cytoskeleton.pptx

The Cytoskeleton and
Cell Motility
Learning Objectives:
Three major cytoskeletal elements
Functions of the cytoskeleton
The terminus of a growing
axon from the sea hare

Eukaryotic cells also possess a “skeletal system”
– a cytoskeleton – which has analogous functions.
The cytoskeleton (CSK) is composed of three
well-defined filamentous structures-microtubules,
microfilaments, and intermediate filaments –
which together form an elaborate interactive network.
Each of the three filaments is a polymer of protein
subunits with distinct mechanical properties.

The cytoskeleton is a cellular scaffolding or
“skeleton system” contained within cytoplasm.
Microtubule Microfilament Intermediate filament
8.1 Overview of the cytoskeleton

Microfilaments are solid, thinner structures
composed of the globular proteins- actin.
Microtubule are rigid tubes composed of
subunits of the globular protein- tubulin.
Intermediate filament are tough, ropelike fibers
composed of a variety of related proteins.
Although the components of the cytoskeleton appear
stationary in micrographs, in reality they are highly
dynamic structures capable of rapid and dramatic
reorganization.
We will consider each of the three major cytoskeletal
elements separately in this chapter, after we briefly survey
their major activities.
Contents of cytoskeleton

Microfilaments, also called actin
filaments, located throughout the cells.
Microtubules are straight, hollow
cylinders that are located inside the
cytoplasm of the cell.
IFs are the most abundant and stable
components of the cytoskeleton. Most
types of IFs are cytoplasmic, but one
type, the lamins, are nuclear.

Key to cytoskeletal functions
microvilli
Motile
machinery
Mitotic spindle

 A dynamic scaffold providing structural support
that can determine the shape of the cell and resist
forces that tend to deform it. The flat, rounded shape
of many cultured cells, for example, dependents on a
radial array of microtubules in the cell’s cytoplasm.
 An internal framework responsible for positioning
the various organelles within the interior of the cell.
This function is particularly evident in polarized
epithelial cells, in which organelles are arranged in a
defined pattern along an axis from the apical to the
basal end of the cell.
Major functions of cytoskeleton

 A network of highways that direct the movement of
materials and organelles with cells. Examples of this
function include the delivery of mRNA molecules to specific
parts of a cell, the movement of membranous carriers from
the endoplasmic reticulum to the Golgi complex, and the
transport of vesicles containing neurotransmitters down the
length of a nerve cell.
 The force generating apparatus that moves cells from
one place to another. Single-celled organisms move either
by “crawling” over the surface of a solid substratum or by
propelling themselves through their aqueous environment
with the aid of specialized locomotor organelles (cilia and
flagella). Multicellular animals have a variety of cells that are
capable of independent locomotion.

 A site for anchoring mRNAs and facilitating their
translation into polypeptides. For example, it was
found that the poly (A) mRNA molecule is localized
between the actin filaments in human fibroblasts.
 An essential component of the cell’s division
machinery. Cytoskeletal elements make up the
apparatus responsible for separating the chromosomes
during mitosis and meiosis and for splitting the parent
cell into two daughter cells during cytokinesis. These
events are discussed at length in further Chapter.

An example of the role of microtubules in
transporting organelles. The peroxisomes were
shown in green; Microtubules appear red.

 The cytoskeletion is currently one of the most
actively studied subjects in cell biology.
 The development of techniques allow investigators
to pursue a coordinated morphological,
biochemical, and molecular approach.
 A few of the most important approaches to the
study of the cytoskeleton will be briefly introduced.
The study of cytoskeleton

 The use of genetically engineered cells
 Knockout animals: most often mice that lack a
particular gene.
 Over express model: usually the gene of a target
protein with a dominant negative mutant is
transfected and high expressed.
 GFP cells and animals: GFP is fused with target
proteins and then induced to express stably in a
cell line or in a whole body.

Expression of a mutant motor protein inhibits the
dispersion of pigment granules in a pigment cells.

Microfilament (MF)
A. MFs are made of actin and involved in cell motility
Using ATP, G-actin polymerizes to form MF, or F-actin
8 nm in diameter and composed of globular subunits of the protein actin (G-actin)

Actin filament structure. a, a model of an actin
filament. The actin subunits are represented in
three colors to distinguish the consecutive
filament. The subdomains in one of the actin
subunits are labeled 1, 2, 3,
and 4. b, EM of a replica of an
actin filament showing its
double-helical architecture.
Actin was identified more than
50 years ago:
Major contractile proteins in
muscle cells.
Major protein in every of
eukaryotic cell.
Higher plant and animal
species possess a number of
actin genes.

