July 19, 2023

CYTOSKELETON

Presenter: Moderator:
Sapana Subedi Sunil Pandey
PG Biochemistry Assisstant Professor
BPKIHS
 OVERVIEW

 Introduction of Cytoskeleton and its major

functions

 Microtubules – Introduction , Structure, Function

 Intermediate Filaments – Introduction , Structure

and Function

 Microfilaments – Introduction, Structure and

Function
 INTRODUCTION
 Skeleton of a vertebrate that support the soft tissues of the body
and play a key role in mediating bodily movements.

 Eukaryotic cells also possess a “skeletal system”—

cytoskeleton—that
cytoskeleton has analogous functions.

Composed 1. Microtubules, Together

of three 2. Microfilaments form an
well- & interactive
defined 3. Intermediate network.
filamentou filaments
s
structures
Polymer of protein subunits held
together by weak, noncovalent
rapid assembly
bonds. and disassembly
 INTRODUCTION
 Network of
fibers
extending
throughout
the
cytoplasm.
 INTRODUCTION

 Each of the three types of cytoskeletal filaments is a polymer of protein subunits

held together by weak, noncovalent bonds.
 Microtubules
• Rigid hollow rods, approximately 25nm diameter
• Composed of tubulin dimer (globular protein)
α-Tubulin
β-Tubulin
Tubulin
Each protofilament is made from dimeric building blocks Dimer
consisting of one α-tubulin and one β -tubulin subunit
In cross sectional microtubules are seen to consist of 13
protofilaments aligned side by side in a circular pattern within the
wall
• Dimer bound by noncovalent bond.
• Evolutionary ancestor of all plants and animal tubulins are seen in
prokaryotes i.e. FtsZ protein which has similar role
Microtubules
 Structure of Microtubules

(c) A ribbon model showing

(a) Electron (b) Electron 3D structure of the
micrograph of micrograph of a cross tubulin heterodimer.
microtubules section through a  β-tubulin subunit has a bound
from brain. microtubule of a GDP, which is exchanged for a
Juniperus root tip cell GTP prior to assembly into a
revealing the 13 polymer.
subunits arranged
within the wall of the  α-tubulin subunit has a bound
tubule. GTP, which is not hydrolyzed
and is non-exchangeable.
 Structure of Microtubules
 One end is known as the plus end and is
terminated by a row of β-tubulin subunits
 Opposite end is the minus end and is
terminated by a row of α- tubulin
subunits.

 The helix is interrupted at one site

where and subunits make lateral
contacts. This produces a “seam” that
runs the length of the microtubule.
 Tubulin - Polymerization
Highly dynamic structures that can undergo rapid cycles of assembly and disassembl

Role of GTP in microtubule polymerization

Tubulin dimers with GTP bound to β-tubulin associate with the growing plus ends

Shortly after polymerization the GTP bound to β-tubulin is hydrolyzed to GDP.

Since GDP-bound tubulin is less stable in the microtubule, the dimers at the minus
end rapidly dissociate. • Hydrolysis of GTP favors Depolymerization.
 Assembly of Microtubules
 In animal cells, most microtubules
extend outward from the centrosome.

(first described by Theodor Boveri

in 1888),
which is located adjacent to the nucleus in
interphase (nondividing) cells.
 Centrosome serves as the initiation site for the assembly of
microtubules in animal cells, which then grow outward toward
the periphery of the cell with their minus ends anchored in
the centrosome.

 The key protein in the centrosome is γ-tubulin, a species of

tubulin that serves to nucleate assembly of microtubules.
 Microtubules Motors and Movement
 Microtubules perform variety of cell movements, including intracellular
transport and positioning of organelles, separation of chromosomes at
mitosis, and beating of cilia and flagella.
 Members of two large families of motor proteins—

kinesins and dyneins—are

responsible for powering the
variety of movements in
which microtubules
participate.
 Dynein and kinesin move along
microtubules in opposite
directions—

 dyneins toward the minus end

and

 kinesins toward the plus end

 Microtubules Motors and Movement
 Schematic illustration of
kinesin-mediated and dynein-
mediated transport of
vesicles, vesiculartubular
clusters (VTCs), and organelles

Transport along microtubule

tracks.
 Microtubules in Mitosis
• Microtubule array of interphase disassemble and reassemble
to form mitotic spindle.

• Duplication of centrosome occur.

