The cytoskeleton and cell movement
actin filaments
intermediate filaments
microtubules
Fig. 11-1
Actin filaments
Cytoskeleton:
1)
2)
3)
provides a structural framework for the cells
serve as a scaffold that determines cell shape and the general organization of the cytoplasm.
the cytoskeleton is also responsible for cell movements.
tight biinding site
tight biinding site
A globular proteins of 375 a.a. (43kd)
All of the actins are very similar in a.a. sequence.
each monomer is rotated by 166o
nucleation
globular [G] actin
actin monomer
filamentous [F] actin
1)
2)
C
N
the + end elongates five to ten times
faster than the minus end
polarity
polarity
7 nm
actin filaments = microfilaments
mm
movie
What gives an actin filament distinct polarity with a plus end and a
minus end?
Reversible Polymerization of Actin Monomers
An apparent equilibrium is reached at the critical concentration of monomer
Cc, where Koff = Cc x Kon
+ end
Koff (小) = Cc x Kon (大) 
small Cc
- end
Koff (大) = Cc x Kon (小)  large Cc
This difference in Cc at two
ends results in
treadmilling
Treadmilling
ATP binding and hydrolysis play a key role in regulating the assembly and
dynamic behavior of actin filaments
hydrolysis of ATP
minus end
plus end
exchange of ATP for ADP
Treadmilling
A dynamic behavior of actin filaments and microtubules in which
the loss of subunits from one end of the filaments is balanced by
their addition to the other end.
Treadmilling
Treadmilling is a behavior of actin filaments (or microtubules) when they
maintain a near-constant length by adding ATP-actin (or GTP-tubulin) at the plus
end and dissociating an equal number of ADP-actins or GDP-tubulins from the
minus end. During this steady-state behavior, subunits hydrolyze their nucleoside
triphosphates after assembly, flux through the filament, and exit from the minus
end.
Drugs Useful in Cell Biology
cytochalasins
phalloidin
X
X
Can you think of a reason that the more rapid growth of actin
filaments at one end (the plus end) compared to the other
(the minus end) is advantageous to the cell?
Which of the following is not true of the assembly of actin filaments?
a. It begins with the formation of an aggregate of three actin monomers.
b. It requires ATP.
c. Polymerization occurs from both the plus and minus ends.
d. Polymerization is faster from the plus end than from the minus end.
Fig. 11-5
1)
2)
Actin-Binding Protein: Arp2/3 complex
Actin-binding proteins also regulate the assembly and disassembly of actin
filaments (in addition to ATP).
The turnover rate of actin filaments is about 100 times faster within the cells than
it is in vitro, and this rapid turnover of actin plays a critical role in a variety of
cell movement
Figure 16-30 Molecular Biolgoy of the Cell
actin
Arp2
A model for actin filament nucleation by the ARP complex
Arp
complex
actin
monomer
nuclear actin filament
Arp3
Actin-Binding Protein: ADF/cofilin complex
ADF/cofilin, profiliin, and Arp2/3 complex (as well as other actin-binding proteins) can thus act
together to promote the rapid turnover of actin filaments and remodeling of the actin
cytoskeleton that is required for a variety of cell movements and changes in cell shape.
Fig. 11-7
closely packed parallel arrays
Organization of Actin Filaments
cross-linked in orthogonal arrays that
form three-dimensional meshworks with
the properties of semisolid gels.
Two Distinct Actin Bundles
Actin filaments are associated into two types of bundles by different actin-bundling proteins
a-actinin
fimbrin
40 nm
14 nm
68-kd
102-kd
Figure 11.9
Actin Networks and Filamin
280-kd
C-
N-
Biconcave Red Blood Cells
Why are erythrocytes good for plasma
membrane and cortical cytoskeleton
studies?
Association of the Erythrocyte Cortical Cytoskeleton
with the
Plasma Membrane
the ERM proteins
The major protein that provides the structural basis for the cortical cytoskeleton in erythrocyts
calponin family-
a large actin-binding protein family
a-actinin
fimbrin
spectrin
fodrin
protein 4.1
ankyrin
dystrophin (Duchenne’s muscular dystrophy)
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
the structural basis for the cortical
cytoskeleton in erythrocytes-spectrin
Figure 11.11
Structure of Spectrin
220-kd
240-kd
N-
220 nm
Figure 16-48
The role of ERM-family proteins in attaching
actin filaments to the plasma membrane
Fig11.13
Attachment of Stress Fibers to the Plasma Membrane
at
Focal Adhesion
Stress fibers are contractile bundles of actin
filaments, crosslinked by a-actinin, that anchor the
cell and exert tension against the substratum.