B. MF assembly and disassembly
Characteristics:
(1) Within a MF, all the actin monomers are
oriented in the same direction, so MF has a polarity

(2) In vitro, (Polymerization) both ends of the MF grow,
but the plus end faster than the minus.
Because actin monomers tend to add to a filament’s
plus end and leave from its minus end---- “Tread-milling”
Cc=0.1umol/L,Mg2+,K+,Na+
─ +

(3) Dynamic equilibrium between the G-actin and
polymeric forms, which is regulated by ATP hydrolysis
and G-actin concentration.
Actin is an ATPase.

(4) Dynamic equilibrium is required for the cell
functions. Some MFs are temporary and others
permanent.
Microvilli
Motile machinery
Contractile ring
Stress fiber

(5)The nucleation of actin filaments at the plasma
membrane is frequently regulated by external signals,
allowing the cell to change its shape and stiffness
rapidly in response to changes in its external
environment.
This nucleation is catalyzed by a complex of proteins
that includes two actin-related proteins, or
ARPs(Arp2 and Arp3).

C. Myosin: the molecular motor for actin
filaments
(1) Myosin move toward the plus ends of microfilaments.

(2) Conventional (Type II) myosins: the best
understood type of myosins.
A pair of globular heads: ATPase
A pair of necks: α helix and 2 light chains
A single, long, rod-shaped tail

Motor activity of myosin: Isolated myosin heads (S1 fragment)
that was immobilized on the surface of a glass coverslip are
capable of sliding attached actin filaments.
In vitro motility assay for myosin:
(a) Schematic drawing
(b) Results of the experiment
depicted in (a). Two video
images were taken 1.5sec apart.
The dashed lines with arrow
heads show the movement of the
F-actin.

Bipolar myosin II filament: Myosin molecules assemble so that
the ends of the tails point toward the center of the filament and
the globular heads point away from the center.
Dimers
Tetramer
Hexamer
Octamer

(3) Unconventional myosins: at least 14 different classes
(type I and type III-XV), 2 of which are unique to plants.
Myosin I: (a) Four light chains; (b) Two are calmodulin; (c)
The head binds to an F-actin; (d) The tail is thought to bind to
plasma membrane.

Microtubules (MTs)
A. Structure and Composition
MTs are hollow, tubular
structure. They occur in
nearly every eukaryotic
cells. MTs are basic
components of diverse
array of structure,
including spindle, cilia
and flagella.
MTs have an outer diameter of 24 nm, a wall thickness of ~5 nm,
and may extend across the length or breadth of a cell. The wall of
a MT is composed of globular proteins (tubulins) arranged in
longitudinal rows, termed protofilaments. MTs are consisted of
13 protofilaments arrayed in a circular pattern within the wall,
when viewed in cross section. (a) EM of MTs in longitudinal view;
(b) EM of MTs in cross section; (c) A ribbon model showing the 3D
structure of the αβ-tubulins heterodimer. (d) Schimetic diagram

B. Assembly and Characteristics of MTs
(1) Each protofilament is assembled from dimeric building
blocks consisting of one α-tubulin and one β-tubulin.
(2) The two types of tubulin subunits have a similar three-
dimensional structure and fit tightly together.
(3) The tubulin dimers are organized in a linear array along
the length of each protofilament.
(4) The protofilament is asymmetric with an α-tubulin at one
end and a β-tubulin at the other end.
(5) All of the protofilaments of a MTs have the same polarity,
and results in the entire polymer has polarity.
(6) One end of a MT is known as the plus end, the other as
the minus end. The endmost row of subunits at he plus
end of MT are β-tubulin, whereas subunits at the tip of the
minus end are α-tubulin.

Dynamic instability
due to the structural
differences between
a growing and a
shrinking
microtubule end.
GTP cap;
Catastrophe:
accidental loss of
GTP cap;
Rescue: regain of
GTP cap

C. MT Associated Proteins, MAPs
(1) Most of the MAPs are found only in brain tissue, but one of
these proteins, called MAP4, has a wide distribution.
(2) MAPs typically have one domain that attaches to the side
of a MT and another domain that extends outward.
 Schematic diagram of a MAP2 bound to the
surface of a MT. Each MAP2 contains 3
tubulin binding sites connected by short arm.
 The binding sites are spaced at sufficient
distance to allow the MAP2 to attach to three
separate tubulin subunits in the wall of the MT.
The tails of the MAP project outward, allowing
them to interact with other components.