• Move opposite side of nucleus forming 2 poles pulling

chromatids on each side.
Cilia and Flagella Are Specialized Organelles Composed of
Microtubules

• Cilia:
• Diameter of 0.25um and length 10um
• Beat in coordinated back and forth motion
• Moves fluid over surface or cell through fluid

• Flagella:
• Similar diameter but 200um length and wavelike pattern of beating
• Sperm contain 1 flagella
Cilia and Flagella Are Specialized Organelles Composed of
Microtubules
• Fundamental unit of both is Axoneme composed of microtubule and
associated protein

• Microtubule arranged in 9+2 pattern (central pair of microtubules

surrounded by 9 outer doublet tubules
Cilia and Flagella Are Specialized Organelles Composed of
Microtubules

• Minus end of microtubules of cilia and flagella are anchored in

basal body, (similar to centriole)

• Movements by outer microtubule doublets relative to another

 Nucleation of Microtubules
• Centrosome : Microtubule organizing center
(MTOC) in animal cell
• Nucleated from centrosome at their minus
ends, so the plus ends point outward and
grow toward the cell periphery
Microtubules keeps
centrosome on center
of cell
• γ-tubulin ring complex (γ-TuRC) originates the 13
protofilaments microtubule
• Centrioles: pair of cylindrical structure at right angle to
each other inside

Fig: Polymerization of tubulin

nucleated by γ-tubulin ring
complexes
 Treadmilling of Microtubules
• Highly dynamic,
• Minus end grow less rapidly than plus end
• Dynamic instability of microtubule is stabilized by
MAPs (Microtubule Associated Proteins)
• Several MAPs are plus end tracking protein
• MAP-1, MAP-2 and tau isolated from neuronal cells
• MAP-4 present in all non-neuronal cells

• Tau protein is extensively studied as main component

of lesion found in brain of Alzheimer's patients.

• Activity of MAPs is regulated by phosphorylation to

control microtubule stability.
 Drugs against Microtubules
 Colchicine and colcemid are examples of drugs that bind tubulin
and inhibit microtubule polymerization, which in turn blocks
mitosis.

 Two related drugs (vincristine and vinblastine) are used in cancer

chemotherapy because they selectively inhibit rapidly dividing
cells.

 Another drug, taxol, stabilizes microtubules rather than inhibiting

their assembly.
Such stabilization also blocks cell division, and taxol is used as an
anticancer agent.
 Intermediate Filament
• 2nd of the three major cytoskeletal elements
• Diameter of 8-11nm (between actin: 7nm and
microtubule 25nm)
• Provide structural role providing mechanical
strength to cell and tissues
• Provide cell to cell attachment
• Phosyhorylation modification is way to regulate
assembly and disassembly
– Eg: phosphorylation of nuclear lamins result in
disassembly of nuclear lamina and break of
nuclear envelope during mitosis.
• Composed of variety of proteins specific to cells
 INTERMEDIATE FILAMENTS

 Strong, flexible ropelike fibers

 that are subjected to physical stress, including neurons,

muscle cells, and the epithelial cells that line the body’s
cavities.

 Unlike microfilaments and microtubules, IFs are composed

of heterogeneous group of proteins.

 In humans, are encoded by approximately 70 different genes.

 INTERMEDIATE FILAMENTS
 No energy in the form of ATP or GTP hydrolysis is required for IF
polymerization.

 IFs have no polarity, whereas microfilaments and microtubules have plus and
minus ends.
 IFs are composed of a
heterogeneous class of
subunits (Table 3–2)
 INTERMEDIATE
FILAMENTS (IFs)
 Intermediate Filament Assembly and Disassembly
FIGURE : A model of intermediate filament assembly and architecture.
Each monomer has a pair of globular terminal domains
(red) separated by a long -helical region (step 1).

Pairs of monomers associate in parallel orientation with their

ends aligned to form dimers (step 2).
Depending on the type of intermediate filament, the dimers
may be composed of identical monomers (homodimers) or
nonidentical monomers (heterodimers).

Dimers associate in an antiparallel, staggered fashion to form

tetramers (step 3), which are thought to be the basic subunit in
the assembly of intermediate filaments.

8 tetramers associate laterally to form a unit length of the

intermediate filament (step 4).

Highly elongated intermediate filaments are then formed from

the end-to-end association of these unit lengths (step 5)
Intermediate Filaments

Lacks
structural
polarity
unlike actin
and
microtubule
 INTERMEDIATE FILAMENTS
• It is important to note
that the keratin
filaments anchored to
both sides of
desmosomes serve as a
mechanical link
between adjacent cells
in an epithelial layer,
thereby
providing mechanical
 Desmosomes and
stability to the entire
hemidesmosomes thus
tissue.
anchor intermediate
filaments to regions of cell–
cell and cell–substratum
contact, respectively.
 INTERMEDIATE FILAMENTS (IFs)

(a) Schematic drawing of a nerve cell

showing the movement of vesicles down the
length of an axon along tracks of
microtubules.

