Funkční diverzita mikrotubulů: její funkce a regulace Karel Souček 5-11-2009 BÍ6051 Molekulární fyziologie živočichů Mikrotubuly ■ struktura ■ dynamika ■ posttranslační modifikace Cy to skelet _______ . j 10 um Figure 16-1. Molecular Biology of the Cell, 4th Edition. Cellular Visions: The Inner Life of a Cell Aktinová filamenta ACTIN FILAMENTS IBÉMEOvVMHh/ - I 1 100 nm "W&^^^^^^^^ff 25 nm Actin filaments (also known as microfilaments) are two-stranded helical polymers of the protein actin. They appear as flexible structures, with a diameter of 5-9 nm, and they are organized into a variety of linear bundles, two-dimensional networks, and three-dimensional gels. Although actin filaments are dispersed throughout the cell, they are most highly concentrated in the cortex, just beneath the plasma membrane. Micrographs courtesy of Roger Craig (i and iv); RT. Matsudaira and D.R. Burgess {ii}; Keith Burridge iiii). Intermediární (střední) filamenta INTERMEDIATE FILAMENTS _______ 100 nm I__________I 25 nm Intermediate filaments are ropelike fibers with a diameter of around 10 nm; they are made of intermediate filament proteins, which constitute a large and heterogeneous family. One type of intermediate filament forms a meshwork called the nuclear lamina just beneath the inner nuclear membrane. Other types extend across the cytoplasm, giving cells mechanical strength. In an epithelial tissue, they span the cytoplasm from one cell-cell junction to another, thereby strengthening the entire epithelium. Micrographs courtesy of Roy Quinlan i\)- Nancy L. Kedersha (\\\; Mary Osborn (iii); Ueli Aebi (iv) jľiV jjp-^ ^*^M^J mm M pí Ml £/*Ssff v*ffi^^^jff&jifoi ŕ$^:* íS%: P§l§t -jSiAřrJ*-■, '^T^Tki.^: 'jf ^* SU! wt*r .** ^í*1 > t./ f« Í3 ■£fe*o^ t*v^ Mikrotubuly MICROTUBULES 25 nm Microtubules are long, hollow cylinders made of the protein tubulin. With an outer diameter of 25 nm, they are much more rigid than actin filaments. Microtubules are long and straight and typically have one end attached to a single microtubule-organizing center (MTOC) called a centrosome, as shown here. Micrographs courtesy of Richard Wade (i); D.T. Woodrow and R.W. Linck (ii); David Shirna { Microtubuly - klíčové složky cytoskeletu - vývoj - udržování tvaru - transport organel - buněčná signalizace - buněčné dělení a mitóza ■ Molecular Biology of the Cell (4th Ed.) interphase early prophase prometaphase lOfirn late prophase Mechanické vlastnosti aktinu, tubulinu a intermediálních filament intermediate filaments actin filaments deforming force Figure 16-17. Molecular Biology of the Cell, 4th Edition. ot-tubulin a ß-tubulin heterodimer - vysoce dynamický polymer Funkční diversita: -Vazba proteinů asociujících s mikrotubuly (MAP); -exprese různých isoforem (6 forem a-tubulinu a 7 forem ß- tubulinu); -posttranslační modifikace. GTP ß-tubulin Molecular Biology of the Cell í4th Fd ) 5„ tubulin heterodimer (= microtubule subunit) protofilament plus end a-tubulin lumen 50 nm, minus j end f«Mfc microtubule 10 nm ■Ml ■ -■ 50 nm Růst mikrotubulu na + konci newly formed microtubules tí ' mikrotubuly rostou rychleji na „plus" konci Figure 16-8. Molecular Biology of the Cell, 4th Edition. (A) rapid growth with GTP-capped end norm accidental loss of GTP