Unit 7 Cytoskeleton Proteins and Cell Movement

UNIT 7

CYTOSKELETON PROTEINS AND

CELL MOVEMENT

Structure

7.1 Introduction
Organisation and Role of Actin
Expected Learning Outcomes Filaments

7.2 Eukaryotic Cytoskeleton 7.5 Structure and Function of

Intermediate Filaments
7.3 Structure of Microtubules
7.6 Structure and Function of
The Centrosome
Cilia and Flagella
Assembly and Disassembly of
7.7 Summary
Microtubules
7.8 Terminal Questions
Role of Microtubules
7.9 Answers
7.4 Structure and Organisation
of Actin Filaments 7.10 Suggested Readings
Actin Filaments Polymerization
and Treadmilling

7.1 INTRODUCTION
In preceding units of Block II you have studied about structure and function of
subcellular organelles; variations in cell wall structure and extracellular matrix
(ECM).You would have realised that various proteins (and other biomolecules)
are organised spatially at multiple levels within cells. It begins with having
functional protein complexes to compartmentalisation in specific membranes
or matrix and other aqueous compartments of organelles and finally a still
higher level of organisation is created and maintained by the cytoskeleton. The
cytoskeleton consists of three types of protein fibres and associated accessory
proteins. They form a network of fibres that establish interconnected paths of
communication. The cytoskeleton is a dynamic structure and reorganizes
rapidly in response to changing demands. These structures help cells to 139
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maintain or change shape; intracellular transport and positioning of subcellular

organelles; cell movement; cell division and above all to withstand stress.

Therefore, in this unit you will study the structure and functions of
microtubules, actin filaments and intermediate filaments. The examples cited
will explain both the structural and dynamic role of cytoskeleton. At the end of
the unit, structure and movement mediated by eukaryotic cilia and flagella are
also discussed. In Block 3 you will study the protein transport.

Expected Learning Outcomes

After studying this unit, you should be able to:

 define the cytoskeleton and its classification;

 describe the structure and organisation of the three major group of

cytoskeleton proteins;

 explain the assembly and disassembly of microtubules and actin

filaments;

 indicate the role of GTP and ATP in polymerization of tubulin and G-actin
respectively;

 explain the role of microtubules (MTs) and microfilaments in cell division;

 describe the families of MTs and Actin based motor proteins;

 describe the structure and function of cilia and flagella and;

 indicate the types of Intermediate Filaments (IF’s); common structural

design; assembly and role.

7.2 EUKARYOTIC CYTOSKELETON

The existence of a network of fibers or cytoskeleton within cells was
postulated in 1928 by a Russian biologist, Nikolai Koltzoff. In eukaryotes the
complex network is created from three types of protein fibers and a variety of
accessory proteins. The interactions between them add to the complexity and
their dynamic nature allows adaptability. A better understanding of their
structure, interactions and dynamics became feasible with advances in fixation
methods for EM and imaging techniques especially fluorescence microscopy.
These techniques were instrumental in locating the protein at any given time
and monitoring their dynamic behaviour in live cells.

It was initially assumed that the cytoskeleton was found only in eukaryotes but
Nikolai Koltzoff in 1992, a bacterial homolog of tubulin was identified. The FtsZ protein of
bacteria resembles tubulin. It binds and hydrolyses GTP and has a seven
amino acid tubulin signature sequence. It can also assemble into
protofilaments. Later bacterial proteins like FtsA, MreB and StbA, related to
actin superfamily were discovered. They can assemble into actin like
filaments. The protein crescentin of Caulobacter crescentus bear homology to

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eukaryotic proteins that assemble as intermediate filament. It is responsible for

the crescent shape of Caulobacter.

In this unit we shall discuss the cytoskeleton of eukaryotes. The cytoskeleton

is a complex network of protein filaments that is highly dynamic and
reorganises continuously as the cell changes shape, divides and responds to
environment. There are three major types of cytoskeleton elements namely,
microtubules, microfilaments and intermediate filaments (Fig.7.1). The
classification is based on their diameter and subunit structure. The diameter of
microtubules, actin filaments and intermediate filaments (IF) is 25nm, 7nm and
10nm, respectively. Microtubules are polymers of tubulin heterodimer; actin
filaments are assembled from G-actin and the building units of IFs varies
among different cell types.

Fig. 7.1: Classification and subcellular localisation of Cytoskeleton proteins.

SAQ 1
Answer the following questions:

i) Enlist three bacterial proteins that bear resemblance to cytoskeleton proteins

of eukaryotes.

ii) What is the basis of classification of cytoskeleton proteins?

