Microtubules
To read: Lodish 5th ed.
Chapter 5.4
Chapter 20
Fig 3-23 and Table 3-2
Molecular Cell Biology. Spring 2006
Nora Ausmees
OUTLINE OF THE LECTURE
MICROTUBULES
I. MT building blocks and assembly
Tubulin subunits, Critical concentration, Nucleotide hydrolysis,
Dynamic instability, Treadmilling
II. Organization of MTs in the cell
III Factors contributing to the dynamic behaviour of MTs.
1. Critical concentration.
2. The presence of GTP or GDP at the plus end.
3. MT-associated proteins
IV. Functions of MTs
1. Motor proteins move on MT tracks and transport cargo
2. MTs in mitosis
INTERMEDIATE FILAMENTS
Microtubules are one of the three main
cytoskeletal structures in eukaryotic cells
•MTs act as beams that provide mechanical
support for the shape of cells,
•as tracks along which molecular motors
move organelles from one part of the cell
to another, and
•separate chromosomes during mitosis
MT building blocks and assembly
MTs are polar filaments.
The two ends have different
structure and different properties.
+ end and – end.
Figs 20-3, 20-7
13 protofilaments per tube → one MT
Kon
the rate constant for
addition of monomers
(M-1s-1)
Koff
the rate constant for
the loss of monomers
(s-1)
The number of monomers that add to the polymer (MT) per second will
be proportional to the concentration of the free subunit (konC).
The subunits will leave the polymer end at a constant rate (koff), that
does not depend on C.
As the polymer grows, subunits are used up, and C is observed to drop
until it reaches a constant value, called the critical concentration (Cc).
At this concentration the rate of subunit addition equals the rate of
subunit loss. At this equilibrium kon C = koff , so that
Cc =
koff
kon
Treadmilling
If both ends of the polymer are exposed, the critical
concentration at both ends is different due to differences in
composition and different GTP hydrolysis ability.
Cc (minus end) > Cc (plus end)
If the concentration of free subunits reaches a value that is
above the Cc for plus end but below Cc for minus end, a steady
state is reached where the net assembly at plus end and the net
disassembly at the minus end occur at similar rates.
The polymer maintains a constant length but grows from one end
and simultaneously depolymerizes from the other end.
This process is called treadmilling.
Dynamic instability
In vitro studies showed that
MTs grow more quickly from the plus end (terminated by the βsubunit) and very slowly from the minus end (terminated by the αsubunit).
•plus ends switch between phases of slow growth and rapid
shrinkage.
conversion from growing to shrinking
conversion from shrinking to growing
The importance of the discovery of dynamic
instability was that it provided for the first time
a mechanism by which microtubules could
reassemble into different structures during the
cell cycle or during development.
Dynamic instability usually involves only the plus
ends of MTs, because the minus ends are mostly
bound to the MTOC (MT organizing center) and
thus stabilized.
Microtubules are dynamic cellular structures
Xenopus kidney epithelial cell line (A6 cells)
GFP was fused to the C-terminus of beta-tubulin (beta-tubulin-GFP) and
expressed in Xenopus A6 cells.
Time-lapse recording of live cells and image analyses was performed using DeltaVision
full spectrum optical sectioning microscope system (Applied Precision, Inc.) equipped
with Olympus IX70 (PlanApo 100x/1.40 NA oil immersion objective) microscope and a
cooled CCD camera (Quantix-LC, Photometrics). The series of images were processed
and converted to QuickTime movie (JPEG compression) with software installed on
Silicon Graphics computer or NIH image software.
The movies show dynamic behavior of MTs in an interphase cell which undergoes less
motility. The MTs in the lamella are magnified in the movie 2. At the edge of cells, the
plus-end of each microtubule repeats very short growth and shortening phases
frequently in an oscillated manner. Occasionally some microtubules are broken at their
middle part, and the newly created short fragments exhibit treadmilling through the
lamella (4).
MOVIE1
MOVIE2
http://www.tsukita.jst.go.jp/kiyosue/cytoskeleton.html
GTP hydrolysis cycle is coupled to structural
changes in the microtubule
(a) Atomic structure of the
tubulin dimer as seen in the wall
of the protofilament
(b) β-tubulin binds the
GTP but does not
hydrolyse it in solution
because the GTP-binding
pocket lacks crucial
residues for hydrolysis.