They are attached to the plasma membrane at
focal adhesion via interactions with integrin.
Attachment of Actin Filaments to Adherens Junctions
Figure 11.16
Electron Micrograph of Microvilli
The best characterized of actin-based cell surface protrusions are microvilli
Organization of Microvilli
Myosin I and
clamodulin
villin and
fimbrin
vinculin
a spectrin rich terminal web
Transient Protrusions
Figure 11.18
Examples of Cell Surface Projections Involved
in Phagocytosis and Movement
pseudopodia of a
macrophage
pseudopodia of an
amoeba
lamellipodia
microspikes/filopodia
Actin, Myosin, and Cell Movement
Fig. 11-19
Structure of Muscle Cell
three distinct types of muscle cells
Structure of the Sarcomere
oMyofibrils are cylindrical bundles of two types of
filaments
oEach myofibril is organized as a chain of
contractile units called sarcomeres
7-nm
15-nm
2.3 mm
What bands or zones of a muscle sarcomere change length during
contraction? Why doesn’t the A band change length?
Titin and Nebulin
Titin acts springs
Keep myosin centered in the sarcomere
maintain the resting tension
acting as rulers that determine their length
Sliding-Filament Model of Muscle Contraction
Andrew Huxley and Ralph Niedergerke,1954. Nature 173:973
Muscle contraction thus result from an interaction between the actin and myosin
filaments that generate their movement relative to one another.
Myosin II (500 kd total)
Myosin II
200 kd
Fig. 11.24
Organization of Myosin Thick Filaments
associated in a parallel staggered array by
interactions between their tails
The Swinging-Cross-Bridge Model
Myosin (in the absence of ATP) binds to actin tightly.
ATP binding dissociates the myosin-actin complex
Hydrolysis of ATP induces a conformational change in myosin.
This change affects the neck region of myosin, which acts as a
lever arm to displace the myosin head by about 5 nm.
The myosin head then rebinds at a new
position on the actin filament, resulting in the
release of ADP and Pi
“power stroke”, the myosin head returns to its
initial conformation
Fig. 18-25 Molecular Cell Biology
The coupling of ATP hydrolysis to
movement of myosin along an
actin filament
movie
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Tropomyosin and Troponin Complex
When the concentration of Ca2+ is low, the complex of troponins with tropomyosin
blocks the interaction of actin and myosin, so the muscle dose not contract.
tropomyosin
troponin complex
Ca2+
myosin binding
sites exposed
TnI
Ca2+
TnC
TnT
tropomyosin
Contractile Assemblies in Nonmuscle Cells
Two examples of contractile assemblies in nonmuscle cells,
stress fibers and adhesion belts
bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules
The most dramatic example of actin-myosin
contraction in nonmuscle cells is provided by
cytokinesis
Toward the end of mitosis in animal cells,
a contractile ring consisting of actin
filaments and myosin II assembles just
underneath the plasma membrane
Regulation of Myosin by Phosphorylation
in Non-/Smooth Muscle cells
MLCK is itself regulated by association with the
Ca2+ -binding protein calmodulin
myosin light chain kinase
Unconventional MyosinMyosin I
unconventional myosins:
1)
2)
3)
transport of membrane vesicles and organelles along
actin filaments
phagocytosis
pseudopod extension
110 kd vs. 500 kd myosin II
no long tail and do not form dimers
Which of the following is true about myosin I?
a. It is involved in muscle contraction.
b. It has a long a-helical tail through which it forms homodimers.
c. It does not act as a molecular motor.
d. It links the actin bundles to the plasma membrane in the microvilli of intestinal cells.
Unconventional MyosinMyosin III - XIV
1)
Myosin V is a two-headed myosin.
2)
It transports organelles and other cargoes (e.g.
intermediate filaments) toward the plus ends of
actin filaments.
Fig. 11-32
Cell Migration
1. The crawling of amoebas
2. The invasion of tissues by WBCs
3. The migration of cells involved in wound healing
4. Spread of cancer cells during metastasis
5. phagocytosis
Cell migration or crawling can be viewed in three stages:
I.
Protrusions such as pseudopodia, lamellipodia,
or filopodia.
I.
These extensions must attach to the substratum
II.