7.3 STRUCTURE OF MICROTUBULES

Microtubules (MTs) are 25nm rigid hollow cylindrical tubules of variable
length.They are found in virtually all eukaryotic cells. The basic building unit of
MTs is tubulin heterodimers (α β) that are closely related and tightly linked
globular proteins. They polymerise to form a protofilament. Both subunits
bind GTP. In mammalian cells a cylindrical MT is formed from 13
protofilaments aligned in parallel with the same polarity (Fig.7.2). The MT is a
polar structure; the two ends are plus (fast growing) and minus (slow growing).
In a microtubule, α-tubulin is present at the minus (-) end of a protofilament
and β-tubulin is at the plus (+) end. The minus ends are stabilised by
embedding them in centrosomes. The plus ends extend throughout the
cytoplasm.
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Fig. 7.2: a) GTP bound tubulin heterodimer b) structure of protofilament c) A

hollow cylindrical microtubule d) Electron micrograph of microtubules

7.3.1 The Centrosome

The centrosome is somewhat shapeless body that nucleates the growth of
microtubules and determines their number and distribution. The MTs in turn
influence the distribution of other cytoskeletal protein fibers. Thus centrosome
is considered as the master architect of cytoskeletal design. In an interphase
cell, the centrosome lies close to the nucleus. It was first described
independently by Theodor Boveri and Edouard van Beneden in 1897.

In animal cells each centrosome has two centrioles at right angles to each
other and surrounded by pericentriolar material or centrosome matrix. Each
centriole is a bundle of nine rods; each consisting of three fused MTs. They
are also present in basal bodies underneath flagella and cilia. Not all
microtubule organizing centres (MTOC) contain centrioles as they are not
indispensable for the assembly or organisation of microtubules. The plus ends
of cytoplasmic MTs emanate from the pericentriolar material. The protein that
nucleates the assembly of microtubules is a highly conserved protein, γ-
tubulin. Generally 10-13 γ-tubulins are complexed to form ring like structures
that have diameter similar to microtubules It is a minor species of tubulin and
may remain bound to the minus end of MTs. γ-tubulinwas first identified in the
spindle pole body of Aspergillus nidulans.

7.3.2 Assembly and Disassembly of Microtubules

Now let us discuss about the assembly and disassembly of microtubules. As
you know both subunits of tubulin heterodimer bind GTP but GTP bound to β-
tubulin is hydrolysed during or shortly after polymerisation. This in turn
weakens the affinity of tubulin for the preceding unit, resulting in
depolymerisation. The dynamic behaviour of microtubules can involve
treadmilling or dynamic instability. The average half life of a MT ranges from
approx. 10 minutes in non dividing animal cells to as short as 20 seconds in a
dividing cell.
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During treadmilling tubulin bound to GDP are continually lost from the minus
end and at the same time they are added bound to GTP from the plus end.
This is more common with actin filaments. Microtubules also have cycles of
growth and shrinkage. A microtubule continues to grow as long as the
concentration of GTP bound tubulin is high and new ones are added more
rapidly than GTP is hydrolysed. A GTP cap will be present at the growing end.
The tubulin dimers are added faster at the plus end of microtubule compared
to the minus end. On the other hand, a MT shrinks due to depolymerisation, if
GTP is hydrolysed more rapidly than the rate at which new subunits are added
(Fig 7.3). This behaviour called dynamic instability was described by
Mitchison and Kirschner and it is due to delayed hydrolysis of GTP after
tubulin assembly.

Fig. 7.3: Polymerization and depolymerisation of microtubules.

It is possible to modify dynamic instability of microtubules to suit specific

needs. Microtubules can undergo post translational modifications after
polymerization. Two of them are acetylation and detyrosination of α-tubulin.
These modifications serve as binding sites for microtubule associated proteins
(MAPs) that stabilize them.Many MAPs have been identified in different cells
which perform a wide range of functions including stabilising and destabilising
microtubules, guiding microtubules to specific cellular locations, and mediating
interactions of microtubules with other cytoskeletal proteins in the cell.

A number of naturally occurring substances interfere with the dynamic

behaviour of microtubules and are used as anti mitotic drugs. One of these is
an alkaloid colchicine. It binds free tubulin and prevents its polymerization.
This results in the disintegration of mitotic spindle in a dividing cell and is
routinely used to induce polyploidy or to study chromosome structure. The
drugs vincristine and vinblastine (similar to colchicine) are used to treat certain
cancers. Another anti cancer drug, taxol binds to MTs and stabilises them. The
cell division is arrested as MTs must shorten to carry chromosomes to the
poles. 143
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7.3.3 Role of Microtubules

In this subsection you will learn about some of the important roles of MTs both in a
resting cell and during cell division.

