ADV CELL BIOLOGY
ADV CELL BIOLOGY BIOL 401
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This 19 page Class Notes was uploaded by Renee Lehner on Wednesday September 9, 2015. The Class Notes belongs to BIOL 401 at University of Washington taught by Staff in Fall. Since its upload, it has received 26 views. For similar materials see /class/192334/biol-401-university-of-washington in Biology at University of Washington.
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Date Created: 09/09/15
Regulation of the Cell cycle In Interphase microtubules grow and shrink at the plus end Overview ofthe Mitosis What regulates entrance into prophase Regulation bl Re lication Com letion Reading Chapter 11 Activities of Cell Division Kinases Regulation by Growth Signals review Localizations of cyclin proteins that activate CDKs Condensation of Chromosomes Dynamics of Microtubules in Prophase and ProMetaphasu Grow and shrink rapidly Capture kinetochores of sister chromosome Functions of microtubule motors in prophase Reading Chapter 6 E D a tea boa 900 i39t i39e 900 o quot508 9 GT quot898 Wow Wm 51 3 0993 We ran PM 73900 90 Wu 0902 C9000 3930 C0900 coone Woo C9000 00093 la h C0090 Co000 W3 mom W qum Q3900 GTPliydrolysis quot3002 cm 0 0 ulGTPcaDDed 3 plusm cacao lasiermzrinew quotM mounnanmuonr I icaiastmvhe o o D o O o C o 0 0 0 0 m o so a 00 CECE OWOOO39 00 pm is 9 00 id 3 000 00000 wow moons maid shrlnkaQE RProteins anchored In the microtubule Organizing center Morgan 2007 Figures 64 and 65 addillun or 00 C GTPtullulm CDon rescue Search and Capture of Chromosomes by the Centrosomal Spindle Microtubule ux in metaphase and anaphase Morgan 2007 Figures 622 CLquot Morgan 2007 h wrux high lensiun avid kineluchnie movemem Figures 628 Malapnassz nulymerizallan llux Anlaphase39 Ananhasa ipalymelizatlnn 232008 Completion of Mitosis 1 Overview ofthe Metaphase Anaphase and Telophase 2 Dynamics of Microtubules in Metaphase and Anaphase 3 Sister chromatids are held together by cohesin and condensin 4 Entrance in to anaphase Securin and cyclin in the cell are tagged by ubiquitin multiubiquitinated amp degraded by Proteasomes 5 Spindle Defect orAnaphase Checkpoint Tension on the kinetochore and loss of kinetochore proteins are required for the cell to enter anaphase 6 Mad2 is activated on the kinetochore in prometaphase to block the Anaphase Eromotion Qomplex a ubiquitin ligase Tension of occupation by MT release Mad2 which is then inactive 232008 as all klnetochores are occupled are pulllngthe chromosomestowards oppOSlte plasma membranes Telnphase MT dlsassemblv Anlphase munmu mmwwmhw Flgure 1427 Karp 5mm ms moska um In Vertebrates Cohesin is Released from Sister Chromatids during Prophase Except at the Centromeres Most cohesin is released in prophase I 1 quot Fluorescent micrograph of metaphase human chromosome DNA is blue Kinetochores green Cohesin is red Fig 1415 Karp wed Scanning electron micrograph of paired human chromatids Fig 1415Karp 4 h ed Checkpoints in Cell Cycle Regulation DNA damage spindle delecl pruphase metaphase anaphase lelophase cylokinesis 1 Cohesin binds sister chromatids in Sphase and holds them together during 62 and partially in mitosis 2 Condensin compacts the chromosomes in prophase SPhase and Interghase 1 After replication DNA helixes of sister chromatids lnterphase are held together by cohesin g Proghase e 2 As the cell enters mitosis condensinis phosphorylated twig con ensm and compaction begins Prophase W i with condensin compressing supercoiled loops 2 3 Topoisomerase II a helix