The Guarantee of Mitosis…
The
2 daughter cells formed are
identical to each other and
identical to the mother cell.

Why is this so important?
1
Essential Features of Cell Division
1. Transmit a complete copy of
genetic information (DNA)
• Transmit materials necessary for
cell to survive and use genetic
information
• Maintains chromosome ploidy of
cell
In Mitosis, each daughter cell
is exactly the same as the
original mother cell.
2
In meiosis, chromosomes replicate
once, nuclei divide twice
3
Comparison of meiosis and mitosis
4
CYTOSKELETON= complex network of filamentous proteins
extending throughout the cytoplasm
Three types of filaments:
Microtubules
Tubulin
Actin Filaments
Actin
Intermediate Filaments
Vimentin, Lamin
5
The eukaryotic cytoskeleton
6
Cell shape
FUNCTION:
Motility:
crawling,
chemokinesis
chemotaxis
endo- and exo-cytosis)
Anchoring of organelles
and cellular structures
Organelle movement
Chromosome movement
Cell polarity
Tensile strength
7
8
 Microtubules provide
an organizational
structure in an
interphase cell and
separate
chromosomes in a
dividing cell
 Centrosome and
spindle fibers
9
10
11
The Orientation of Microtubules in a Cell
12
13
14
15
16
Il centro organizzatore dei microtubuli (MTOC)
Cellula ovarica di criceto
cinese trattata con anticorpi
per la tubulina e per una
proteina del centrosoma.
MTOC di una cellula animale. I
due centrioli (C) sono
perpendicolari e circondati
dalla matrice pericentriolare; i
microtubuli (M) sono in giallo.
17
Microtubules are long hollow cylinders
made of the protein tubulin
Le subunità globulari di tubulina (ognuna
delle quali è lunga circa 8 mm) formano le
pareti di questa struttura cilindrica
18
An α, β-tubulin heterodimer
is the basic structural unit of
microtubules.
The heterodimer does not
come apart, once formed.
α
GTP
β
GTP
tubulin
heterodimer
The α & β tubulins, each about 55 kDa, are homologous
but not identical. Each has a nucleotide binding site.
 α-Tubulin has a bound GTP, that does not hydrolyze.
 β-Tubulin may have bound GTP or GDP.
Under certain conditions, β-tubulin can hydrolyze its
bound GTP to GDP plus Pi, release Pi, and exchange
bound GDP for GTP.
19
A microtubule is a hollow
cylinder, about 24 nm in
diameter.
Along the microtubule axis,
tubulin heterodimers join endto-end to form
protofilaments, with
alternating α & β subunits.
Staggered assembly of 13
protofilaments yields a helical
arrangement of tubulin
heterodimers in the cylinder
wall.
β-GTP
α-GTP
β-GDP
α-GTP
+
−
20
A
A
B
A
B
C
In doublet & triplet microtubules the wall of one
microtubule partly consists of the wall of an attached
microtubule.
The A tubule is a complete microtubule cylinder, made
of 13 protofilaments. “Piggyback” B or C tubules are
made of less than 13 protofilaments, usually 10.
21
α
GTP
β
GTP
tubulin
heterodimer
GTP must be bound to both α and β subunits for a
tubulin heterodimer to associate with other
heterodimers to form a protofilament or microtubule.
During in vitro microtubule assembly, heterodimers
join end-to-end to form protofilaments. These
associate laterally to form sheets, & eventually
microtubules.
22
α
GTP
(−)
β
GDP
α
GTP
protofilament
β
GTP
(+)
Heterodimers can add or dissociate at either end of a
microtubule in vitro, but there is greater tendency for
subunits to add at the plus end, where β-tubulin is
exposed.
Subunit addition brings β-tubulin that was exposed at
the plus end into contact with α-tubulin.
This promotes hydrolysis of GTP bound to the now
interior β-tubulin. Pi dissociates, but β-tubulin within a
microtubule cannot exchange its bound GDP for GTP.