cap CATASTROPHE mm^ rapid shrinkage mm*« regain of GTP cap RESCUE rapid growth with GTP-capped end etc. Figure 16-11 part 1 of 3. Molecular Biology of the Cell, 4-th Edition Dynamická nestabilita ■ Rostoucí MT má na konci ß podjednotku s GTP ■ pokud dojde k hydrolýze dochází ke smršťování GTP-tubulin dímer r (B) ot ß GTP i \ exchangeable GTP )le GTP J straight protofilament I GTP HYDROLYSIS CHANGES SUBUNIT CONFORMATION AND WEAKENS BOND IN THE POLYMER curved protofilament DEPOLYMERIZATION GDP-tubulin dimer GDP-GTP EXCHANGE J Hydrolýza GTP Zeslabení vazby v polymeru depolymerizace Figure 16-11 part 2 of 3. Molecular Biology of the Cell, 4th Edition. Růst a smršťování MT GTPcap less stable region of microtubule containing GDP-tubulin dimers GROWING SHRINKING ÍC) Figure 16-11 part 3 of 3. Molecular Biology of the Cell, 4th Edition. Dynamická nestabilita ■ ■ Rat Vascular Smooth Muscle Cells EB1 a a-Tubulin značení ■ Rychlé změny v přestavbě cytoskeletu během vývoje embrya Drosophily a Caenorhabditis distance from cell surface Ojmm 2 um 4 um 6 um 0 min interphase - * - 1.5 min •V. J prophase ♦.•••1 P * * «I 4.5 min metaphase 50 pm Figure 16-13. Molecular Biology of the Cell, 4th Edition. Call, Vol. OS, 517-521, Fatjruaiy 19, 1999. Copyright 81909 by Cell Prass CLIP-170 Highlights Growing Microtubule Ends In Vivo Franck Perez,* Georgien S. DiamantopDulosr Romaine Stal derr and Thomas E. Kreist Department of Cell Biology Sciences III University of Geneva CH-1 204 Geneva Switzerland bound GTP polymerizes at the plus ends, andr as microtubules groWr the GTP bound to the ß subunits is hy-drolyzed so that normally only a small segment ot GTP-tubulin should remain (discussed by Caplowr 1992: Desai and Mitchisonr 19971. This GTP cap is thought to be necessary tor elongation of the polymer and to prevent microtubules from d epolymerizationr while hydro lysis ot the nucleotide is a prerequisite for disassembly Call, Vol. 06, 517-S2J, Fabruaiy 19, 19S9. Copyright 31999 by Cell Prasi CLIP-170 Highlights Growing Microtubule Ends In Vivo Franck Perez..1 Georcpos S. Diamantopxxilosr Romaine Staid er and Thomas E. Kreist Department of Cť-ll Bioloc Si io| Unk CH-j Swrtl bound GTP polymerizes at the plus endsr andr as microtubules grown the GTP bound to the ß subunits is hy-drolyzed so that normally only a small segment ot GTP- small soluble large filamentous subunits polymer (A) * • ľ • * / signal, such as a nutrient source DISASSEMBLY OF FILAMENTS AND RAPID DIFFUSION OF SUBUNITS REASSEMBLY OF FILAMENTS AT A NEW SITE {B> Cytoskelet a změna tvaru Figure 16-2. Molecular Biology of the Cell, 4-th Edition Formování filament z podjednotek malých proteinů rychlá reorganizace cytoskeletu v odpovědi na vnější signál ____ - o H3C-C-0 0 O H3< 15|im Figure 16-21. Molecular Biology of the Cell, 4th Edition. Microtubuly - cíle protinádorové léčby Jordan M.A. and Wilson L. Nature Rev. Cancer, Vol 4, 2004, 253-265 Table 1 | Antimitotic drugs, their diverse binding sites on tubulin and their stages of clinical development Binding domain Related drugs or analogues Therapeutic uses Stage ot clinical development References Vinca domain Inhihipp Vinblastine {Vel ban) Hodgkin's disease, testicular germ-cell cancer In clinical use; 22 combination