 Intracellular transport of cell organelles and vesicles

Virtually all membrane bound organelles have a close association with

microtubules. The ER extends almost to the edge of the cell whereas the Golgi
apparatus is present close to the centre of the cell, near the centrosome. MTs are
involved both in the intracellular transport and positioning of organelles. Similarly
they transport vesicles to their destination. The motor proteins, kinesin and dynein
are involved in microtubule based motility. The cytosolic face of organelles has
receptors on their membranes through which they interact with specific motor
proteins. These proteins require ATP to generate force and transport either along
the plus (kinesin) or minus (dynein) end of MTs (Fig.7.4). The motor proteins
belong to two large families of related proteins. The microtubules function as tracks
for transport or positioning of cargo.

The motor protein kinesin consists of two heavy chains and two light chains; it
has a rod-like structure composed of two globular heads, an α-helical coiled –
coil stalk involved in dimer formation and a fan like tail domain while dynein
comprises of two or three heavy chains with multiple light and intermediate
chains. The heavy chains in both form globular ATP binding motor domain that
facilitate movement along microtubules. They are MT-activated ATPase. The
base of both proteins interacts with organelles and vesicles via a connector.
The ER membrane has kinectin that binds kinesin. Similarly the connector
complex, dynactin binds vesicles to dynein.

Fig.7.4: Microtubule associated motor proteins.

 Cell division

The dynamics of microtubule assembly and disassembly changes

dramatically as the cell enters mitosis. The cytoplasmic microtubules
become very unstable and the interphase array depolarises. The
centrosome duplicates to form the mitotic spindle and it nucleates and
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promotes the growth of many dynamic microtubules (MTs).The MTs

emanating from the spindle are kinetochore MT, polar MT and astral MT
(Fig 7.5).

The kinetochore MTs attach to specialised regions on condensed

chromosomes called kinetochore. As MTs from both poles of mitotic
spindle attach to kinetochore they will stop growing or shrinking. It is as if
the centrosome sends out “feelers” in search of chromosomes. The net
outcome is the alignment of all replicated chromosomes at the metaphase
plate. The polar MTs are stabilized by overlapping with each other while
the astral MTs extend towards the cell periphery.

Fig. 7.5: The arrangement of MTs in a non-dividing and mitotic cell.

(Credit image: M. Cooper, The Cell: A Molecular Approach)
The separation of sister chromatids and movement to opposite poles is
dependent both on the dynamic behaviour of MTs and kinetochore associated
motor proteins. The kinetochore MTs are shortened and the polar MTs elongate.
We will come back to this subject again in unit 11 Cell Cycle (Block 4).

In most cells cytokinesis follows nuclear division. In animal cells an actin and
myosin based contractile ring is assembled precisely half way between the poles
and perpendicular to the spindle. Even here MTs are implicated in indirectly
influencing the site of cleavage furrow.

 Intracellular organisation

The main function of microtubules is to provide intracellular framework and

maintain cell shape. In non dividing cells, network of microtubules helps to stabilize
subcellular organelles in their proper position.

 Cell motility

The structural components of eukaryotic cilia and the flagella are bundles of
microtubules. They are present on the surface of many kinds of cells and are
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used for multiple purposes such as locomotion, to collect food, move mucus
along with trapped particles consisting of air pollutants, dust or
microorganisms up towards the mouth to be swallowed and to propel eggs.
The structure and movement of cilia and flagella will be described in section
7.6.

 Cell polarity

The cytoskeleton can be reorganised to polarise a cell transiently. Generally actin

filaments and MTs act together. To demonstrate how these events are
coordinated, we will consider the killing of a target cell by cytotoxic T lymphocyte. A
cytotoxic T cell expresses antigen specific T cell receptor that looks for processed
foreign antigen presented on target cells. Once antigen is recognised by a T cell it
signals local reorganization of underlying actin cortex and then the centrosome
reorients, moving MTs along with it. The MTs in turn position the Golgi body near
site of contact allowing transport of materials needed for cytotoxicity from Golgi
along MT tracks to the target cell for focused secretion.