The GTP on α-tubulin does not hydrolyze.
23
α
GTP
(−)
β
GDP
α
GTP
protofilament
β
GTP
(+)
The minus end of α-tubulin may contribute an
essential residue to the catalytic site of βtubulin.
Thus the minus end of an α subunit may serve as
GAP (GTPase activating protein) for β-tubulin of
the adjacent dimer in a protofilament.
24
L’assemblaggio dei microtubuli è mediato
dalla γ-tubulina
Complessi ad anello (γ-TuRC)
isolati da Xenopus
γ-TuRC (γ-Tubulin Ring Complex)
Due modelli di assemblaggio
mediato da γ-tubulina
25
26
β
27
28
Polymerization of tubulin
29
Microtubule dynamics
Microtubule polymerization/depolymerization depends upon:
•Temperature
•Concentration of αβ tubulin dimers
30
Effect of [α/β tubulin dimers] on
microtubule assembly
31
32
Fraying or curving of protofilaments is observed at the ends of rapidly
disassembling microtubules.
This may be due to a change in conformation when β-subunits of
heterodimers at the plus end have bound GDP instead of GTP.
33
34
Dynamic Instability In Vivo
Dynamic instability of microtubules in vivo may
be regulated by interaction with other proteins
at the (+) end.
E.g., during prophase of mitosis, microtubules
grow out from the centrosome. If the (+) end of
a
microtubule
makes
contact
with
a
chromosome, the end becomes stabilized.
Otherwise rapid disassembly at the (+) end
ensues, and the tubulin dimers are available for
growth of another microtubule.
35
Allungamento ed accorciamento in vivo di singoli microtubuli.
Tubulina fluorescente microiniettata in fibroblasti umani;
cicli di depolimerizzazione (a freddo) e polimerizzazione
(37°C) e analisi a tempi differenti.
36
Polymerization of microtubules
37
Dynamic instability of microtubules
38
Instabilità dinamica
Durante la transizione Interfase/Metafase si osserva:
• aumento della emivita media (da 5 minuti a 15 secondi)
• aumento del numero di microtubuli
• aumento del ritmo nucleazione
39
Toxins & Drugs
Some toxins and drugs (all of which inhibit mitosis)
affect polymerization or depolymerization of
tubulin:
 Taxol, an anti-cancer drug, stabilizes
microtubules.
 Colchicine binds tubulin & blocks polymerization.
Microtubules depolymerize at high [colchicine].
 Vinblastine causes depolymerization and
formation of vinblastine-tubulin paracrystals.
 Nocodazole causes depolymerization of
microtubules.
40
La colchicina e il taxolo
interferiscono con
l’assemblaggio e il
disassemblaggio dei
microtubuli.
Questi farmaci hanno
un effetto antimitotico
efficace soprattutto
per le cellule a rapida
divisione, come le
cellule tumorali.
41
Stabilizzazione microtubuli
 Estremità protetta da MTOC
 Estremità +
inizialmente libere, protette poi
da proteine di corteccia cellulare
 Cellula non polarizzata
polarizzata
42
43
Organizzazione dei microtubuli (A) e delle
proteine associate ai microtubuli MAP (B) in
cellule HeLa in interfase
A
B
Colorazione con anticorpo anti-tubulina (A) e anti-MAP4 (B).
La disposizione colineare della MAP4 e dei microtubuli indica
una loro interazione.
44
PROTEINE ASSOCIATE AI MICROTUBULI (MAP)
1)ALTO PM: 200-300 Kda (MAP1-2)
sequenza aa conservata: LYS-LYS-GLU-X
2)BASSO PM: 55-62 Kda (tau)
2 domini funzionali
BASICO:
per i microtubuli
Diverse
ACIDO:
per altri componenti
cellulari
stabilizzano microtubuli formati
inibiscono dissociazione tubulina
se fosforilate non legano i microtubuli
funzioni
formano reti citoscheletriche
45
MAPs speed up nucleation and
stabilize microtubules
46
Dimostrazione che la distanza fra i
microtubuli (MT) dipende dalle MAP
 Cellule di insetto
transfettate con
DNA codificante
MAP2 o Tau
sviluppano MT.