These residues are
donated by the α-subunit
when it docks to the end
and triggers hydrolysis.
Thus hydrolysis is
coupled to
polymerization.
The GTP dimer has a straight
conformation and fits nicely into the
straight wall of the microtubule.
Hydrolysis of GTP induces a bend in
the subunit. Within the lattice the
bend is constrained, but places stress
on the lattice.
Many proteins bind to MTs and regulate MT dynamics
MT-associated proteins or MAPs modulate MT dynamics
MAPs were originally isolated from bovine brain as proteins that copurify with MTs. Most of them bind all along the MT lattice.
But the most dynamic part is the microtubule end...
A significant step forward in understanding the dynamics of the
plus end was taken with the introduction of green fluorescent
protein (GFP) technology to describe proteins that specifically
target microtubule ends and in many cases mediate their dynamics.
Two distinct classes of end-binding proteins have been described:
the MCAKs (for mitotic centromere-associated kinesins), which
bind to MT ends and destabilize them.
the plus-end-binding proteins (or +TIPs), which bind to the
growing end of the microtubule and at least in some cases
stabilize the MT during its growth phase).
MT end-binding proteins are most important
factors controlling the dynamic behavior of MTs
GFP–MCAK bound to MT ends in vitro.
MCAKs (also called Kin I kinesins) are
unusual kinesins. Rather than moving
along MTs like other motor proteins,
they use energy from ATP hydrolysis to
bind to the ends of microtubules, remove
tubulin subunits and thus trigger
depolymerization and catastrophies
Model for MCAK (green) binding to the lattice.
CLIP-170 (a +TIP protein) promotes rescue
GFP–CLIP-170 bound to the ends of growing
MTs in cells. The yellow segments represent
GFP–CLIP-170 at MT ends, and the red is MTs.
Model for CLIP-170 (green) binding to MT
From Howard & Hyman (2003) Nature 422 (753-758)
Organization of MTs in the cell
MTOC – MT organizing center
Centrosome in most animal cells
Centrioles (MT-based structures)
pericentriolar matrix
(γ-tubulin and pericentrin)
Fig. 20-13
γ-tubulin – an unusual form of tubulin
Important for MT formation. Treatment of cells with anti- γ-tubulin
antibodies blocks MT assembly.
This complex is localized at only one end of the MTs, not
along the sides.
8 subunits form a ring-like structure
of 25 nm in diameter. This structure
can nucleate MT assembly at
subcritical concentrations (lower than
the normal Cc).
Fig. 20-15
Fig. 20-14
Life is movement
Motor proteins move on MT tracks and transport cargo
(organelles, vesicles).
Motor proteins are molecular machines — they transduce
chemical energy derived from ATP hydrolysis into mechanical
work used for cellular motility.
Kinesin and Dynein are MT-associated
motor proteins.
Different kinesins are either (+) end directed or (–) end
directed motors.
Dynein is a (-) end directed motor.
Kinesin
Different kinesins are either (+) end
directed or (–) end directed motors.
Kinesins are divided also into
cytosolic and mitotic kinesins,
depending on the type of cargo they
carry. The sequence of the unique tail
domain determines the cargo.
Fig. 20-20
Step size – 8 nm,
Force – 6 piconewtons
Speed – 3 m/s
Kinesin is a highly processive motor that can take several
hundred steps on a microtubule without detaching.
Fig. 20-21
How does kinesin move?
catalytic core
ATP binding to the
leading head initiates
neck linker docking
tubulin heterodimer
α-tubulin
tightly docked
neck linker
detached
neck linker
β-tubulin Neck linker docking is completed by the leading head,
which throws the partner head forward by
160 Å toward the next tubulin binding site
The trailing
head
hydrolyzes ATP
to ADP-Pi
The new
leading head
docks tightly
onto the
binding site
The trailing head, which has released its Pi and
detached its neck linker (red) from the core, is
in the process of being thrown forward.
Adapted from: Figure 1 in Vale & Milligan (2000) Science, Vol 288, Issue 5463, 88-95
Dynein
Very large multimeric complex
(–) end directed movement
Dynein needs dynactin to link
vesicles and chromosomes to the
dynein light chain
What can be transported by kinesins and dyneins?
