GLI EVENTI CRITICI DELLA MITOSI SONO PRINCIPALMENTE

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1 GLI EVENTI CRITICI DELLA MITOSI SONO PRINCIPALMENTE
RIFERIBILI ALLE DINAMICHE DEL DNA E DEI MICROTUBULI 1

2 I centrioli

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4 3 tipi di microtubuli Astrali= tengono il fuso attaccato alla membrana Polari= tengono il fuso in asse Del Cinetocore=agganciano i cromosomi Il FUSO Mitotico

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6 COME SI ALLINEANO I CENTROSOMI? KINESIN RELATED PROTEINS

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9 Schematic depicting Eg5 activity in the mitotic spindle
Schematic depicting Eg5 activity in the mitotic spindle. Tetrameric Eg5 motors (red) help organize microtubules (green) to form the mitotic spindle. (A) At the onset of mitosis, the duplicated centrosomes (blue) separate and nucleate two microtubule asters. Processive Eg5 motors may translocate to the plus-ends of microtubules, located distal to the centrosomal organizing center and by crosslinking antiparallel microtubules, may promote bipolarity. (B) By metaphase, a stable bipolar spindle has formed. Eg5 motors likely provide structural integrity and also slide microtubules toward the centrosomes, contributing to the generation of poleward flux. (C) A close-up depiction of Eg5 motors walking to the plus ends of antiparallel microtubules, moving both poleward simultaneously.

10 COME SI SEPARANO I CENTROSOMI? KRP BimC DINEINA

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14 I motori proteici

15 CHINESINE E DINEINE REM NMR

16 PROPRIETA’ DELLE CHINESINE
passo = 8 nm forza = 6 pN 3 tipi: N - motore N-terminale >>> estremità + M - motore centrale >>> estremità + C - motore C terminale >>> estremità - 2 classi: chinesine citosoliche KIF1A, KIF1B chinesine del fuso CENP-E, ncd, BimC

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21 PROPRIETA’ DELLE DINEINE
peso molecolare elevato funzionano insieme a MBP 2 classi: dineine citosoliche dineine dell’assonema

22 Prometafase: scompare la membrana nucleare
                                                                                                                                                                                                                                                     Spindle MTs capture chromosomes e i cromosomi si fanno catturare

23 COME I MICROTUBULI SI LEGANO AI CROMOSOMI?
cromosoma metafasico centromero cinetocore M.del cinetocore cromatide

24 Il passaggio da due gruppi di microtubuli astrali organizzato dal centrosomi (a sinistra) alla tipica struttura fusiforme dela metafase (a destra) richiede la creazione di connessioni tra i cromosomi e poli del fuso attraverso microtubuli dinamici. (B, C) modello di ricerca e cattura per la congressione cromosomica. Nella formulazione originale di ricerca e cattura (B), lo spazio attorno cromosomi passivi viene saggiato da microtubuli che si allungano e si accorciano (rosso) che si originano dal centrosomi (centrioli in verde). I successivi eventi di cattura (1) sui cinetocori fratelli risultatno nel in bi-orientamento e nella congression (2). Ulteriori meccanismi di ricerca e cattura (C) avviata dai cromosomi. fibre K nucleate dai cinetocori attraverso un gradiente RanGTP (blu) si allungano verso la periferia e cattura i microtubuli astrali (3). Essi sono successivamente inserito nel polo attraverso un trasporto dipendente dalla dineina.

25 COME VENGONO ‘AGGANCIATI’ I CROMOSOMI?

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27 cinetocore centrosoma

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30 COME I MICROTUBULI SI LEGANO AI CROMOSOMI?
CROMATIDI CROMATINA corona fibrosa microtubulo CINETOCORE piastra esterna piastra interna 30

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34 COME SI MUOVONO I CROMOSOMI? The pushing and pulling forces drive the chromosomes to the metaphase plate MT behavior during formation of the metaphase plate. Initially,MT from opposite poles are different in length. 

35 COME SI MUOVONO I CROMOSOMI?

36 Movimento della tubulina M.del cinetocore M.polare M.astrale centrosoma cromosoma cinetocore Disassemblaggio Assemblaggio

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39 Postional cues for chromosome congression may be derived by integrating two force gradients in the spindle, the polar ejection force and the traction fiber mechanism. A chromosome (blue) moving from left to right is shown. Red arrows indicate translocation of the traction fiber with the number of arrows proportional to the length and therefore the forces acting along the kinetochore fiber. The blue arrows correspond to the polar ejection force. This force is predicted to decrease as the distance from the pole increases. The kinetochores (red) are under tension and stretched (pulled apart) due to forces acting on chromosomes. The magnitude of tension at kinetochores can regulate the movement of a chromosome.

40 rimozione veloce rimozione lenta
METAFASE ANAFASE rimozione veloce rimozione lenta rimozione aggiunta

41 COME SI SEPARANO I CROMATIDI?
ANAFASE A ANAFASE B

42 Laser Ablate Kinetochore Mono-oriented Chromosome
I cinetocore generano segnali di controllo 20 min. Variable 20 min Laser Ablate Kinetochore from last Mono-oriented Chromosome

43 Monitoring correct attachment to spindle
Sister chromatids are held together by cohesin proteins… Any kinetochore not experiencing tension  block destruction of cohesins So, no sister separation until all chromosomes are ready! Separase: can destroy cohesins Unattached kinetochore: blocks separase