 La distanza fra i
microtubuli in a) è
maggiore di quella
visibile in b).
47
48
49
Two main families of microtubule motor proteins carry out
ATP-dependent movement along microtubules:
1. Kinesin: Most members of the kinesin family of
motor proteins walk along microtubules toward
the plus end, away from the centrosome
(MTOC).
2. Dynein: The dyneins walk along microtubules
toward the minus end (toward the centrosome).
In each case there is postulated to be a reaction cycle
similar (but not identical) to that of myosin.
The motor domain undergoes conformational changes as
ATP is bound and hydrolyzed, and products are released.
50
Kinesins
Kinesins are a large family of proteins with diverse
structures. Mammalian cells have at least 40 different
kinesin genes.
The best studied is referred to as conventional
kinesin, kinesin I, or simply kinesin.
Some are referred to as kinesin-related proteins
(KRPs).
Kinesin I has a structure analogous to but distinct
from that of myosin.
There are 2 copies each of a heavy chain and a light
chain.
51
Motor protein: Kinesin
100 nm
52
microtubule
Cargo
proteins
bound by
kinesins are
diverse.
scaffolding
protein
kinesin
cargo
vesicle
receptor
Some organelle membranes contain transmembrane receptor proteins that
bind kinesins. Kinectin is an ER membrane receptor for kinesin-I.
Scaffolding proteins, first identified as being involved in assembling signal
protein complexes, mediate binding of kinesin light chains to some cargo
proteins or receptors.
53
scaffolding
protein
kinesin
microtubule
In absence of cargo, the
kinesin heavy chain stalk
folds at hinge regions,
bringing heavy chain tail
domains into contact with
the motor domains.
cargo
vesicle
receptor
inactive kinesin
In this folded over state kinesin exhibits decreased ATPase activity
and diminished binding to microtubules.
This may prevent wasteful hydrolysis of ATP by kinesin when it is not
transporting cargo.
54
Unfolding of kinesin into its more
extended
active
conformation
is
promoted by:
 phosphorylation of kinesin light
chains, catalyzed by a specific
kinase, or
 binding of cargo.
55
Per muoversi lungo i microtubuli, motori
proteici usano energia derivata da
idrolisi ATP:
 il legame di ATP sposta la proteina
motrice dalla conformazione 1 alla 2
 ATP legato viene idrolizzato ad
ADP+Pi (da 2 a 3)
 rilascio ADP legato e Pi (da 3 a 1)
 la proteina si muove in avanti
 ciclo irreversibile
56
Ciclo di idrolisi della chinesina
57
Kinesin-mediated movement (+)
Observations of conventional kinesin transporting
elongated particles have demonstrated that cargo
particles do not roll along the microtubule.
Instead kinesin walks along, maintaining the
orientation of a cargo particle.
58
Kinesin-mediated movement of
a microtubule
59
60
Strutture cristallografiche di teste di miosina
e di chinesina
I domini centrali di attacco al nucleotide della miosina e
della chinesina (ombreggiati in giallo) sono strutturalmente
molto simili
61
Kinesin and myosin
may cooperate in
vesicle transport
(−)
nerve cell body
axon with
microtubules
(+)
axon ending
with actin
cytoskeleton
Vesicles in extruded nerve axoplasm were found to attach
to and move along both microtubules and actin filaments.
Kinesins and myosin V are both associated with precursors
of synaptic vesicles.
Kinesin transports vesicles along microtubules in the axon
to the plus ends, where the axon ending begins.
Axon endings instead have an extensive actin cytoskeleton.
