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PDF Printing 600 Cellular distribution of microtubules: Mitotic cell (red: metaphasechromosomes) ( tt.irJ,^t*trt f,or,^ / Interphasecell: tal r*'.,t't,'. sgi.It. ) Centro s ome (Microtubule organ izing center) ("MTO C" ) In an interphasecell (G2, S, G2) microtubules(MT's) are arrangedin a dynamic array of long fibers that "$ PHASE =*--- radiateout from the centrosometo fill the entire i mitosis -\\ , {nuclear cytosol. I divisionl cytokinesis\ {T:Fx:\* {cytoplasmic \ In mitotic cells ("M phase")(upper left insert),the cytosolic arrayof MTs reorganizesto form the mitotic spindleapparatus. S PHASE {DNA replication} Figure li*3. MDlPcr,larBiology of th6 Cell, +th Erjition. r31 Structure of tubulin and its organization in microtubules: Tubulin = α + β polypeptides. (α & β bind tightly Tubulin has a (+) end and a (-) end. Subunits come to each other, don’t come apart; act as a single unit.) on and off at both ends, but at concentrations of tubulin exceeding the critical concentration, net th Fig 20-3 (5 Ed.) “Tubulin” addition of tubulin onto the (+) end is more rapid than at the (-) end. Fig 20-7 (5th Ed.) ( - ) ( + ) ( - ) ( + ) Fig 18-9 (6th) (microtubule = 13 protofilaments aligned side by side, slightly staggered upward) Fig 18-3 (b) (6th Ed.) This figure shows the seam along the length of the microtubule: (in vitro experiment, showing that tubulin adds more rapidly to (+) end than to (-) end 140 Orientation of cellular microtubules: Interphaseanimal cell interBhare --".-f\ Basalbody cell Flagellun or cilium Nucleus Centriole MTOC (qke "c e*t ro sc*ac") centrosome Mitotic animal cell romosome A:6fi Centrio le Nerve cell +| - Dendrite -) Axon +) (*) (+) (-) t+)t*) Nucleus MTOC,,' \-r Cellbody ll{l Two well-known drugs that affect microtubulesare Colchicineand Taxol At high concentrations,colchicine (and a syntheticderivative, colcemide) causes depolymerization of microtubules;at low concentrations,it"freezes" microtubules. When addedat low concentrationsto growing cells, cells accumulatein M phase,where they are blocked in metaphase.(After washing away the colchicines,cells resumemitosis.) CH.,S-"' -\d".*F"', ../---r '. Y ,Nt"{**C -.Cll? (^\lo cotc'a*'J' D"Jr"t ' [, a.s i. li ..! ,l t CI t1T', cH*O-*v'\"***rf\ N ocoJ" e"l ") cH,s''(r'- ! i \ i \o***y'r"* i *{HN *c{chi*in* " o..rer-5t^1, l,trg" HT,' laol Taxol hasthe oppositeeffect: it hlper-stabilizesmicrotubules, driving the equilibrium in the cell such that all tubulin in polymerized,and dynamic instability is lost. This is just a bad for the eventsof mitosis as MT depolymerizationor "fteezing" causedby colchicines. Taxol has proved to be a very effectiveanti-cancer drug. {.} ll H 3C*f *{3 f'13{ P^id-t Y.-*' ,r-*h l O*1*Ct"l3 r' tr., (-\-g-r-' ., (6"tk : !orerc< {A} taxnl t) - t f".1"1) 1"****J {8} 1Sgm Figure 1S*21.Moleeular Biology of the Cell, ilth €ditisn. tl{1 The centrosome(microtubule organizingcenter) contains a pair of orthogonal centrioles,and many y-tubulin ring complexesimbedded in an amorphous pericentriolar matrix: Fig20-13 Fig5-33 ###ffi , o'5Pm , Centrosome(yellow) Centriolepair (locatedin centrosome) Fig 16-23(Alberts et al, Molec Biol Cell, *.d.) L nueleatingsites {ltubulin ring complexesl .