Fig. 20-23
Nerve cells
Transport of material along
the axon towards and away
from the synaptic terminal.
Fig. 20-14
Anterograde transport – from the
cell body to the synaptic terminal
transports material needed for
growth and synaptic function, mostly
in the form of synaptic vesicles.
(Kinesin).
Retrograde transport – to remove
used material, old synaptic
membranes, from the synapse.
(Dynein).
Fig. 20-2
Movie at the Lodish website
MTs in mitosis
The main purpose of mitosis is to segregate the of replicated
chromosomes (sister chromatids) into two nascent cells, so
that each daughter cell inherits one complete set of
chromosomes.
Microtubules form the mitotic
apparatus, the mitotic spindle,
which is able to move the
chromosomes.
G0
M
Cellular microtubules undergo
dramatic reorganization to form
the mitotic spindle
G1
G2
S
Mitosis
The main purpose of mitosis is to segregate the of replicated
chromosomes (sister chromatids) into two nascent cells, so
that each daughter cell inherits one complete set of
chromosomes.
Mitosis in newt cells movie
http://www.bio.unc.edu/faculty/salmon/lab/mitosis/mitosismovies.html
Fluorescence micrographs of mitosis in fixed newt lung cells stained with
antibodies to reveal the microtubules (green), and with a dye (Hoechst 33342)
to reveal the chromosomes (blue).
Drawings of mitosis in newt cells found in a book by
Walther Flemming from 1882.
W. Flemming. Zellsubstanz, kern und zelltheilung. (Verlag Vogel, Leipzig,
1882).
Dynamic instability of MTs is used by the cell to
create the basic machinery of the mitotic spindle.
How the long MTs of the interphase array are reorganized into a mitotic
spindle remains a central question in the field of mitosis.
MT plus ends are more dynamic in interphase cells than in solutions of
purified tubulin and GTP, suggesting that….
… cellular factors regulate MT turnover in vivo
Mitotic MTs turn over 5-10 fold faster than interphase MTs,
suggesting that…..
… some of the cellular factors are cell cycle-regulated
Modulating the ability of MAPs to bind to MTs by reversible
phosphorylation can be used as a tool to regulate the length of MTs.
Phosphorylated stabilizing MAPs are unable to bind to MTs, thus
phosphorylation promotes disassembly.
MAP kinase takes part in many signal transduction pathways. Some
MAPs are also phosphorylated by CDKs.
Interphase (G2)
Interphase – time between two cell divisions.
Centrosome is a microtubule (MT)organizing centre. Consists of a
collection of MT-associated proteins.
Contain a pair of centrioles.
G0
M
G1
G2
S
Centriole – A short, barrel-like array of
microtubules that organizes the centrosome and
contributes to cytokinesis and cell-cycle
progression.
In the end of G2 centrioles replicate and form
daughter centrosomes.
Prophase
Spindle poles
Interphase MTs disappear, new
centrosomes nucleate new MTs,
which are:
•more numerous,
•shorter,
•less stable than interphase MTs.
Centrosomes migrate to opposite sides of the nucleus
Spindle starts to form
Nuclear envelope starts to disassemble
Chromosome condensation begins
MT dynamics in mitosis
More…
MT nucleation at spindle poles increases 4-fold at the
G2/prophase transition.
Shorter…
Catastrophy rates increase in mitosis.
Two factors that increase catastrophy rates in vivo are
Op18/stathmin – mechanism not clear
KinI kinesins (XKCM1) – bind to MT ends and destabilize
More dynamic…
Increased catastrophy rates are counteracted by plus end
stabilizing MAPs.
CLIP170, EB1 stabilize and reduce catastrophes.
XMAP215 binds specifically to MT + ends and increases MT
growth rates, but does not reduce the rate of catastrophes.
MAP activity can be suppressed by phosphorylation.
XMAP215 is phosphorylated in a cell cycle-dependent manner by the cyclindependent kinase 1 (Cdk1).
Prometaphase
Sister chromatid
Kinetochore
Spindle formation.
A large protein complex.
Specialized attachment
site for MTs on the
chromosomes.
Chromosome condensation is
completed. Each chromosome
contains two sister
chromatids.