winding enzyme amp Condesin curl the DNA into tight loops 4 Cohesin is released from chromosomes except at Coh esin llti n etoc ho res Fig fgqmak fuusnu Emma we 5m mu m min a sum Degradation of Cohesin in Anaphase allows separation of sister ch romatids Morgan 2007 Figures 53 Initiation ofAnaphase 2 Active APCCchO ubiquitinates securin which leads to it 3555ng degradation by a proteasome i seculin sefaarase senarase sepavase 7 in mug unos o denim senamse Incanzanon pharyuiinn WWW was phalylalmn sewm niacine sepavase inaDIlVel laclive precursor Inacllvel Ana 39 Prophase and Metaphase 1 Securin secures the protease enzyme separase which degrades Cohesin Morgan 2007 Figures 711 Sisteisepaiallnn conesm EYEIlii Cllk m2 n 5 cycim desmmion securin I desiruciiun 1 232008 The anaphase checkpoint for aligned chromosomes 1APC is blocked by kinetochoreassociated proteins 2 Kinetochore occupation and tension biorientation release the checkpoint proteins Activated by the kinetochore are M Cdk CchO M Cdk Plk Spindlecheckpoint protein chromosome bioiienlalimi l m 0 In Emi1 desiruciiaii 11 quotin min Emit High activity of Emi1 in 62 and G1 blocks Cdc20 proptiase pmmataphase melaphase Morgan 2006 Figure 73 What are these spindle checkpoint proteins or kinetochoreassociated proteins Antibodies fluorescently label proteins on kinetochores Madl 317311 Hula Buth v o Shannon at al 2002 Mol Biol Cell 13 3706 Figure 5 23 C Alexa488 Mad2 injected in to cells shows Mad2 at unoccupied kinetochores iltl 39l lzli 22 Shannon at at 2002 Mol Biol Cell 13 3706 Figure 3 230 MadZ Ab injected into cells accelerates anaphase 232008 Madl FlO fragment injected into Cells at 23 C is a dominant negative and binds to Mad2 preventing Mad2 from blocking APC Bule Ab injection into cells accelerates anaphase Shannon et al 12002 MOI Biol Cell 13 3705 Figure 5 2350 The Big Picture APC Inhibitory Complexes at Kinetochores a Recruitment b Rearrangement and turnover 6 Cdc20 APC inhibition by activated Mad2 mum pool uh Do mm Winch Nicklas RB Waters JC Salmon ED Ward sc12001 J Cell Sci 114 23 4173723 Note 50le Mad 3 in yeast Entrance into Anaphase What happens to sister chromatids 1 Cohesinholds sister Metaphase Enter Anaphase chromatlds together chromatlds In metap ase 2 In anaphase securin that 1 quot inhibits separase is Smpm 9am i Amtm i polyubiquitinated l i J 5 by activated APC 5 l I 39 39 smn 3 Proteasomes degrade securin and cyclin K 5 quot I t 4 Separase a protease hi i39 i is no longer inhibited 1 I t E 5 Separase cleaves cohesin CoheSiquot 6 Sister Chromatids Figure 21719 Lodish et al 2003 232008 Entrance into Anaphasea What happens to MPF 1 Securln ls ublqulnnated bv cdczo actlvated APC 2 Securln releases separase whlch degrades mnesln 3 Mltmla cyalln IS ublqultmated bv thrl actlvated APC 4 Mltetle Cvclln ls degraded MPFlsii 7 All prmelns pnaspnmylated hv MPF are depnospnmylated MTrassoClate nrotems Nuclear Envelonrassoclated nrotems Condensm R N Separase Caczu ajja a 3 calm 1 Mlluhl LyleL l 3amp9 J 9 Muluuhns Kern ngure 14 27 gm ed Sum arv Onset of Prophase and Anaphase 1 Sister chromatlos are held together by cohesln 3 conoensln ounng repllcatlon 3 prophase 2 synthesls of cyclln onves the entrance lnto prophase MPF becomes an actlve sennythreonlne klnase 3 ln lheAnaphase Eromollon Qomplex a ublqultln llgase 4 Tenslon or occupatlon by MT release Maoz Whlch becomes lnactlve 5 APC onves the entrance lnto anaphase Securln ano Cyclln are tagged by ublqultln 3 degraded by Proteasomes 25 Cell enters anaphase Intellectual teasers What happens