The trailing edge of the cell must dissociate from
the substratum and retract into the cell body.
Figure 18-42 Molecular Cell Biology
Myosin I
A model of the molecular events at the leading
edge of moving cells
The polymerization of actin filaments at the (+) end,
stimulated by profilin located at the leading-edge
membrane, pushes the membrane outward
profilin
Arp2/3
Arp2/3 and actin cross-linking proteins stabilize
the actin filaments into networks and bundles
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Simultaneously, cofilin induces the loss of
subunits from the - ends of filaments
cofilin
myosin I is thought to link actin filaments to
the leading-edge plasma membrane
focal adhesion
Reconstruction of focal adhesions occurs
in two steps:
lamellipodia
Step I:
appearance of small focal complexes
(microfilaments attach to integrin)
and subsequent growth of them
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Step II: retraction of the trailing edge;
ARF
myosin II
movie
Figure 16-49
Focal contacts and stress fibers in a cultured fibroblast
reflection-interference microscopy
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
focal contacts appear as dark patches
Immunofluorescence staining of actin
Intermediate filament
Intermediate
Filaments
50 different proteins classified into 6 groups
hard keratins 
hair, nails, and
horns
abundant in
axons of motor
neurons
Would you expect mutations of keratin genes to affect fibroblasts?
Figure 19-57
A diagram of desmin filaments in muscle
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Intermediate Filament Proteins
Intermediate filaments are not directly involved in cell movements. They play
basically a structural role by providing mechanical strength to cells and tissues.
10 nm
Presumably determine the specific functions of the
different intermediate filament proteins.
Assembly of Intermediate Filaments
coiled-coil sturcture
tetramers in a staggered
anti-parallel fashion
apolar
eight protofilaments
Lehninger Fig. 4-11
Figure 19-51 Molecular Cell Biology
Levels of organization and assembly of
intermediate filaments
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Intracellular Organization of Intermediate Filaments
keratin and vimentin
position and anchor the nucleus
Fig. 11-36
Attachment of Keratin Filaments to
Desmosome and Hemidesmosome
Desmosomes and hemidesmosomes are junctions.
Keratin filaments
dense plaque
desmoplakin
keratin filaments
Desmosones and hemidesmosomes anchor intermediate filaments to
regions of cell-cell and cell-substratum contact, respectively
plectin
Figure 22-9
Adhesion molecules in junctions involved in
cell-matrix adhesion
stress fiber
keratin
F actin
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
integrin
focal adhesion
hemidesmosome
Figure 22-5
Adhesion molecules in junctions involved in
cell-cell adhesion
F actin
keratin
cadherin
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
adherens
junction
desmosome
Keratin filaments
anchored to dense
plaque
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
cytoplasm
desmoplakin
Intermediate filaments
Plectin bind those three filaments
intermediate filaments
plectin
microtubules
Fig. 11-38
Experimental Demonstration of Keratin Function
Stephen Hawking’s ALS
(amyotrophic lateral sclerosis)
Accumulation and abnormal assembly of
neurofilaments

Progressvie loss of motor neuron

Muscle atrophy, paralysis, and death
Microtubules
Cell shape
Cell movements
Intracellular transport of organelles
Separation of chromosomes in mitosis
Beating of cilia and flagella
Fig. 11-39
Structure of Microtubules
55 kd each (vs. 43 kd actin monomer)
dimer of a- and b-tubulin
protofilaments
microfilaments vs. microtubules
1)
microtubules are polar structures with
two distinct ends
2)
Tubulin dimers polymerize at the plus
ends in a flat sheet which then zips up
into the mature microtubule
3)
Microtubules undergo treadmilling
Fig. 11-40
Dynamic instability of microtubules
In microtubules, GTP hydrolysis results in the behavior
known as dynamic instability, in which individual
microtubules alternate between cycles of growth and
shrinkage
If GTP is hydrolyzed more rapidly than new subunits are then added
The rapid turnover resulting from dynamic instability is critical for the
remodeling of the cytoskeleton that occurs during mitosis.
Whether a microtubule shrinks or grows is determined by
a. the rate of GTP-bound tubulin addition relative to the rate of tubulin GTP hydrolysis.
b. the phosphorylation state of b-tubulin.
c. the rate of ATP hydrolysis relative to the rate of ATP-bound tubulin addition.
d. the presence or absence of -tubulin.
