a preto elektróny trávia viac času v blízkosti atómu kyslíka. Výsledkom je, že koniec molekuly vody, kde sa nachádza kyslík, má relatívne záporný náboj, zatiaľ čo koniec s vodíkmi je nabitý relatívne kladne. Kladné konce molekuly vody sú priťahované k záporným koncom susedných molekúl vody, ako je znázornené na obrázku nižšie, a to umožňuje zlúčeniu molekúl vody. V dôsledku tohto javu ste mohli vidieť aj kvapky vody na čelnom skle auta.

Lipidy, tj mastné molekuly, sú na druhej strane nepolárne, čo znamená, že distribúcia náboja je rovnomerne rozložená a molekuly nemajú kladne a záporne nabité konce. Nepolárne molekuly sa v polárnych roztokoch dobre nerozpúšťajú Rovnako ako voda v skutočnosti, polárne a nepolárne molekuly majú tendenciu sa navzájom odpudzovať rovnakým spôsobom, ako sa olej a voda nemiešajú a oddelia sa od seba, aj keď sú silne pretrepané v snahe ich premiešať. Toto rozlíšenie medzi polárnymi a nepolárnymi molekulami má dôležité dôsledky pre živé veci, ktoré sa skladajú z polárnych molekúl aj nepolárnych molekúl. Nasledujúce časti budú ilustrovať dôležitosť tohto.


The biology of cancer cell shape and why it’s important

Long before modern medicine, doctors and philosophers learnt about cancer by simply looking at tumours taken from people who had died. This was coined pathology, which loosely translates to the study of disease.

Fast-forward to the 1800s and doctors began inspecting tumours removed during surgery more closely, thanks to the invention of microscopes. They soon realised that cells in tumours look very different from healthy tissue and they could even tell apart different types of cancer.

Studying samples of tumours (biopsies) in this way still plays a vital role in diagnosing cancer patients today. It reveals information about the type of cancer and how aggressive it’s likely to be, which helps doctors offer patients the best treatments.

But until now it has remained a mystery as to why the shape of cancer cells is so good at predicting the disease’s behaviour.

In a study published today in the journal Genome Research, researchers led by Dr Chris Bakal from The Institute of Cancer Research, London and funded by Cancer Research UK, reveal some key information about this strong link between cancer cell shape and patients’ outlook.

This in-depth account of the genes behind cancer cell shape, and how they’re linked to the likelihood of a tumour spreading, could help develop new treatments that make cancer less aggressive and easier to destroy with other therapies.

And Bakal’s team has produced a map to help navigate these next steps.

Constructing the map

The team started building a map using huge amounts of data from breast cancer cells grown in the lab. Some of these breast cancer cells grow aggressively, others more slowly.

They had images of more than 300,000 individual cells that were matched to genetic data showing how active different genes were.

Different shaped cancer cells Different shaped cancer cells Different shaped cancer cells
Different shaped cancer cells

“We looked at features including the size of the cells, how closely cells were packed together, how much contact they have with their neighbours, and whether the outside of the cell is smooth, ruffled, or spiky,” says Bakal. “And then we matched it to their genetic data.”

The team combined the information into a map, much like the London Underground map, with the cells’ appearance forming the stations and the genetic information revealing the tube lines connecting these shapes.

The team’s map that connects genetic data with the shape of cells. Credit: Dr Chris Bakal

“We already had lists of genes where there are significant differences between aggressive and more slow-growing breast cancers,” Bakal says. “But we were surprised that we found so many of these genes play an important role in determining cancer cells’ shape.”

On closer inspection, the activity of many of these genes was already known to affect cell shape, for example genes that are needed to make cells’ internal ‘skeleton’. But there were also some surprises.

“We found some unexpected results, for example some genes that matched with very rounded cells are involved in processes that happen in the mitochondria – the energy-producing machinery in cells,” says Bakal.

What came first – the genes or the shape?

The next thing the researchers wanted to know was whether cell shape was affecting the levels of genes, or whether those genes being switched on or off was controlling cell shape.