trials in progress 75-77,131 II II IIUIOC polymerizace Vine Istine (Oncovin) Leukaemia, lymphomas In clinical use; 108 combination trials in progress 132-134 Vinorelbine (Navel bine) Solid tumours, lymphomas, lung cancer In clinical use; 29 Phase l-l II clinical trials in progress (single and combination) 135-137 Vinflunine Bladder, non-small-cell lung cancer, breast cancer Phase III 131,138 Cryptophycin 52 Solid tumours Phase III finished 139,140 Hallchondnns {such as E7389) - Phase 1 58,141-143 Dolastatins (such as TZT-1027) Potential vascular-targeting agent Phase 1; Phase II completed 144 Hemiasterllns (such as HTI-286) - Phase 1 145,146 Colchicine domain Colchicine Non-neoplastic diseases (gout, familial Mediterranean fever) Appears to have tailed trials, presumably because of toxicity 89-90 Inhibice polymerizace Combretastatlns (AVE8062A, CA-1 -R CA-4-R /V-acetylcolchicinoT 0-phosphate,ZD6126) Potential vascular-targeting agent Phase 1, II 91,147 2-Methoxyestradiol - Phase 1 148,149 Methoxybenzene-sulph on amide (sucnasABT-751,E7010) Solid tumours Phase 1, II 150 Taxane site Stablizace Paclltaxel (Taxol), TLD0139 and other analogues of paclitaxel Ovarian, breast and lung tumours, Kaposi's sarcoma; trials with numerous other tumours In clinical use: 207 Phase l-lll trials in the United States; TL00139 Is In Phase I trials 82,151-153 polymerizace Docetaxel (Taxctere) Prostate, brain and lung tumours 8 trals In the United States (Phases l-lll) 154,155 Epothilones (such as BMS-247550, epothilones B and D) Paclitaxel-resistant tumours Phases l-lll 156-159 Discoderrrtolide - Phase I 160-164 Other microtubule binding sites Estramustlne Prostate Phases l-lll, in numerous combinations with taxanes, epothilones and Vinca alkaloids 122,165-168 Information on clinical trials was obtained from the National Institutes of Health Clinical Trials web site (www.clinicartrials.gov), the European Organisation for Research and Treatment of Cancer web site (www.eortc.be) and lhe Proceedings of the American Association for Cancer Research meeting in 2033 (www.aacr.org|. CA-4-R combretastatin-A-4 3-t»-phosphata; CA-1 -P. combrastatin A-1 -phosphate Pirotenin - specifický pro a-tubulin (váže kovalentně Lys) Usui T. et.al. Chem. Biol., Vol 11, 2004, 799-806. Shrnutí -1 ■ Mikrotubulyjsou klíčové komponenty cytoskeletu ■ Hydrolýza GTP je důležitá pro dynamiku MT ■ Mikrotubulyjsou cílem protinádorové terapie ^tubulin cc-tubulin ß-tubulin Reg (A) accessory proteins in y-tubulin ring complex _..J L 100 nm Figure 16-22. Molecular Biology of the Cell, 4-th Edition. tvorby filament y-tubulinový prstenec tvoří počátek - nukleační místo MT centrosom -organizační místo MT nucleating sites (y-tubulin ring complexes) A pair of centrioles (A) (B) microtubules growing from y-tubulin ring complexes of the centrosome Figure 16-23. Molecular Biology of the Cell, 4th Edition. Centrioly jsou součástí centrosomu válcovitě uspořádané krátké MT ■ stathmin it ■O ár free tubulin sequestered by stathmin to tubulin subunit pool shrinks stathmin izoluje vždy dvě podjednotky a a ß tubulinu © subunit addition stops © GTP hydrolysis catches up microtubule shrinks Figure 16-31. Molecular Biology of the Cell, 4th Edition. . MAP mohou regulovat