The microtubule behavior in which tubulin adds at the plus end, fluxes through a
constant-length microtubule, and comes off the minus end is called
a.
b.
c.
d.
an equilibrium state.
dynamic instability
treadmilling.
recycling.
Table 16-2 Molecular Biology of the Cell
Drugs That Affect Actin Filaments and Microtubule
ACTIN-SPECIFIC DRUGS
Phalloidin
Cytochalasin
Swinholide
Latrunculin
binds and stabilizes filaments
caps filament plus ends
severs filaments
binds subunits and prevents their polymerization
MICROTUBULE-SPECIFIC DRUGS
Taxol
Colchicine, colcemid
Vinblastine, vincristine
Nocodazole
binds and stabilizes microtubules
binds subunits and prevents their polymerization
binds subunits and prevents their polymerization
binds subunits and prevents their polymerization
Table 16-2 Molecular Biology of the Cell
Drugs That Affect Actin Filaments and Microtubule
ACTIN-SPECIFIC DRUGS
Phalloidin
Cytochalasin
Swinholide
Latrunculin
binds and stabilizes filaments
caps filament plus ends
severs filaments
binds subunits and prevents their polymerization
MICROTUBULE-SPECIFIC DRUGS
Taxol
Colchicine, colcemid
Vinblastine, vincristine
Nocodazole
binds and stabilizes microtubules
binds subunits and prevents their polymerization
binds subunits and prevents their polymerization
binds subunits and prevents their polymerization
Colchicine, an alkaloid derived from plants, binds tightly to
tubulin and inhibits its polymerization
colchicine
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
taxol
centrosome
determines the Intracellular Organization of Microtubules
The microtubules in most cells extend outward from a microtubule-organizing center
(MTOC)
Growth of Microtubules from the Centrosome
The distribution of microtubules in a normal interphase cell
new microtubules growing out
of the centrosome
centrosome
mouse fibroblast
Centrosome and Centriole
transverse section of a centriole
nine triplets of microtubules
Fig. 11-44
Structure of a Centriole
The two centrioles are conncected by one or more fibers
that contains centrin
Centrioles are not necessary for the microtubule-organizing
functions of the centrosome.
However, removal of centrioles results in dispersion of the
centrosome contents and a decline in microtubule turnover.
Figure 16-23 Molecular Biology of The Cell
The centrosome
-tubulin is specifically localized to centrosome, where it plays
a critical role in initiating microtubule assembly.
+
+
+
nucleating sites
(-tubulin ring complexes)
+
+
+
ª›®œ•Œ QuickTimeý ©M
+ (LZW)°®
°ßTIFF
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
pairs of centiroles
+
+
Microtubules growing from -tubulin
ring complexes of the centrosome
Figure 16-22
Polymerization of tubulin-tubulin
nucleated by -tubulin ring complexe
_
+
accessory proteins in -tubulin ring complex
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Which of the following is not correct?
a) The centrioles nucleate assembly of microtubules
b) The centrosome is the major MTOC of animal cells.
c) centrosome consists of an amorphous matrix of protein
containing the -tubulin ring complexes
d) Centrioles are not necessary for the microtubule-organizing
functions of the centrosome
The proteins Rab, Ran, and Tubulin are all
a.
b.
c.
d.
involved in cell motility.
nuclear proteins.
G proteins, regulated by bound GTP or GDP.
parts of signal transduction pathways.
Microtubules are not the basis for
a.
b.
c.
d.
movement of chromosomes during mitosis.
transport of membranous vesicles in the cytoplasm.
cytokinesis of animal cells.
movement of cilia and flagella.
Fig.11-45
Electron micrograph of the mitotic spindle
Formation of Mitotic Spindle
The centrioles and other components of the
centrosome duplicated in interphase cells
The two centrosomes separate and move to
opposite sides of the nucleus, forming the two
poles of the mitotic spindle.
As the cell enter mitosis, microtubules
assemble and disassemble begins.
Figure 19-42 Molecular Cell Biology
polar microtubules
- end – directed motors
late prophase
pushing forces
ª›®œ•Œ QuickTimeý
in ©M
overlap
zone
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
pulling forces
on asters
cytosolic dynein located at
the cortex may pull asters
toward the poles
(+) end – directed KRPs,
probably BimC kinesins,
associated with the polar
microtubules
is involved in separation of
the poles
The dynamic behavior of microtubules can be modified by the
interactions of microtubules with proteins
Strathmin:
a protein overexpressed in leukemia, highly
proliferative breast cancers, and malignant ovarian
cancers.