D. MTs primarily act as structural supports and
organizers
(1) MTs are stiff enough to resist forces that might compress
or bend the fiber. The distribution of cytoplasmic MTs in a
cells helps determine the shape of that cell.

(2) MTs play a key role in
maintaining the
extended shape of
the axon as it slowly
grows out of the
central nervous
system into the
peripheral embryonic
tissues.

MTs as supporting rods: (a) This protist has tentacles projecting from its cell
body. (b) The tentacles are supported by an elaborate spiral array of MTs.

(3) In plant cells, MTs play an indirect role in maintaining
cell shape through their influence on the formation of
the cell wall.
 To influence the movement of cellulose-synthesizing
enzymes located in the plasma membrane.
 To determine the orientation of these cellulose
microfibrils.
(3) MTs are also thought to play a role in maintaining the
internal organization of cells, for example, the
location of membrane organelles, particularly the
Golgi complex.

E. MTs as Agents of Intracellular Motility
(1) Axonal Transport
(a) Schematic drawing of a
nerve cell showing the
movement of vesicles
down the length of an
axon along tracks of MTs.
(b) Schematic drawing of the
organization of the MTs
and intermediate
filaments within an axon.

F. Motor Proteins That Traverse the MTs
(1) Kinesins: (a) structure of a kinesin molecule; (b) schematic
diagram of a kinesin moving a vesicle along a MT track. (c) the
conformational changes of the head and neck portions of
kinesin that drive the movement of the protein along a MT.
To convert chemical energy into mechanical energy, to
move cellular cargo attached.

Kinesin mediated organelle transport: (a,c) cell’s
mitochondria are located along MTs; (b,d) lack of kinesin protein
caused that the Mit clustered in the central region of the cell.

(2) Cytoplasmic Dynein:
Dynein is the first microtubule-associated motor protein, which
was discovered in 1963 as the protein responsible for the
movement of cilia and flagella.
The cytoplasmic dynein was purified and characterized from
mammalian brain tissue, and was soon identified in a variety of
nonneural cells. It is now known to be a ubiquitous motor protein
in eukaryotic cells.
Cytoplasmic dynein is a huge protein composed of two
identical heavy chains (acts a force-generating engine) and a
variety of intermediate and light chains.
Cytoplasmic dynein moves along a microtubule toward the
polymer’s minus end.
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 vesicles and organelles through the
cytoplasm.

(2) Cytoplasmic Dynein: (a) structure of dynein; (b) two
vesicles moving in opposite directions along the same MT; (c)
kinesin mediated and dynein mediated transport of vesicles.

G. Microtubule organizing centers, MTOCs
(1) Two phases of MT assembly: a slower phase of
nucleation, in which a small portion of the MT is initially
formed, and a more rapid phase of elongation.
(2) Microtubule organizing centers: The nucleation of MT in
vivo is associated with a variety of specialized structures
that, because of their role in MT nucleation and organization,
are called MTOCs.
Major types of MTOCs:
 Centrosomes: the sites of the MT nucleation in animal cells.
MTs typically form in association with the centrosome, a
complex structure that contains two centrioles and
surrounded pericentriolar material (PCM).
 Basal bodies: MTs in a cilium or flagellum originate from a
structure called a basal body.
Other MTOCs: in plant cells, MTOC consists of patches of
material situated at the outer surface of the nuclear envelop.

Structure of the
centrosome:
paired centroles and
the surrounding
pericentriolar materials
(PCM), where
nucleation occurs

H. The dynamic properties of Microtubules
The dynamic character of the MT cytoskeleton is well
illustrated by plant cells. If a typical plant cell is followed
from one mitotic division to the next, four distinct arrays
of MTs appear, one after another.

1) During most of interphase, the MTs of a plant cell are
distributed widely throughout the cortex.
2) As the cell approaches mitosis, the MTs disappear from
most of the cortex, leaving only a single transverse band,
called the preprophase band.
3) As the cell progresses into mitosis, the preprophase band
is lost and MTs reappear in the form of the mitotic spindle.
4) After the chromosomes have been separated, the mitotic
spindle disappears and is replaced by a bundle of MTs
called the phragmoplast, which plays a role in the
formation of the cell wall that separates the two daughter
cells.