lrnelther secunn or cyclln are degraded What happens Ifonly secunn I egraoeo but not Cyclln What happens If only cyclln ls oegraoeo but not securln 232008 Meiosis Events and Aneuploidy in Mammalian Eggs 1 Meiosis Events Crossover and reduction of chromosome number 2 Restriction of replication in meiosis 3 Crossing over in diplotene 4 Structures of the chiasmata 5 Errors in spindle attachment and condensin formation 6 Aneuploidy in mammalian eggs Initiation of V V Glyeast I v Phases Of meiOSis gt v Meiosis Sphase Imel Imez Srcdk meioticSphase lltv ase meiotic 39 61 Sr dkl pmphase meiotic pmphase V homologous I Spolw gt gt V V recom ination 39 menus gt Name MCdk metaphase g A homolog segregation APC Z d quot Replication anaphasel I D block M Cdk gt meiosis l lt l metaphase 1 x Morgan haplmdgametes APCCMD gt V Fig 9392 8 9393 D anaphase 322008 Meiotic replication block requires MCdkl Wee1 blocks MCdk activity Wildtype Wee1 CY5 Fluorescent dUTP In Xenupus oocytes in the presence o f Weel sence of MCdkl activity DNA replicates after Meiosis 1 Noncrossover versus Crossover Meiotic prophase 1 Homologous recombination Double strand breaks by 5po11 n strandextension o strandextension r H mammalian i mm overhang capture xtension murmurrim DNA ligation 9 double Holiday iunetion in nunmm n mull DNA ligation Cleavage amp repair 9 resolution Holiday iunetion crossover strand stabi on at designated sites 5 unumnm Morgan Figure 95 Zygoten 322008 322008 Stages of Meiotic Prophase I 39 i iei Homoiog axes aiign in pairs 400 nm separation amp doubie stmnded breaks Zygotene 5 na sis or aciltin of homoiog pairs to 100 nm ormation ofthe synaptonemai compiex SCI the sites of crossing over in yeast 39 a all 39 pou 4 J 39 r PachyteneThe synaptonemai compiex in compiete and hLepmtequote1Emene homoiogs tightiy iinilted their entire iength 1 The synaptonemaicompiexdisassembies and the chromatin decondenses Diplotene Chromosome become highiy condensed and crossovers visibie as chaisma a Entry into the first meiotic division Spindie poies separate and homoiog pairs bioriented o Stages of Meiotic Prophase 8L selection of crossover sites 5pu11 mutant quot ZVEOtEHE Wild type Zygotene Pachytene m Synaptonemal Tm complexes Unaligned homologs Diplotene chiasmata In grass upper y 3 2 Morgan Fig 97 99 amp 910 322008 Spindle attachment during Prometaphasel amnnilelin l l l I Tension but separation prevented by sisterkinetochore fusion Anaphasel A phosphatase is required after anaphase to Disassemble the spindle synlalic nu hlurlanlaliun l l 4 7 x No tension unstable Matapl lasequot Here there is synlelic hiurientatinn and homologous chromosomes do not se arate l i p Anaphase i l Tension stable Cohesion regulates successful Anaphase a Cohesin distal to centromere stabilizes linkages b Premature separation lb U z 39 77 l 7 7 r c Crossovers too close insufficient linkage Lack of Checkpoint Control at the MetaphaseAnaphase ransition Meiotic Nondisjunction in Mammalian Female Mice Metaphase l Conclusion No Block at metaphaseanaphase transltlon for Chromosomes LeMaireAdkins R at al J Cell Biol 19971391611 l g a Lack of Checkpoint Control at the MetaphaseAnaphase Transition Meiotic Nondisjunction in Mammalian Female Mice Prometaphase XrChromosome Green Fluorescent Probe Metaphasel Metaphase ll Adkins R et al mm 11411 rim polar body 322008 322008 Lack of Checkpoint Control at the MetaphaseAnaphase Transition Meiotic Nondisjunction in Mammalian Female Mice Oocytes from 50 X0 female mice 0A 0 t f Amphase 10 ocv es mlquot XX female mlce Onset 30 