44 Monitoring correct attachment to spindle (cont’d)

45 Monitoring correct attachment to spindle (cont’d)
Anaphase begins! Correct attachment

46 The anaphase entry checkpoint
Unattached kinetochore separase active! cohesins Sister chromatid separation

47 The anaphase entry checkpoint—genetic analysis
separase (non-functional) mutation*… phenotype? cells stuck in metaphase cohesin (non-functional) mutation*… phenotype? premature sister separation Double mutant phenotype? premature sister separation! *how to keep the strains alive? …use temperature sensitive mutants

48 Checkpoints Examples:
Cellular surveillance systems to monitor the integrity of the genome and of cellular structures Enforce the correct order of execution of cellular events. Examples: Chromosomes not attached to spindle  block onset of anaphase DNA is damaged  halt the cell cycle to allow repair Irreparable DNA damage  trigger cell death

49 Potential Checkpoint Targets:

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51 Unattached Kinetochores Cause a Checkpoint Delay

52 The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547

53 APC is the Target of the Mitotic Checkpoint
From Zhou et al. (2002) J. Cell Sci. 115:3547

54 Both Attachment and Tension are Monitored
3F3 Epitope Phosphorylation of unknown proteins -dims upon attachment -disappears upon tension

55 Spindle Assembly Checkpoint Pathways
Securin Separase Adapted From Susan Forsburg, Salk Institute

56 Mitotic Exit is a Spindle Checkpoint Target
Bub2 active: NO Cdc14 Release Mitotic Exit Network (MEN) Bub2 inactive: Cdc14 Released From Susan Forsburg, Salk Institute

57 APC is the Target of the Mitotic Checkpoint
Cdc2/Cyclin B Mad2 Mad3/BubR1 Bub2 Cdc2/Cyclin B Adapted from Peters (2002) Mol. Cell 9:931

58 Spindle Assembly Checkpoint Pathways
Securin Separase From Susan Forsburg, Salk Institute

59 Checkpoint is Mediated by Rearrangement of
Mad2 Complexes Mad2 Inhibits APCCdc20 From Millband et al. (2002) Trends Cell Biol. 12:205

60 Mad2 Cycles Through Kinetochore and Inhibits Cdc20
Shah and Cleveland (2000) Cell 103:997

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65 BubR1 (vert) Protein Kinase
Spindle Assembly Checkpoint Components Gene Molecular Function Checkpoint Function Bub1 Protein Kinase Transduction Bub2 Tem1 GAP (Mitotic Exit) Bub3 Kinetochore Scaffold Mad1 Generates Mad2/Cdc20 Complex Mad2 Inhibits Cdc20 Mad3/BubR1 BubR1 (vert) Protein Kinase Mps1 Ipl1 Corrects Mono-orientation CENP-E Molecular Motor Trasduction, Congression Dynein Congression, Checkpoint Off

66 In profase le proteine del cinetocore sono fosforilate
SPINDLE CHEKPOINT In profase le proteine del cinetocore sono fosforilate Bub1 e 3 (proteine del checkpoint) si legano al cinetocore Mad2 si lega alle pp del cinetocore Mad2 inibisce CDC20 CDC20 attiva APC APC degrada Pds1(Cut2) (securina) Pds1 inibisce Esp1(Cut1) (separasi) Esp1 degrada Scc1 (coesine) Scc1 tiene uniti i cromatidi

67 METAFASE MPF attivo MPF PROFASE Ciclina B cdk poliubiquitinazione proteasoma ATP ADP sintesi di ciclina B APC inattivo APC attivo ANAFASE TARDIVA INTERFASE MPF inattivo TELOFASE

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73 The cohesin cycle. During telophase or G1, cohesin loading depends on Scc2 and Scc4. During S phase, establishment of cohesion requires Ctf18, Eco1 and other proteins, as well as cohesin. At the onset of anaphase, APC/C–Cdc20 promotes degradation of securin, thereby activating separase, which cleaves cohesin.

74 Overview of the licensing and cohesion cycles
Overview of the licensing and cohesion cycles. A small segment of chromosomal DNA, encompassing three replication origins, is shown during G1, S and G2. Mcm2–7 and cohesin are loaded during G1. During S phase, the Mcm2–7 complex is displaced from DNA as it replicates, and cohesion is established. During anaphase, cohesin is cleaved, thereby allowing segregation

75 Figure 17-24 Molecular Biology of the Cell (© Garland Science 2008)

76 COHESIN

77 METAFASE Inibitore dell’anafase coesine coesine

78 METAFASE Inibitore dell’anafase coesine APC attivo coesine

79 APC attivo METAFASE Inibitore dell’anafase coesine coesine
ubiquitinizzazione

80 METAFASE Inibitore dell’anafase coesine coesine

81 METAFASE Inibitore dell’anafase coesine coesine

82 METAFASE Inibitore dell’anafase coesine coesine

83 ANAFASE

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85 19_18_envelope breaks.jpg 19_18_envelope breaks.jpg

86 CYTOKINESIS - The plasma membrane begins to invaginate due to the action of the contractile ring of actin and myosin fibers inside the cell. This ring always orientates itself along the metaphase plate 19_19_cleavage_furrow.jpg 19_19_cleavage_furrow.jpg

87 LA CITOCHINESI DEVE AVVENIRE NEL POSTO GIUSTO E NEL MOMENTO GIUSTO
Microtubuli del fuso mitotico Filamenti di actina dell’anello contrattile

88 Actina e miosina

89 COME SONO RIPARTITI GLI ORGANELLI
TRA LE CELLULE FIGLIE? Gli organelli della via secretoria vengono frammentati in vescicole che formano clusters e vengono ripartiti tra le cellule figlie.