Myosin V may take over to transport vesicles along actin
filaments to near the plasma membrane at the synapse.
62
Dyneins are minus end-
MTOC
directed motor proteins.
They were first studied
in cilia & flagella.
Many cytoplasmic
dyneins have now been
discovered.
nucleus
(−)
(+)
microtubules
Cytoplasmic dyneins mediate ATP-dependent
retrograde movements of vesicles and organelles
along microtubules, toward the centrosome
(MTOC).
63
Dynein is large and
complex. Its structure
has not been fully
determined.
Cytoplasmic dynein has
a MW exceeding 106.
head
domain that
interacts with
microtubule
stalk
Dynein
(approximate
structure)
motor
domain
Dynein includes 2 or 3 heavy chains. Each is about 4600
amino acid residues long and includes a globular motor
domain.
There are also multiple intermediate and light chains.
Dynein also requires large complexes of other proteins to
mediate binding to cargo such as membrane vesicles.
64
domain that
Extending out from each
interacts with
motor domain is a narrow stalk
head
microtubule
that ends in a small globular
domain.
stalk
It is this domain at the end of
the stalk that interacts with
Dynein
microtubules.
motor
(approximate
structure)
domain
The stalk may help avoid
steric interference when
multiple dyneins interact with a microtubule.
The stalk is thought to be an intra-molecular coiled coil,
formed by interaction of α-helical segments on either side
of the microtubule-binding segment in the primary
sequence of the dynein heavy chain.
65
Motor protein: Dynein (-)
66
Modello per
l’attacco della
dineina ad un
organello
racchiuso da
membrana
67
Flagellar
Dyneins
68
Motor
Proteins
 Motor proteins bind to
microtubules and move
by cycles of
conformational changes
using energy from
ATP.
 One end of the protein
can bind to specific
cellular components.
69
Motor Proteins Transport
Cargo
70
71
Microtubules are conveyer
belts inside the cells. They
move vesicles, granules,
organelles like mitochondria,
and chromosomes
72
Microtubules Provide Tracks for
Transport
73
Vesicle transport in two
directions
74
Coinvolgimento del
citoscheletro nella mitosi
PROCESSO
AGENTE
MECCANICO
COMPOSIZIONE
PROTEINA
PRINCIPALE
PRINCIPALE
SOSTANZA
DISTRUTTIVA
CARIOCINESI
FUSO
MITOTICO
MICROTUBULI DI
TUBULINA
COLCHICINA,
NOCODAZOLO
CITOCINESI
ANELLO
CONTRATTILE
MICROFILAMENTI
DI ACTINA
CITOCALASINA
B
75
CARIOCINESI / CITOCINESI
 Divisione mitotica del
nucleo
 FUSO MITOTICO:
microtubuli composti di
tubulina
 Detta anche
“citodieresi”
 ANELLO
CONTRATTILE:
microfilamenti di actina
76
MTOC=CENTROSOMA
•Principale centro organizzatore dei
microtubuli
•MATRICE DEL CENTROSOMA:
contiene γ-tubulina e pericentrina
•Ha una coppia di CENTRIOLI
•Duplicazione, divisione, formazione dei
due poli del fuso mitotico
77
• The centrosome
provides nucleating
sites from which
microtubules can
grow.
78
Centrioles are cylindrical
structures, usually found in
pairs orientated at right
angles to one another.
The wall of each centriole
cylinder is made of nine
interconnected triplet
microtubules, arranged as
a pinwheel.
Centrioles at right angles,
as in the centrosome.
cartwheel structure
at one end
The interior of each centriole appears empty, except for a
"cartwheel" structure at one end.
In isolated preparations, electron micrographs show fibrous
structures connecting the two centriole cylinders.
79
During centriole duplication prior to mitosis (G1 - S
phase), the two centriole cylinders separate, and a
daughter centriole grows from a short disk-like
structure at right angles to each parent centriole.
80
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Formazione del fuso mitotico
83