Gerald Karp
Cell and Molecular Biology
Fifth Edition
Chapter 9:
The Cytoskeleton and Cell Motility
Copyright © 2005 by John Wiley & Sons, Inc.







The study of the cytoskeleton
Molecular motors
Microtubules
Intermediate filaments
Actin filament
Muscle contraction
Non-muscle motility



Skeleton: movement and support
Cytoskeleton: movement and support
Highly dynamic structure: m-RNA fixed
on the cytoskeleton to process the
transcription
9.1 Overview of the major
function of the cytoskeleton






Structural support that can determine
the shape of the cell
Positioning the organelles (polarity)
Delivery the materials or organelles (mRNA)
to the specific part of a cell
Vesicles movement
Cell locomotion
Cell’s division machinery
9.2 The study of the
cytoskeleton



The use of fluorescence microscopy
The use of video microscopy and laser
beams for in vitro motility assays
The use of cells with altered gene
expression
(a) The use of knockout animals
(b) The use of cells over-express a
dominant negative mutant protein
9.2 The study of the
cytoskeleton



The use of fluorescence microscopy
The use of video microscopy and laser
beams for in vitro motility assays
The use of cells with altered gene
expression
(a) The use of knockout animals
(b) The use of cells overexpress a
dominant negative mutant protein
Dominant negative mutant



Cells produce large amounts of a
nonfunctional protein.
Cells are transfected to take up the altered
DNA and incorporating it into their
chromosomes.
Once the cells have been genetically modified,
the mutant protein either competes with the
normal protein or interferes in some other
way with its function, causing the cell to
exhibit the mutant phenotype.
A pigment cell from the xenopus
Which treated with a hormone that
induces dispersion of the pigment
granules-lightening the skin
A pigment cell that is overexpressing
a gene for a mutant motor protein
(kinesin II). The failure of the
pigment granules to disperse in the
cell indicating the Kinesin II as the
motor protein responsible for the
outward movement of the granules.
(c). The use of small, double-stranded RNA
molecules (siRNA) that complementary to the
mRNA that encodes the particular protein
being investigated.
(Fig. 11.38)
This RNA interference has become a common
strategy in recent years to investigate the
effect of a missing protein
9.3 Microtubules





Structure and composion
Hollow, tubular structure
Mitotic spindle, flagella, cilia
25 nm (outer diameter)
13 protofilaments
Functions pf microtubules


As structural supports and organizers
As agents of intracellular motility:
axonal transport
Functions pf microtubules


As structural supports and organizers
As agents of intracellular motility:
axonal transport
Motor proteins that transverse
the microtubular cytoskeleton


It has been known for several decades
that microtubules are primarily
structures that serve as track for a large
number of motor proteins that
generate the forces required to move
objects within a cell.
Convert chemical energy (ATP) to
mechanical energy


Motor protiens can be grouped into kinesins,
dyneins (move along microtubules), and
myosins (move along microfilaments)
As the protein moves along, it undergoes a
series of conformational changes that
constitute a mechanical cycle coupled to the
steps of a chemical cycle.
The steps of chemical cycle include





1. binding of an ATP molecule to the motor
2. the hydrolysis of the ATP
3. the release of the proteins (ADP, Pi) from
the motor
4. the binding of a new molecule of ATP
5. The binding and hydrolysis of a single ATP
is used to drive a power stroke that moves
the motor a precise number of nanometers
along its track.
A body of evidence suggests at least two
roles for cytoplasmic dynein


1. a force-generating agent in the positioning
of the spindle and movement of
chromosomes during mitosis
2. a minus end-directed microtubular motor
for the positioning of the Golgi complex and
the movement of organelles, vesicles, and
particles through the cytoplasm
Microtubule-organizing centers
(MOTCs)



microtubule assembly in vitro:
a. slow phase of nucleation
b. elongation
In vivo, nucleation of microtubules
takes place rapidly inside the cell--microtubule-organizing centers
The best-studied MOTC is the
centrosome
Centrosomes (in animals)



2 centrioles + pericentriolar materials
(PCM)
PCM: nucleation occurs
Is the major site of microtubule
initiation in animal cells and remain the
cell’s microtubular network.