SAQ 2
a) Fill in the blanks:

i) The microtubules are made up of …………………

ii) Microtubules are polar structures because …………………….

iii) The dynamic instability of MTs was described by ………… and ……………

iv) The MTs are used as …… for transport and positioning of organelles and
vesicle; the movement is dependent on ……………….

v) The kinetochore microtubules are attached to …………..during mitosis.

vi) The plus end directed motor protein of microtubules is ………….

vii) The hydrolysis of GTP from β-tubulin causes …………. of microtubules.

viii) The cellular function of γ-tubulins is …………………….

ix) Colchicine binds to …………… that results in ……………

x) The cilia of eukaryotes are………….

b) The diagrams show growing and shrinking microtubules. Label the (+)
and (–) ends of MTs in (i) and (ii).

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7.4 STRUCTURE AND ORGANISATION OF

ACTIN FILAMENTS
An actin filament as the name suggests is made up of actin (43Kd). It was
discovered and isolated from muscle cells by Brunó Ferenc Straub in 1942. It
is a major globular protein of muscle cells. An actin monomer is called G
(globular) actin that binds ATP. It polymerises to form actin filaments (or F-
actin). The filament has a polar structure as each subunit faces in the same
direction. It has a slow growing minus or pointed end and a fast growing plus
or barbed end. The polarity of actins is important in establishing the directional
movement of myosin relative to actin and in their assembly. Brunó Ferenc Straub

As compared to other cytoskeletal protein fibres they are thinner (7-9nm) and
hence they are also referred to as microfilaments. Electron micrograph of actin
filaments shows that they consists of two twisted chains of G-actin molecules
arranged head-to-tail which makes them look like a double helix (Fig. 7.6).

Fig.7.6: a) Molecular structure of Actin and b) Actin filament (F-actin)

Actin filament is a major cytoskeleton element of all eukaryotic cells;

vertebrate skeletal muscles are a rich source (approx. 20% of total protein)
and the usual source of actin for experimentation. In eukaryotes actin is
encoded by a multigene family and they are divided into three classes (α-β-
and γ-actins) based on their isoelectric point; α-actin is found in skeletal and
cardiac muscles whereas beta (β) and gamma isoforms (γ1-cytoplasmic) are
present in non muscle cells. Inspite of the occurrence of multiple forms they
are highly conserved proteins. Actin filaments are generally cross linked to
form actin networks and bundles which are much stronger than individual
filaments.

7.4.1 Actin filament Polymerization and

Treadmilling
In this subsection you will learn about reversible polymerisation of actin
monomers. Look at Fig. 7.7 (a) for an overview of filament assembly and
disassembly. The first step of actin polymerization is nucleation to form a small
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complex consisting of three actin monomers. Then actin filament grows by

addition G-actin from both ends. The two ends of the filament grow at different
rates; the plus end polymerises five to ten times faster than the minus end.
During the elongation phase there is a net increase in filament length. The
actin subunits in a filament can hydrolyse ATP and depolymerise. The rate at
which actin monomers are added into filaments is dependent on the critical
concentration of free monomers.

Actin filaments engage in an ATP dependent dynamic behaviour called

treadmilling. In this ATP-actin monomers are added from the plus end with
concomitant loss of ADP-actin from the minus end (Fig. 7.7b). There is no net
change in filament length but it allows a rapid exchange of subunits. This
occurs at monomer concentration intermediate between the critical
concentration for plus and minus ends.

Fig 7.7: a) Assembly & disassembly of actin filaments b) Treadmilling.

The rate of polymerisation and depolymerisation is tightly regulated in the cell

by actin-binding proteins. As expected these proteins can either enhance
depolymerisation or stimulate the incorporation of monomers into actin
filaments. In general the turnover of actin filaments is much faster in the cell
and more than 40% of actin in animal cells is unpolymerised.

Look at Fig. 7.8 in which some actin binding proteins are shown to control
polymerization. A G-actin sequestering protein, profiling stimulates exchange
of ATP for ADP and its polymerisation to F-actin. A capping protein, CapZ then
binds to the growing plus end to fix actin polymerization. On the other hand,
cofilin protein binds to the minus end of actin filaments and enhances the
dissociation of actin monomers. In addition cofilin can sever actin filaments
and then each end can be disassembled, enhancing the overall rate. Another
protein gelsolin fragments actin fragments in presence of Ca+2.The Arp 2/3
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proteins can nucleate the growth of new actin filaments. It also caps the minus
end. The activities of these proteins are regulated in the cell.

Fig. 7.8: Control of actin polymerisation by actin binding proteins

(Image source: Albert: Molecular Biology of the Cell).

Some products of biological origin are known that affect actin polymerisation.
The drug cytochalasins are fungal products which bind to plus end of actin
filaments and block further polymerisation. It is used to study cell locomotion.
Similarly phalloidin from Amanita mushroom binds actin filaments tightly and
stabilise them. It is generally labeled with fluorescent dyes to track actin
filaments by fluorescence microscopy. These drugs result in dramatic changes
in actin based dynamic structures and functions dependent on them.