MTs start the search-and-capture game for chromosomes.
Kinetochores are MT landing pads on the chromosome.
Finally all sister chromatids become tethered to the spindle.
MTs in prometaphase
Centromeric DNA sequence
and attached proteins
(kinetochores) are not very
well conserved in evolution.
However, the proteins that
link MTs to kinetochores
are well conserved from
yeast to humans.
MCAK, CLIP170, CENP-E.
Fig. 20-36
MTs in metaphase
Chromosomes align at the equatorial plane
+ ends more dynamic
K-fibers: bundles of
kinetochore-attached
MTs
Astral MTs are
important for
positioning of the
spindle
Fig. 20-31
MTs in metaphase
Metaphase spindle is a very dynamic structure
Fig. 20-37
Chromosomes align at the equatorial plane in a
tug-of-war between different forces.
MT length remains the same in metaphase, but
there is a constant poleward flux of subunits
Poleward MT flux is driven in the metaphase half-spindle by a
spindle-pole-associated Kin I kinesin. MT length is maintained
by the activity of the kinetochore-associated CLASP protein
that induces plus-end polymerization.
How to visualize mitotic microtubule dynamics in vivo?
Mark a region of the mitotic spindle, follow its movement
I. Fluorescence-speckle microscopy. Label the MTs with a
substoichiometric amount of fluorescent subunits so that the MT
appears as “speckled”. Follow the movement of fluorescent dots in a
timelapse series.
Fig. 20-38
II. Photobleaching experiments
Follow the movement of the photobleached region
Fig. 20-39
MTs in anaphase
The glue holding sister chromatids together
dissolves and they take off to opposite
spindle poles
Anaphase A
Chromosomes separate and move towards opposite spindle poles due to
the shortening of kinetochore MTs
Anaphase B
The two spindle poles move apart bringing the chromosomes into new
daughter cells
Shortening of MTs in anaphase A
In fruitfly embryo two Kin I-type proteins are responsible for MT
shortening and chromosome movement.
1. Marked region moves
polewards: depolymerization is
occurring at the minus end.
KLP10A is at spindle
poles and is responsible
for the reeling in.
2. The disappearance of the
marked region as the
chromosome moves past it. The
kinetochores are chewing up the
MT plus ends as they move
towards the pole.
KLP59C found at kinetochores
and is the molecular reason
behind Pac-Man.
Rogers et al (2004) Nature
Spindle elongation in anaphase B
Plus-end directed cross-linking
motors (kinesins) decrease the
overlap of antiparallel microtubules
and contribute to spindle pole
separation.
Lengthening of the polar
MTs at their plus ends.
Cytoplasmic dynein (dark green)
in the cortex can pull on astral
microtubules
Depolymerization of astral
MTs. In yeast MTs from one of the
Fig. 20-40
spindle pole bodies attach to the bud
cortex. Depolymerization at the
cortex may reel in the spindle into
the bud.
MTs as molecular machines
Microtubules themselves, in the absence of motors, can move
cellular structures around inside cells by maintaining
attachments as they grow or shrink.
The force generated by polymerization/depolymerization is
enough (up to 4 pN) to move chromosomes during mitosis or to
position the spindle in the cell.
During metaphase of mitosis in yeast, movement of the chromosome (to
the right) is associated with polymerization of microtubules on one side
(left) and depolymerization on the other (right).
Bacterial cell division protein (FtsZ) and metazoan tubulin are
structurally very similar and believed to have a common ancestor.
Methanococcus jannaschii
(from Erickson, H.P. (1998)
Trends Cell Biol. 8:133)
Bacterial tubulin-like protein FtsZ forms a cytoskeletal
structure at the cell middle, the so called Z-ring.
Many proteins, including those involved in cell-wall synthesis, are
sequentially recruited to the Z-ring.
Z-ring contracts, and
creates a gradually
developing cell-wall
constriction in the
cell, until finally the
cells divide.
Telophase
Chromosomes decondense
Nuclear envelope reforms
Cytoplasm divides - cytokinesis
Cleavage furrow
Contractile ring creates
cleavage furrow where the
mitotic chromosomes had lined
up in metaphase.
MTs in telophase
What determines the location of the cleavage
plane during cytokinesis?
Fig. 20-41