(b) Schematic drawing of the organization of the microtubules

and intermediate filaments (neurofilaments) within an axon.

Cytoplasm of neurons contains loosely packed bundles of intermediate filaments whose

long axes are oriented parallel to that of the nerve cell axon.
 Microfilaments: Actin Filament
• 2 stranded helical polymer of actin.
• Diameter of 5-9nm
• Organized in variety of linear bundles, in 2D and 3D
structure
• G-actin (globular actin), with bound ATP, can
polymerize to form F-actin (filamentous)
 Microfilaments: Actin Filament

Function: Determine shape of cell’s surface & cell

locomotion
•Actin subunit assemble head to tail to generate
filaments with structural polarity
•Actin filament considered as 2 parallel
protofilament twist around each other in right hand
helix
•Filament flexible and easily bend compared to
microtubules but assemble in bundle to form
strong structure
 MICROFILAMENTS
Actin filament structure.
(a) A model of an actin filament.
The actin subunits are represented in three
colors to distinguish the consecutive
subunits more easily.
The subdomains in one of the actin
subunits are labeled 1, 2, 3, and 4 and the
ATP-binding cleft in each subunit
is evident.
Actin filaments have polarity, which is
denoted as a plus and minus end.
The cleft (in the upper red subunit) is
present at the minus end of the filament.

(b) Electron micrograph of a replica of an

actin filament showing its double-helical
architecture.
 Microfilaments - Identification
 Identification of actin filaments in a given cell Cytochemical test

 Actin filaments (regardless of their source) will interact in a highly specific

manner with the protein myosin.
 Myosin (obtained from muscle tissue) is cleaved into fragments by a
proteolytic enzyme.
One of these fragments, called S1 binds to the actin all along the
microfilament.
When S1 fragments are bound, one end of the microfilament appears pointed like
an arrowhead, while the other end looks barbed.
 Microfilament Assembly and Disassembly
 Before it is incorporated into a filament, an actin monomer
binds a molecule of ATP.
 The ATP associated with the actin monomer is
hydrolyzed to ADP at some time after it is incorporated into
the growing actin filament.
 As a consequence, the bulk of an actin filament consists of
ADP-actin subunits.
 MICROFILAMENTS
Actin filament polymerization occurs over three phases:
1. A nucleation phase,
2. an elongation phase and
3. a steady state phase.
 Polymerization of Actin Filaments

https://www.researchgate.net/figure/Polymerization-of-actin-
filaments
 First, actin monomers linked to adenosine triphosphate (ATP) (G-actin,
green) form an aggregate (violet) that
 Grows exponentially by the addition of monomers to both ends of the
filament (elongation phase).
 In the end, actin filaments reach a stationary state with G-actin, and actin
filaments have a double-helical conformation.
Formin and the Arp2/3 complex (actin-related protein)
 Principal proteins that stimulate the initiation and elongation of actin
filaments
 The activities of these proteins are regulated in response to a variety of signals
to determine where filaments are formed within the cell.

 Formins are a family of proteins that bind ATP-actin and nucleate the initial
polymerization of actin monomers (Figure 14.5A).

(A) Formins initiate and stimulate the elongation of actin filaments.

Formin and the Arp2/3 complex (actin-related protein)
(B) The Arp2/3 complex
binds to actin filaments near
their plus ends and initiates
the formation of branches.

 Formation of branched
actin filaments is
regulated by the
physiological
needs of the cell.

Branched actin filaments play a key role in driving

cell movement at the plasma membrane
 MICROFILAMENTS
Dynamics and Treadmilling
 Actin filaments may undergo treadmilling, in which filament length
remains approximately constant, while actin
monomers add at the (+) end and dissociate from the () end.

Because subunits are

being added to the
plus ends and
removed from the
minus ends of each
filament at steady
state, the relative
position of individual
subunits within each
filament is continually
moving—a process
known as
“treadmilling”
 MICROFILAMENTS
Actin Binding Proteins
 Assembly and disassembly of actin filaments is regulated by Actin
binding proteins which regulate formation and stability of actin
cytoskeleton.
Stabilization of actin filaments
 Many actin filaments are relatively
stable within cells due to

 Capping proteins that

bind to their ends

 filament-stabilizing
proteins, such as
members of the
tropomyosin family,
which bind lengthwise
along the groove of
actin filaments (Figure
14.6).
Cofilin and Profilin role
Stress fiber & Focal adhesion
Adhesion of actin filament with
extracellular matrix mediated by
-Talin
-Vinculin
-Integrin
 Actin filament & Cell to Cell Junction

• Cadherin forms complex with

cytoplasmic protein catenin which
associtate with actin filaments.
Drugs affecting Actin filaments and microtubules

• Actin specific Drugs

– Phalloidin binds and stabilize filaments
– Cytochalasin caps filament plus ends
– Latrunculin binds subunits and prevents
polymerization
 Protrusion of cell surface

• Seen in
– Microvilli on brush border cell
– Pseudopodia
– Lamellipodia (for cellular motility)
– Filopodia (microspikes)
Cell Movement
Association of actin filaments with the plasma membrane
Human red blood cells
(erythrocytes)

 Lack other cytoskeletal

components
(microtubules and
intermediate filaments).