++ * microtutrulesgrowlng lrom y-tubulinring ccmplexe* tA] {E} of the centrosome Figure 16-23"Moleculsr Eiology o-fthe Cell, 4th Edition. h5 Microtubules exhibit oodvnamicinstabilifv" in cells: Fig 20-10: Fluorescentlylabeled MT's in a living cell; time intervals Fig l6-l I uq\ ' YTnto' lrit rc. i*rl. t;h{i Fig20-9 50 Catastrophe E g- 4o v -30 0) -e #*sfr- a20 Ass*nrbly .x 10 ]ii;., = 0 lessstable regionof W Time (min) microtubule c0ntaining GDP-tubulin dimers ffi GNOWING SHRINKING {c) Figure16-11 part 3 of 3. MolecularBiology of the Cell,4th fdition. t+T RESEARCH ARTICLES globulin G), was found to be conformation- microtubules (Fig. 1, B and C),which suggests microtubules were stained by MB11, which sug- specific. It did not recognize denatured tubu- that it recognized a conformation and not the gests that some microtubules possessed confor- lin by immunoblotting and seemed not to bind nucleotide itself. mational defects under these conditions. to native nonpolymerized tubulin. However, We then used hMB11 to stain by immu- Detection of tubulin in GTP conformation hMB11 cosedimented specifically with microtu- nofluorescence a mixture of microtubules po- in cellular microtubules. We next used hMB11 bules polymerized in the presence of guanylyl lymerized from pure tubulin in the presence of to localize GTP-tubulin in cellular microtu- 5′-(b,g-methylenediphosphonate) (GMPCPP), a GTP or GMPCPP (Fig. 1D). Under these con- bules by immunofluorescence. Because of its nonhydrolyzable GTP analog, and not with con- ditions, hMB11 stained only GMPCPP micro- conformational binding, hMB11 staining was trol microtubules assembled in the presence of tubules [representing 68.6 T 17.3% (SD) of very sensitive to structural alterations occurring GTP (Fig. 1A). In this experiment, low concen- MB11-positive microtubules] and not control after fixation (10). It was best to use unfixed trations of taxol (0.1 to 1 mM) were used to pre- microtubules (1.8 T 0.9%). The remaining 29.7 T cells permeabilized in the presence of glycer- vent depolymerization of control microtubules. 16.6% were bundles of both GMPCPP and ol and/or low taxol concentration to prevent When a higher concentration of taxol was used, control microtubules. Despite varying experi- microtubule depolymerization. In three repre- hMB11 bound to both control and GMPCPP mental conditions, not all GMPCPP-containing sentative cell lines (HeLa, Ptk2, and MDA- on November 27, 2008 www.sciencemag.org Downloaded from Fig. 4. A GTP-remnant model for microtubule dynamic instability. (A) kymographs show the dynamics of the microtubules highlighted in red Model for microtubule dynamics showing GTP-tubulin (red) in a GTP cap and yellow (top) aligned with hMB11 staining (bottom). Note the good during polymerization (P) and in inner microtubule regions. Upon cap coincidence of rescue position and GTP remnants. (C) Quantification of loss, the probability of catastrophe (C) increases and the microtubule experiments done as in (A), showing the proportion of polymerizing depolymerizes (D) until its end reaches a GTP-tubulin remnant. A GTP end microtubules stained by hMB11 at their plus ends and the proportions of is restored and the probability of microtubule polymerization increases, GTP-tubulin remnants that colocalized with rescue locations in Ptk2 cells allowing its rescue (R). (B) Ptk2 cells stably expressing GFP-tubulin were (means T SEM). The proportion that would be expected in stochastic con- imaged at the