I. Cilia and Flagella: structure and function
 Cilia and Flagella are hairlike, motile organelles that
project formt eh surface of a variety of eukaryotic cells.
 Cilia and flagella are two versions of the same structure
and are best distinguished by their patterns of movement.
 Cilia tend to occur in large numbers on the cell surface,
and their beating activity is usually coordinated. Flagella
are usually longer than cilia, and cells have fewer of them.
Flagella exhibit a variety of different beating patterns.

Intermediate Filaments (IFs)
A. Definition
1) For many years, electron microscopy had revealed the
presence in numerous cell types of solid, smooth-surfaced,
unbranched filaments that had a diameter of approximately
10 nm, which is intermediate between that of microtubules
and microfilaments. Because of their relative dimensions,
these structures were called intermediate filaments (IFs).
2) IFs are a chemically heterogeneous group of structures
that, in humans, are encoded by at least 50 different genes.
The polypeptide subunits of IFs can be divided into six
major classes (I to VI) based on their tissue distribution
as well as biochemical, genetic, and immunologic criteria.
Most, if not all, of these polypeptides have a similar
arrangement of domains that allows them to form similar
looking filaments.

B. IF assembly and disassembly
 The basic unit of IF assembly is thought to be a tetramer
formed by two dimers that become aligned side-by side in
a staggered fashion with their N- and C- termini pointing in
opposite (antiparallel) directions.
 Tetramers associated with one another both side to side
and end to end to form the final filament.
 IFs are highly resistant to tensile forces, and tend to be
more resistant to chemical disruption than other types of
cytoskeletal elements and more difficult to solubilize.
 IFs can be depolymerized and repolymerized repeatedly
in vitro, indicating that the subunits possess all of the
information necessary for their self-assembly.

Function of IFs: Confer mechanical strength on tissues
Disruption of keratin networks causes blistering

Muscle contractility
A. Skeletal muscle cell, Muscle fiber
 Skeletal muscles derive their name from the fact that most
of them are anchored to bones that they move.
 Skeletal muscle cells have a ighly unorthodox structure. A
single, cylindrically shaped muscle cell is 10-100um thick, up
to 40m long, and contains hundreds of nuclei. Because of
these properties, a skeletal muscle cell is more appropriately
called a muscle fiber.
 Muscle fibers have multiple nuclei because each fiber is a
product of the fusion of large numbers of mononucleated
myoblasts (premuscle cells) in the embryo.
 Fusion of mononucleated myoblasts is readily demonstrated
in the culture dish, even when the myoblasts are derived
from distantly related animals.
 Skeletal muscle cells may have the most highly ordered
internal structure of any cell in the body.

The structure of skeletal
muscle

B. The Sliding Filament Model of Muscle
contraction
 All skeletal muscles operate by shortening.
 The units of shortening are the sarcomeres, whose
combined decrease in length accounts for the decrease in
length of the entire muscle.
 The banding pattern of the sarcomeres at different stages
in the contractile process evidenced the mechanism
underlying muscle contraction.
 A band remained in length, while H band and I bands
decreased in width and then disappeared altogether.
 As shortening progressed, the Z lines on both ends of the
sarcomere moved inward until they contacted the outer
edges of the A band.

The shortening of the sarcomere during
muscle contraction

Tropomyosin, Tm and Tropnin, Tn
Ropelike molecule
Regulate MF to bind to the
head of myosin
Complex, Ca2+-subunit
Control the position of Tm on
the surface of MF

Summary of cytoskeleton
1. Three types of cytoskeletal filaments are common to many
eucaryotic cells and are fundamental to the spatial
organization of these cells.
2. The set of accessory proteins is essential for the controlled
assembly of the cytoskeletal filaments(includes the motor
proteins: myosins, dynein and kinesin)
3. Cytoskeletal systems are dynamic and adaptable.
Nucleation is rate-limiting step in the formation of a
cytoskeletal polymer.
2. Regulation of the dynamic behavior and assembly of the
cytoskeletal filaments allows eucaryotic cells to build an
enormous range of structures from the three basic
filaments systems.

cytoskeleton.pptx

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cytoskeleton.pptx