“We had a classic chicken and egg situation,” Bakal says. “A huge data set from the Broad Institute in the US helped us answer this question.”

We discovered that shape was having the bigger effect on genes, rather than the other way round

By analysing publically available data, the researchers could figure out which way information was flowing in their map.

“We discovered that shape was having the bigger effect on genes, rather than the other way round,” says Bakal. “And this could be relevant when it comes to thinking about potential new treatments.”

Cells can sense their environment and the forces around them that push, pull, stretch and squash them.

“We know this environment is important because tumours like their surroundings to be stiff. This stiffness helps cancers grow, spread and resist treatments. And it turns out that the stiffness of their surroundings also affects the shape of cancer cells and in turn changes their genetic profile.”

One of the genes central to the map is called NF-kB. “The close association between highly active NF-kB and aggressive cell shape is really interesting,” says Bakal, “because there are rarely mistakes in this gene in cancer. The gene itself often isn’t faulty in solid tumours, but shape changes can alter its activity, and help the cancer spread.”

On course for potential new treatments

Doctors use pathology data and the shape of cancer cells to diagnose cancer because it gives them reliable information on how aggressive the cancer is likely to be. Bakal’s team wanted to know if the genetic map they’d created could predict the same outcomes.

“We compared our shape-based genetic map, drawn up from lab-grown cells, with genetic and medical information from women who’ve had breast cancer,” says Bakal.

The researchers used data from our METABRIC study, which includes genetic information and clinical records from nearly 2000 women with breast cancer.

More stunning images from our labs

“We found that our gene profiles could be used to predict how aggressively breast cancers behaved, telling us the genetic map we’d created was relevant to cancer in the clinic too,” Bakal says.

This is the first glimpse into why – at the genetic level – the ancient practice of studying cells’ shape is a tell-tale sign of a cancer’s behaviour. And Bakal hopes their genetic data map will be mined for information leading to new treatments.

For example, researchers are studying whether drugs that stop cancer cells producing a molecule called LOX can be effective at treating some types of cancer. The LOX molecule works by making the tumour’s surroundings stiffer, “and this will probably change cancer cells’ shape and genetic profile as we’ve seen in our studies of breast cancer,” says Bakal.

As well as drugs targeted to the tumour’s environment, this research could lead to new drugs that change the shape of the cancer cells, re-routing the activity of their genes and hampering their ability to spread.

“Many of the chemotherapy drugs we use today, like paclitaxel, change the shape of the cells,” says Bakal. “Now we’re getting to the bottom of what’s happening at the genetic level, there’s scope to make shape-altering drugs more potent.

“Changing the shape of cancer to make it less aggressive could be effective in combination with other therapies – giving them a better chance to work.”

It’s early days yet, but this research is a big step forward in understanding a fundamental, age-old mystery: why the shape of cancer cells is so important in predicting how the disease will behave.


Scientists just discovered a strange new DNA shape lurking in human cells

The discovery of DNA as a double helix is a hallowed story of scientific triumph—the work of four researchers merging together to solve one of science’s biggest mysteries, giving birth to what we know as the field of modern genetics. But decades later, we’re still learning that DNA is a more furiously complicated piece of biological machinery than we ever knew.

For the first time ever, scientists have just discovered a new shape of DNA lurking inside human cells. In a study published Monday in Prírodná chémia, a team of researchers from the Kinghorn Centre for Clinical Genomics at the Garvan Institute of Medical Research in Sydney describe finding DNA as a four-stranded knot-like structure—called an i-motif—in human cells, upending much of what we previously thought could and could not exist in living humans, and eliciting a slew of questions about what the role of this structure might be, if it even has one.

We already know DNA can come in other forms, such as triple helices or cruciforms. And the i-motif is not the first four-stranded structure to be found in human cells scientists already did that with the discovery of G-quadruplex DNA in humans in 2013. But this is the first time i-motifs have ever been found in human cells. The i-motif structure was first observed around two decades ago, in lab conditions that were quite acidic, and most assumed the i-motif would probably never be found in nature.