Conclusion No delay of anaphase o onset for unaligned 6h 8h 10h 12h 14h 16h chromosomes in oocytes Time in culture In contrast Monovalent chromosomes in male mice results in metaphase arrest and spermatocyte degeneration LeMaireAdkinS R et al J Cell Biol 19971391611 Cohesin deficient female mice provide evidence for age related nondisjunction Wild type SMCl deficient Wild type SMCl deficient b c Pachytene cells stained for synaptonemal complexes Unpaired univalent chromosomes Shorter synaptonemal complexes and distal chiasmata ln cohesmdeflclent mlce in cohesmde cient mice diakinesisimetaphase chromosomes Hodges et al 81 PA Hunt 2005 Nature Genetics 37 1351 322008 Loss of cohesin inhibits chromosome condensation Wildtype females b W39ldultp igigg gfs centromere coheisin centrom ere SMClde cient females centromere Hodges et al amp PA Hunt 2005 Nature Genetics 37 1351 Futher scewing of chiasmata placement increased with age of females with deficient cohesin Sites of chromosome exchange 2 months old 4 months old in oocytes becomes distal with age female mice female mice b i 17 day mice Peiceiilage oi Iolai exchanges Proximal lriiersliiial Distal Placemenl of exchanges u chiasmala Hodges et al amp PA Hunt 2005 Nature Genetics 37 1351 Human and mouse oocyte exhibiting congressional failure in Meiosis amp II in age induced females Unaligned Chromosomes Human Mll Polal body Human Mll Mouse Mouse in latel Mouse light p olllelaphage plomelaphase Metaphase I Melaphase spindle wilholll Melaphase Melaphase spindle willle Hodges CA et al a PA Hunt 171171 2002 Meiosis Checkpoints but lack of Anaphase metaphase Checkpoint Mammalian Oocytes cause Ameuploidy 1 DNA replication is a checkpoint mediated by MChk l 2 Crossing over requires regulator proteins to allow sister chromatid separation 2 Lower amounts of cohesins in agesimulates mice cause aneuploidy 5 Mitotic spindle detects occur in ageSimulated mice causing aneuploidy 322008 Cytokinesis The Separation of the Segregated Chromosomes and Cytoplasmic Organelles How is cytokinesis accomplished What special structures are used Where does it occur What are the signals actin and myosin laments of the contractile ring Alberts 5m ed Figure 1749 actin nunvmusnle myosin ll Mechanism l moi culled call all imam of I l aclln aciln heavy cilaln liglli heavy chai contra Ctlle monomer lilamenl Clerminus chains Nlermlrlus c H 53quot Actinmyosin nlemblane Sliding Mechanism assembly ol aclln mycsln bundles 18point type D I cantraclinn n l M 39 b barbed end p pointed end Morgan Fi ure 83 20point type Examples of Cytokinesis Mechanisms in malauhasb auaphase m aciIrHrlyoslrl mg myosin mg S cerevisiae medial rmg aclirHrlynslll rlrlg i i l I K aDlInmyuslll mlg l central splndla 00 n1 mshphase anaphasa n1 5 llamlla i i i i animal cells Morgan Figure 8 1 Evidence for Actin and Myosin at the Contractile Ring Myos39l n H Dlviding Slime mold Actin goes into the plane of the cortex remaining inlerpclar microluhules Irom central spindle 05 pm Alberts 5quot ed Fig 1750 contractile ring nlaclin and 2182008 2182008 In large cells and in Bacteria and Plants the In large animal cells the membrane must be added membrane and cellulose must be added to the plane of the cell division to the plane of the cell division Cleaving Frog Xenopus Egg Cleaving Xenopus Egg lb 7 central spmme memmane vesmle lurmw nucmlubula array I ll zoo m 3338 minim Aberts 5th ed Figure 1749 a o In plants membrane and cellulose must be In plants membrane and added to the plane of cell division cellulose must be added l 6 