Microtubule associated proteins (MAP):
plus-end-tracking proteins
MAP-1,MAP-2, and Tau:
neuronal cells
MAP-4:
nonneural cells
Figure 11.47
Organization of Microtubules in Nerve Cells
MAP-2 and tau distribution are responsible for the distinct
organization of stable microtubules in axons and
dendrites.
MAP-2
MAP-2
tau
Figure 16-36 Molecular Biology of the Cell
The effects of proteins that bind to microtubule ends
MAP
stabilization
destabilization
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
catastrophin
longer, less
dynamic
microtubules
shorter, more
dynamic
microtubules
Figure 16-33 Molecular Cell Biology
Organization of microtubule bundles by MAPs.
microtubule
microtubule
MAP-2
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
tau
Figure 11.48
Microtubule Motor Proteins
the motor domain
ATP
The heavy chain have long
a-helical regions that wind
around each other in a coilcoil structure.
380 kd
Bind to other cell components that are transported along
microtubules by the action of kinesin motors
X-ray crystallography of motor domains indicates that kinesin
and myosin evolved from a common ancestor.
motor domain 850 a.a.
myosin
actin-binding sites
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
ATP
kinesin
microtubule-binding sites
ATP
motor domain 340 a.a.
Figure 11.48
Microtubule Motor Proteins
ATP
2000
kd
kinesin
heavy chain
coiled-coil complex
globular
head
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
ATP hydrolysis;
biinding to
microtubules
light chain
binding to
transport
vesicle
1)
18 different kinesins are encoded in the genome of C. elegans
2)
As many as 100 different members of the kinesin family in humans
3)
Different members of the kinesin family vary in the sequences of their
c-terminal tails and are responsible for the movements of different
types of cargo, including vesicles, organelles, and chromosomes,
along microtubules.
Figure 19-24 Molecular Cell Biology
Model of kinesin-catalyzed anterograde transport.
vesicle
kinesin
receptor
kinesin
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Figure 11.49
Transport of Vesicles along Microtubules
Particullary in nerve cells;
proteins, membrane vesicles, and organelles (e.g. mitochondria) must be
transported from the cell body to the axon
How are kinesin and myosin II similar and how are they different?
1)
3)
Both kinesin and myosin II are motor proteins that bind ATP and move toward the plus end of
filaments.
Both are composed of two large heavy chains with a globular head containing an ATPbinding site and a filament binding site, and a long, coiled-coil tail.
Both have light chains associated with each heavy chain.
4)
They are different in that kinesin binds to microtubules and myosin binds to actin filaments.
5)
Kinesin’s light chains are at the tail, whereas myosin’s are at the neck.
6)
Myosin II associates tail-to-tail to form bipolar filaments, whereas kinesin functions as an
individual molecule.
2)
Figure 11.50
Association of the Endoplasmic Reticulum
with Microtubules
ER stained with a fluorescent dyeStained with tubulin specific Ab
Figure 11.51
Anaphase A Chromosome Movement
spindle poles
spindle poles
Kinetochore motor proteins
dynein
Figure 11.52
Spindle Pole Separation in Anaphase B
kinesin
cytoplasmic dynein
Figure 11.53
Examples of Cilia and Flagella
ciliated epithelial cells lining the surface of trachea
Figure 11.54
Structure of the Axoneme of Cilia and Flagella
The fundamental structure of both cilia and flagella is the axoneme
The microtubules are arranged in a characteristic “9+2” pattern
Figure 11.55
Electron Micrographs of Basal Bodies
Basal bodies thus serve to
•
initiate the growth of axonemal microtubules
•
anchor cilia and flagella to the surface of the cell.
Figure 11.56
Movement of Microtubules in Cilia and Flagella
ª›®œ•Œ QuickTimeý ©M
°ßTIFF (LZW)°®
—¿£¡Yæš
®”¿Àµ¯¶ššœµe°C
Adjacent microtubule doublets in cilia and flagella produce a bending
movement because
a.
b.
c.
d.
tubulin is contracting on one side of the microtubules.
dynein is contracting on one side of the microtubules.
kinesin is contracting on one side of the microtubules.
nexin links between microtubule doublets convert a sliding movement
Into a bending movement.
Key Experiment 11.1
Expression of Mutant Keratin Causes
Abnormal Skin Development
Key Experiment 11.2
The Isolation of Kinesin