 Major protein that provides

the structural basis for the
cortical cytoskeleton in
erythrocytes is the actin-
binding protein spectrin.
 Association of actin filaments with the plasma
membrane

• Filamin link actin filament with plasma

membrane of blood platelets.
• Dystrophin link actin filament with
transmembrane protein of muscle cell plasma
membrane
– X linked inherited disease related to Dystrophin
• Duchenne’s muscular dystrophy (protein unit absent)
• Becker’s muscular dystrophy (abnormal protein)
Cell surface protrusions and cell movement
Myosin Motors
Actin filaments, often in association with myosin, are responsible for many types
of cell movements.
muscle contraction

Myosin is the prototype of a molecular motor—a protein that converts chemical

energy in the form of ATP to mechanical energy, thus generating force and
movement.
Myosin Motors
 Sliding Filament Model
 Is the basis for understanding muscle contraction.
 First proposed in 1954 both by Andrew Huxley and Ralph Niedergerke and by
Hugh Huxley and Jean Hanson.

During muscle contraction

 Each sarcomere shortens, bringing

the Z discs closer together.

 No change in the width of the A band

 Both I bands and the H zone

almost completely disappear.

These changes are explained by the actin and

Here myosin function as a
myosin filaments sliding past one another so
motor that drives actin
that the actin filaments move into the A band
filament sliding.
and H zone.
Sliding Filament Model
Myosin present in muscle is myosin II, is a very large protein (~500 kd) consisting of
two identical heavy chains (~200 kd each) and two pairs of light chains (~20 kd each)
Contraction of skeletal muscle is:
Triggered by nerve impulses, which stimulate the release of Ca2+ from
sarcoplasmic reticulum—that stores high concentrations of Ca2+ ions

This ↑es conc. of Ca2+ in cytosol from approximately 10-7 to 10-5M

↑es Ca2+ conc. signals muscle contraction via the action of two actin filament
binding proteins: tropomyosin and troponin

In striated muscle, each tropomyosin molecule is bound to troponin, which is a

complex of three polypeptides: troponin I (inhibitory), troponin C (Ca 2+
binding), and troponin T (tropomyosin-binding)

When conc. of Ca2+ is ↓ , the complex of troponins with tropomyosin blocks the
interactions of almost all actins with the myosin head groups, so the muscle does
not contract

At high concentrations,Ca2+ binding to troponin C shifts the position of the
complex, allowing access of the myosin head groups to an increasing number of
actins and allowing contraction to proceed
ATP Hydrolysis Is Necessary for Interactions with Thin
Filaments
 Skeletal muscle contraction requires interactions of myosin II head
groups with the thin filaments.

 These interactions are governed by binding and hydrolysis of the high-

energy molecule ATP by the ATPase activity resident in the globular
myosin II head domain.
ATP Hydrolysis Is Necessary for Interactions with Thin
Filaments
Binding of ATP to a myosin head
group causes release from the actin
filament (step 1).

Hydrolysis of ATP to ADPPi readies

the myosin head to contact an actin
filament (step 2)

The initial contact of the myosin with an

actin filament causes the release of Pi
and a tight binding of the actin
filament (step 3).

This tight binding induces a change in

conformation of the myosin head, such
that it pulls against the actin filament, the
powerstroke (step 4).

This change in conformation is accompanied with the release of ADP.

The binding of an additional ATP causes a release of the actin filament and a return of
the myosin head to a position ready for another cycle.
Non muscle myosin
ATP Hydrolysis Is Necessary for Interactions with Thin
Filaments
 If no ATP is available to the muscle (e.g., after death), the muscle
will remain rigid, owing to the tight myosin–actin interactions.
This condition is referred to as rigor mortis.

 Under normal circumstances, a molecule of ATP will displace the

bound ADP, causing release of the actin filament from the myosin
head group, effectively relaxing the muscle and returning to step 1 of
the cycle.

 The hydrolysis of the newly bound ATP then prepares the muscle
for
further rounds of myosin–actin interactions.
 References
1. Molecular Biology of THE CELL, 5th Edition
2. N. V. Bhagavan, Medical Biochemistry
3. Harper’s Illustrated Biochemistry, 31st Edition
4. Internet sources