Actin and MT mark cell plate site GI Vesicles deposit cellulose precursor n2 celliwzn ceumemnm aletelnphase phnfmoplaslmlcrmubules to the plane of cell division A plant cell in telophase 9mm and almkmmhukx rlnnlnl nllnlupuhr main quotmy 1 and mu Illmania mm mluolnbulg lntnphue mm qu A l mamnran cell plale assembly 20 plala VBSlClES malnx muwpha e band lale annphas 5th and 1757 Morgan Figure 88 The plane of cleavage is determined by the position of the spindle fibers W mmiumn BI m j EE L m j C EEIEIWEI Aiberts Figure 1753 and Morgan Figure 8715 The plane of cleavage is determined by the position of the spindle fibers chromosomes centrosome glass bead QM 1 into the ell displaces on one side of cell the spindle produdng a binucleate X ra C eavage p ane 999 y 39 lt 4 g 39 59 cleavage otcurs both between the entrosomes linked by mitotk spindles and between the two centrosomes that are simply adjacent and four daughter ells are formed both nuclei enter mitosis Aiberts Figure 1754 The plane of cleavage is determined by the position of the spindle fibers 150 minutes 4 may live Rackciciiur Universiiy Press 159 minutes Positioning of cytokinesis in a sand dollar egg 39 153 minutes Cytokinesis is earlier r amp not cente Mor u Mm gan Figure 8714 Mechanism of cleavage plane determination 39 Rho erGEF Travel Slgnls on the RhoGAF gt PebbleEel spIndle fIbers Rho GTPase likely Rho travels on the MT to lormm Rhuvaclivaled kinase the Plasma Membrane momm unigssgi ialasa RhoGTP stImulates myosin activation aclin iilamenl RMLC pnuspncrylauon matiun myosin Ii aciivalmn Aiberts Figure 1752 and Morgan Figure 876 mg cammwon 2182008 Mechanism of cleavage plane determination Rho GTPase likely travels on the spindle fibers Cytokinesis regulators at the central spindle of human cells Rho GTPase red and Cyk4 green Control of cytokinesis by RhoGEF Pebble in a Drosop embryo a wild type b Pebble mutanl 0 wild lyire Nuclei in GREEN Plasma membrans in RED HhuGEF Pebble mutants have more PebbleE512 than one nucleus per cell Morgan Fig876 amp 877 g HhuGAP gt n embryos the spindle rotates in metaphase to determine the next plane of cleavage IC elegans Dynein motors may rotate the spindle complex a rquot quotw m quot E Morgan Figure 819 A Beginning of cytokinesis 8 Crosssection at Cyt4 controls Rho activity the contractile ring at the furrow Rho coincides with the actin ring Alberts 5m ed Figure 1755 2182008 The final fusion event is not K understood 39 Aberts Fig 17 51 rpolarmicmtubules dy J gt Ir m ufrgals a 399 at 39Ww dense matrix material plasma membrane The rst 13 division without cytoplasmic division 1 lenlllled egg DIVI5ONS w Embryo NUCLEAR MIGRATION 1 T0 contzx mary widen ulitle plasma a syucyuum memb39l Variations in The Drosophila Embryo cellularizes after cycle 13 Cytokinesis in Early Embryos The Drosophila n Alberts 1759 Morgan 816 quot mm m m nucleus myosm ll basal lull Morgana17 a d o o l The Drosophila Embryo cellularizes after cycle 13 1 Rain militant c was Mature micmtubules Golgi bodies merge Wild we Microtubules in GREEN and Golgi protein in RED Dynein is required for movement of the Golgi vesicles To the apical surface and membrane formation Morgan 818 Cytokinesis The Separation of the Segregated Chromosomes and Cytoplasmic Organelles How is cytokinesis accomplished What special structures are used Where does it occur What are the signals r if 39 Reading Chapter 8 39 Morgan 2007 actln and myosm filaments of the contractile ring Alberts 5 h ed Figure 1749 2182008
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