indicated times. Rescue events (colored arrows) and the tip ditions is shown for reference at the right (Monte Carlo simulation, table of a growing microtubule (arrowhead) are indicated. After cytosol extrac- S1) (9). The table shows that the rescue frequency varies with the dis- tion, cells were stained with hMB11 (red) and imaged again, often show- tribution of GTP-tubulin remnants (means T SEM, comparison of Ptk2 and ing GTP-tubulin remnants at rescue locations. Scale bar, 10 mm. The two RPE1 cells) (9) (table S1). www.sciencemag.org SCIENCE VOL 322 28 NOVEMBER 2008 1355 GTP cap modelfor dynamicinstability: Hllx.l.:+r_J_r_d (gl.D_U_LL lfl.k- --]l/1st-owonowrx) (o{olol iIi\ ll Phase transition + GDP ffi ar"^rr,-"LossoF,^r"^rr(r' /:./l W ' !\ ffi ffffi I @ *drn / ffi G]Ff!D g*vE Growing phusc Shrinkingphirsc ffi Fig. 5 Modcl for micotubule ends exchanging subunits with Figure6. GTP Cap Modot tor Dynamic hstabitity soluble dimer. Thc opcn circlcs rcprcscnt tubulin ligandcd with Miclotubul€snlth e cap ot GTp-containlngsubunits, domtod by T, aro GDP, and the closcd circlcs reprcscnt GTP-tubulin. Thc growing growing shorn skarly by lhe addition o{ T.containingsubunlls, The phasc of microtubules is stabilizcd by a GTP cap. This cep is of free T subunlls ar3 stx)ln b bo in oquitibriumwith the T-containing fluctuating lcngth, bur thc avcragelcngth incrcascc with frce tubutin end. with a wry lov, t(d$. At somo probebllity,GTp hydrot).siscatches conccnration. The shrinking phasc has lost this cap. lntcrconver- up wlth asgombly,th€ T cap dlsappears,and th€ polytnor transits to sion between thc two phascs is rclativcly rare. the'np1d rhrtnkagc'phsso (a[ D] s]roiln at the bottom,This polymer npktly losos subunlts from its cnd (hlSh KdJ, The GDp-contalning subunib thai ar€ reloascd,denot€d bV e erctrange with frse GTp in solution aod h,rm T subunits.lt is not knoronwhelher T 6ubunitscan add to a depdymerizing ond b recap the shrinking polymer. How cells might usedynamic instability to orientthe microtubulecytoskeleton: K Figure 11. Morghogen€sis by S€l€ctivo Stabilization In (A)' t|s depict an unpolarized c€ll wilh microtubules growing lhat are out rvlth no pretened direc{ion and that ar€ spontaneously shorlening by dynamic ins{ability. In (B)' a local oxtracellulsr signal adivates some capping structures near the cell periphery. In (C), the sete"d"" etabltiraiion ot those capping slrucluros gredually leads to a reorientalion ot th€ microtubule arra!r6. In (D), the polarization is completo, I,vithcapped microtubules much less dynamic than unpolarized micrcttubul$. Here we also shorv chemical modilication ot th6 less dynamic microtubules (as indicat€d by lhe dicts). tkx Microtubule AssociatedProteins (MAPs): Fig20-12 Sf9 insectcells expressingeither MAP2 (left) or Fig l6-31 (Albertset al, 4rt Ed) stathmin %. #ml* +:.;{-a='- """'r. i.- $ffi &-8 fr*e tuhulin trrbulinsubunit O subunitaddi tion sropr ops e soquastered pool shrink* hy *tathmin GTPI,.tr f hydr'drollysis catch:chessup #i I' @qb tb) Microtubule tu;q"s@ MAP2 Figure 1S-31.Molecular Eiology of the Cell,4th Edition. jii:i .{irr i.j!' 'i.:ttfii iitJ 25 nm 25 nm t-J Tau (greenstaining) is confined to the axon of a (ott-f) hippocampal neuron (long branched structure on ,l4q ( / the left), whereasMAP2 staining (orange)is t* de* J" iit.
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