In unpolarized cell, the centrosome is
situated near the center of the cell.
In polarized cell, microtubules are
anchored by their – ends near the
apical surface as + ends extend toward
the cell’s basal surface.
Basal bodies and other MOTCs




1. centrosomes are not the only MOTCs in
cells
2. microtubules in a cilium or flagellum are
generated in the structure called a basal body
(identical in structure to centrioles)
3. basal bodies and centrioles can give rise to
one another (centriole → basal body → flagellum
during spermiogenesis, or sperm basal body → a
centriole during the 1st mitotic division of the
fertilized egg).
4. Plant cells only have MOTCs.
Microtubule nucleation


MOTCs
γtubulin, 0.005 % of total cell protein
The dynamic properties of microtubules




Mitotic spindle fiber: liable (sensitive to
disassembly)
Mature neurons: less liable
Cilia, flagella: highly stable
Noncovalent association of dimeric
building blocks
Disassembly the microtubules




Cold temp, hydrostatic pressure, elevated
Ca 2+ conc., colchicine, vinblastine, vincristine,
nocodazole
Taxol: bind to polymer, inhibit disassembly,
preventing from the assembling new microtubular
structure
Death of cancer cells due to the drugs effect on the
assembly of spindle fiber in the cell division
Recent research has revealed that normal cells arrest
division until drugs eliminated. However, the cancer
cells lack this mitotic check point and attempt to
complete their division even in the absence of a
functioning mitotic spindle → cell death
The study of microtubule dynamics in
vitro




1972, R. Weisenberg of Temple Univ.
Crude brain homogenates at 37oC
Adding Mg 2+, GTP, EGTA (bind to Ca2+,
an inhibitor of polymerization)
Found the microtubules could be
disassembled and reassembled over
and over by lowering and raising the
temperature of medium
The study of microtubule dynamics in vivo

By microinjecting fluorescently labeled
tubulin into a cultured cell
Cilia and flagella: structure and
function (only limit to the eukaryotes)
How does a cell organize and maintain a
construction site at the outer tip of an axoneme
situated um from the cell body ?

Intraflagellar transport (IFT)
for assembling and maintaining the
flagella

1. the protein responsible for
conversion of the chemical energy of
ATP into the mechanical energy of
ciliary locomotion was isolated by Ian
Gibbons of Harvard in 1960s
9.4 Intermediate filaments





Antiparallel associate each other
Highly dynamics in vivo
Disappear before cell division, reappear
in the daughter cell
Assembly and disassembly related to
phosphorylation and dephosphorylation
(phosphorylation of vimentin cause
disassembly)
Functions of IFs




Found around the nucleus
Terminated in the cytoplasmic plaques
of desmosomes and hemidesmosomes
In nerve cells, called neurofilaments
Japanese quail bearing mutation of
neurofilaments possess axons that are
markedly thinner than normal
Functions of IFs


Mice carrying deletions in type 1 keratin are
so sensitive to mechanical response, can
cause severe blistering of the skin or tongue
An inherited human disease “desmin-related
myopathy” patients suggested from skeletal
weakness, cardiac arrhythmias and
congestive heart failure
9.5 Microfilaments




7-8 nm, phalloidin or actin-Abfluorescent
Migration, contraction, motility
Wound-healing, axon growth, WBC
migration, phygocytosis, cytokinesis
G-actin → F-actin filament in the
presence of ATP




Highly ordered form (brush boarder)
Ill-defined network
Tightly anchored bundle (focal adhesion)
Highly conserved
Microfilament assembly and
disassembly



In vitro: ATP-actin monomers →
growing end
Barbed (+) end is the fast-growing end
(high affinity to ATP-actin)
Pointed (-) end is the slow-growing end
(low affinity to ATP-actin)
Cells maintain a dynamic equilibrium
between the monomeric and polymeric
forms of actin



Cytochalasin (from mold) binds to +
end of the actin filament allowing
depolymerization at the – end.
Phalloidin (from a poisonous mushroom)
binds to intact filament and prevents
their turn over.
Latrunculin (from a sponge) binds to
free monomers
Myosin: the molecular motor
of actin filaments



Myosin superfamily (40+), generally
divided into two groups: conventional
(type II) and non-conventional.
Move toward the + end of an actin
filament
Exist in mammalian cells, plants,
nonmuscle cells, protists, vertebrate
cardiac and smooth muscle cells
9.6 Muscle contraction




The sliding filament model of muscle
contraction
The composition and organization of
thick and thin filaments
The energetics of filament sliding
Excitation-contraction coupling




T-tubules and SR
Ca2+ and SER
Ca2+ in SR 10-2 M
Stimulus → T-tubules → Ca2+ releasing
channel on SR→ Ca2+ release to the
cytoplasm (10-5M) →troponin C →
tropomyosin release from actin filament →
exposure myosine binding site → combine
with actin filament → contraction
9.7 Nonmuscle motility



Contractile proteins are present in less
ordered, more liable, transient
arrangements.
They are restricted to a thin cortex just
beneath the plasma membrane.
Phygocytosis, cell division, cell
movement etc.