“This raised a scientific debate concerning the biological relevance of this motif,” says Daniel Christ, director for the Centre for Targeted Therapy at the Garvan Institute and a co-author of the new study. “We are providing first direct evidence that the i-motif structure exist in cells under physiological conditions.”

Here’s how the i-motif works: imagine a small section of the DNA double helix where the hydrogen bonds that connect the two major strands come apart while the helix suddenly untwists. If one of the strands is chock-full of cytosine (one of the four major nucleic acids that comprise DNA), it will loop outward like a tied shoelace. Hydrogen bonds form within the loop itself, binding those cytosines to one-another (instead of to guanine, as is normally the case in the double helix).

“They essentially form a scaffold, where each C-C bond is 90 degrees to its corresponding C-C pair,” says Laurence Hurley, a medical chemist at the University of Arizona who has also studied i-motifs.

i-Motif structure. Garvan Institute of Medical Research

To confirm the presence of these i-motifs in human DNA and pinpoint their locations, the Sydney team created a special fragment of an antibody molecule capable of binding to the i-motif structure. They then used fluorescent techniques to highlight the antibody molecules under the microscope. It’s a very tried-and-true method in chemistry and biology, and should wash away lingering doubts about the veracity these i-motifs can arise in nature.

But what does the i-motif do? There’s quite a bit of evidence to suggest it plays a role in transcription (when the cell uses DNA as instructions to make different proteins). The Sydney team studied the presence of i-motifs during all phases of the cell cycle, and found that the motifs appear most commonly when DNA is being actively transcribed, but disappear when DNA is being replicated.

They also found that the motifs often appeared in parts of the promoter regions of genes, which are not read and expressed as protein products, but instead can turn on and off the expression of other genes and prevent or promote the production of certain proteins. “We consider it likely that the formation of i-motifs in promoter regions fine-tunes the expression of corresponding genes,” says Christ.

According to Hurley, there are specific proteins and mechanisms that work to unwind the DNA, create the i-motif folds and stabilize them during transcription, and then unfold these knotty loops and zip things back up into a double helix when it’s time for cell division. And this can be accomplished without the super-acidic setting typically needed for these motifs. “That’s where the power of these structures are,” he says. “They are highly dynamic, and you can fold them and unfold them, in order to activate transcription.”

“It’s clear that these structures [i-motifs] are implicated in gene expression,” says Hurley. “This paper provides the icing on the cake.”

Hurley and others have previously found evidence that i-motifs are related to a number of cancer-related genes, like MYC (expressed in over 80 percent of cancers), KRAS (which controls cell growth and proliferation signaling), and BCL-2 (which prevents cancer from undergoing apoptosis, or programmed death). Hurley himself recently founded a new company, Reglagene, that is seeking to use these i-motifs as potential targets for new cancer drugs, and prevent oncogenesis at the genetic level itself, as opposed to “undruggable” protein targets.

While the new findings pretty solidly show i-motifs can show up in living human cells, Hanbin Mao, a biochemist at Kent State University who was not involved with this study, notes there is room for more convincing evidence that i-motifs are a natural phenomena. The Sydney researchers cannot say for sure the antibody wasn’t binding to other targets, and more importantly, that the antibody’s binding to the DNA did not promote the formation of i-motif itself. Needless to say, there are years or even decades’ worth of follow-up research in store to learn more about what i-motifs are, how they work, why they exist, and how we might be able to harness their powers.

Nearly 65 years after Watson, Crick, Franklin, and Wilkins, the DNA plot continues to thicken.


Pseudostratified Columnar Epithelia

Two types of cells are present in a pseudostratified columnar epithelium: 1) round-looking basal stem cells, which continually divide, and 2) taller, columnar cells that resemble tiny cylinders. This type of epithelium is found within the lungs and trachea. The top of each columnar cell is decorated with tall filaments called mihalnice. These cilia beat in synchrony with each other and propel mucus out of the lungs and towards the throat. The function of this mucus is to trap particles and other inhaled material so that they don’t enter the lungs. Cilia move the mucus towards the mouse and esophagus, so it can be swallowed and destroyed in the stomach.



Komentáre:

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