stabilitu MT - MAP2 = prostornější MT -tau = kompaktní MT -catastrophin = destabilizuje MT 300 nm Cel Figure 16-33. Molecular Biology of the Cell, 4th Edition. Regulace stability MT (A) MAP 1 STABILIZATION frequency of catastrophes suppressed and/or growth rate enhanced GTP cap on plus end of microtubule DESTABILIZATION J catastrophin frequency of catastrophes increased RESULT: longer, less dynamic microtubules RESULT: shorter, more dynamic microtubules Figure 16-36 part 1 of 2. Molecular Biology of the Cell, 4th Edition. Molekulové motory spojené s mikrotubuly - Kinesiny (^+) a dyneiny (-* -) N KINESIN KIFC2 KIF2 N :Cř c( KIF1B (A) motor domain N[ i----------1 N 10 nm N Nl I I I I I I I I I I I I I I I I I I i_________i 500 amino acids cytoplasmic dynein ciliary dynein 25 nm Figure 16-56. Molecular Biology of the Cell, 4th Edition. Figure 16-55. Molecular Biology of the Cell, 4th Edition. ankyrin spectrin Arpi filament __dynein microtubule 25 nm Figure 16-63. Molecular Biology of the Cell, 4th Edition. trailing leading head head kinesin MINUS END {A) KINESIN microtubule PLUS END hydrolysis cycle ADP myosin POWER STROKE MINUS END actin filament IN ISOLATED DOUBLET MICROTUBULES: DYNEIN PRODUCES MICROTUBULE SLIDING + + ffi-J) linking proteins + .+ IN NORMAL FLAGELLUM: DYNEIN CAUSES MICROTUBULE BENDING Figure 16-79. Molecular Biology of the Cell, 4th Edition. -igure ABC 100 nm (B) Figure 16-80. Molecular Biology of the Cell, 4th Edition. Organizace mikrotubulů ve fibroblastu a neuronu (A} FIBROBLAST 60% pacientů přežívá >5let. Ve stadiu 4 (metastáze) má pouze 30% naději na dožití >5let. Léčba Chirurgická, rádioterapie, anti-androgenní terapie, chemoterapie. -v ■ ' rtnost ti nejčastější příčinaúmrtriia nádorové onemocnění. (JÄäte^) 1 1 tí t V~- Ol f— ■m i.T r- Ol r- o Líl r- Ol ■■- CM r* - S. r^ CO CO CO CO CO CT) o; CT) cd Ol O O c t) o> Ol CD O) 0) CTI CT) - T: CT) CT) '-■ O o OJ CM Female Non-Hodgkin Lymphoma ID ľ- m *— CO LO l^ Ol T— m LT K Ol i- CSJ r- r~- K m on m m m m m m Ol m o o e» en Ol m tt en oi ai ai ľh en Ol Ol o o tN C\j Year of Diagnosis Year o1 Diagnosis Cancer Statistics, 2006 A Comer It A CancerJosin laijor Clii lia'aw Stages 1 and 2 (Cancer that is only in the prostate gland, PSA) Between 65 - 98% with stage 1 and 2 prostate cancer will live for more than five years after they are diagnosed. Stage 3 (Cancer cells have spread outside the covering (capsule) of the prostate gland to tissues around the prostate but not to the lymph nodes.) About 60%) diagnosed with stage 3 prostate cancer will live for more than five years after diagnosis. Stage 4 (Cancer cells have spread (metastasized) to lymph nodes (near or far from the prostate gland) or to organs and tissues far away from the prostate such as the bone, liver, or lungs.) About 20 -30%) have cancer spread to another part of their body when they are diagnosed with prostate cancer. About 30%) men with advanced prostate cancer will live for more than five years after diagnosis. On average, men in this situation can expect their cancer to respond to treatment for about 12 to 18 months. Average survival after that is about another two years. www.cancerhelp.org.uk, www.phoenix5.org/staging.html Future Diagnostic tools •Gene Chip Analysis (prostate specific genes) •Proteomic (tissue, serum, urine) ■^specific ~ "fingerprint" ~ "signature" Tyr-Tub ■ PGIu-Tub Ac-Tub 1.0 0.62 0.65 <0.01 ■ a-Tub p-Tub ß-Actin Post-translational modification of a-Tubulin in prostate epithelial cells i-ysi- I I M ^^ ^m. E E E E H^M Ao I ■^FFV 40 Souček, K. et al, 2006 PrEC Pz-HPV-7 LNCaP PC3 Glu-Tubulin a-Tubulin merge + DAP I Glu-Tubulin ^ & ^ ^ _r2, HnN' -"^ '.' r (ŕ > S V fjWI 3EE Souček, K. eř a/., 2006 Tubulin Tyrosine Ligase - potential tumor supressor? Journal ol Cell Science 111: 171-131 {1998) Primed in Great Brilain © The Comoany of Biologists Limited 1998 JCS9S95 Suppression of tubulin tyrosine ligase during tumor growth Laurence Lafanechěre1'*, Celine Courtay-Cahen2'1^, Toshiaki Kawakamľ3, Michéle Jacrot4, Manfred Rüdiger5. Jürgen Wehland6, Didier Job1 and Robert L. Margolis2'* [CANCER RESEARCH 61, 3024-ÄE7, July 1, 2001] Tubulin Detyrosination Is a Frequent Occurrence in Breast Cancers of Poor Prognosis Agues Mialhe,2 Laurence Lafaueckere,2 I s a h e lie Treilleux, Nadine Peloux. Charles Dmnontet, Alani Brémond, Metig- Hong Pauli. Raoul Pay an, Jürgen Wehland, Robert-Louis Margolis, and Didier Job3 lilt J CaflCPf" 112 ^6^—375 Í2004] t-ii'"'"1--**-- Publication of the Internal onal Union Aaainst Canoer © 2004 Witey-LiHB, Inc. ^StS ' LOW EXPRESSION OF HUMAN TUBULIN TYROSINE LIGASE AND SUPPRESSED TUBULIN TYROSINATION/DETYROSINATION CYCLE ARE ASSOCIATED WITH IMPAIRED NEURONAL DIFFERENTIATION IN NEUROBLASTOMAS WITH POOR PROGNOSIS Cliíakí Kato1-2, Kou Miyazaki1, Ateuko Nakagawa^ Miki Qhira1, Yohko Nakamtjra1, Tos hí norí Ozaki\ Tos hí o Imai2 and Akira Nakagawara1* tubulin tyrosine ligase expression in prostate epithelial cells A ^" £ ^ ŕ / /V <£ 1.0 1.1 0.1 0.6 V TTL ß-Acti n Tyroansted «-tubulin fTyMub) ...EEY .EE Dstyrtrainated ct-tJDulin (Glu-tub) A2-tubulin Glu-Tubulin Souček, K. et al, 2006 u-Tubulin and Tubulin - Tyrosine Ligase expression in prostate epithelial cells - proof of the concept 1.0 43.2 B.7 3Ô.1 43,3 3Ü.6 2.9 TTL 1.0 *0jO1 ŮjDŮ Ů.Ů1 Üjffi Q25 0.1 ■■■ Tub HnN------------------------------------------GEE Ac-Tub H£N-----JL- Souček, K. £ŕ ah, 2006 SUMMARY •Normal and prostate cancer cells display distinct molecular profiles of a-Tubulin posttranslational modifications •Low expression of tubulin tyrosine ligase is characteristic also for prostate cancer cells •Different profile of post-translation modifications a-Tubulin in various prostate epithelial cell lines show the possibility to distinguish the stages of cancer disease and has the potential to establish a novel tool to diagnose and treat prostate cancer. Souček, K. et ah, 2006 Shrnutí - III ■ Postranslační modifikace MT mohou ovlivňovat jejich funkci ■ detyrosinace ot-tubulinu je evolučně konzervovaný proces - klíčovým enzymem je tubulin tyrosin ligáza (TTL) ■ detyrosinace ot-tubulinu koreluje s rozvojem některých nádorových onemocnění Shrnutí přednášky ■ struktura ■ dynamika ■ posttranslační modifikace Na konci dnešní přednášky by jste měli: 1. znát základní strukturu mikrotubulů (MT); 2. umět popsat způsob jakým řízena dynamika a stabilita MT; 3. znát základní postranslační modifikace MT. ____________________________________________________________________________________