7.4.2 Organisation and role of actin filaments

As mentioned earlier actin filaments are organised into bundles or networks
with the help of cross linking proteins. The actin bundles may be closely
packed or loosely crosslinked bundles (Fig.7.9). Some of them are also
contractile (contractile ring; stress fibres).

Fig. 7.9: Types of actin binding proteins. 149

Block 2 Structure and Function of the Cell

The two cross linking proteins shown are α-actinin (a dimer) and fimbrin (a
monomer). The spacing in the contractile bundle allows motor protein, myosin
to interact with the filaments. An actin binding protein filamin has an actin
binding domain at one end and a dimerisation domain at the other end of each
monomer. The cross linking of actin filaments by a flexible V-shaped filamin or
similar proteins results in a loose gel like 3D network of actin cortex beneath
the plasma membrane (Fig. 7.10). The cortex determines cell shape and
surface activities including movement as it is also linked to cell membrane that
receives and signals the cell to modify the underlying cortex.

Fig. 7.10: Various actin filament based structures.

In many cells there are discrete regions of cell membrane that make contacts
with adjacent cells or to the extracellular matrix. The sites of attachment
include focal adhesion and adherens junctions (Refer to unit 6).Similarly
microvilli are abundant on the apical surface of intestinal epithelial cells
involved in absorption. It has parallel actin filaments held together by a
bundling protein, vilin.

The actin bundle is linked to the overlaying plasma membrane by lateral

bridges of myosin I. In addition there are structures that are transient such as
pseudopodia that are formed in cells during phagocytosis and movement by
amoeba.

The motor protein for ATP dependent roles of actin filament belongs to myosin
super family whose members are present in both muscle and non muscle actin
based structures. The best studied among them is myosin I and II.

A myosin II protein is composed of two identical heavy chains and two pairs of light
chains. Each heavy chain has a globular head that forms the motor domain and a
long α-helical tail. The tails of both heavy chains are twisted around each other to
form a coiled coil dimer. A pair of light chains is present close to the head domain
of each heavy chain (Fig. 7.11).
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Fig. 7.11: Structure of myosin II.

Now we will learn about some examples of contractile assemblies of actin-myosin

in non muscle cells. Muscle contraction will be covered in Human Physiology
course of semester IV (BBCCT-115). During cytokinesis an actin –myosin II based
contractile ring is assembled transiently in animal cells just below the plasma
membrane. The contraction of the ring pulls the membrane inwards producing two
daughter cells. The actin filaments disassemble as contraction progresses. The
other examples include the intracellular transport of organelles and vesicles along
actin filaments and cell crawling movements used by a variety of cells such as
embryonic cells and migration of white blood cells to sites of damage.

In addition to conventional bipolar myosin II, several other types of myosin such as
myosin-I are found in nonmuscle cells. The myosin--I is much smaller and does
not form dimers. Its tail can carry cargo along actin filaments; forms the lateral
arms of microvilli and movement of plasma membrane during phagocytosis.

SAQ 3
a) Indicate whether the following statements are true and false. If false, point
out the error:

i) Actin filaments are also known as microfilaments. [ ]

ii) The bundling protein in microvilli is α-actinin. [ ]

iii) There is no net change in actin filament length during treadmilling. [ ]

iv) Dynein is the motor protein of actin. [ ]

v) Actin filaments display “dynamic instability”. [ ]

vi) Myosin-I forms dimers like myosin II. [ ]

b) Which one of following figures shows the distribution of actin filaments in a

cell?

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7.5 STRUCTURE AND FUNCTION OF

INTERMEDIATE FILAMENTS
As the name suggests intermediate filaments have a diameter (10nm) that is
intermediate between actin filaments (7nm) and microtubules (25 nm).The
term cytoskeleton was originally coined for the stable and insoluble fibres left
behind when cells are treated with concentrated salt solutions and non ionic
detergents. This fraction was actually represented by intermediate filaments
(IFs).They do not bind nucleotides to regulate their assembly and
disassembly. Unlike actin filaments and microtubules IFs constitute a
heterogeneous group of fibrous proteins with variable expression profile. They
are ubiquitous in multicellular eukaryotes. The intermediate filaments are
classified based on their sequence (Table 7.1).

Table 7.1: Types of Intermediate filament (IF) proteins

Type IF proteins Expression Remark(s)

profile

I Acidic keratins Epithelial cells Type I and II always

copolymerise.

II Basic or neutral keratins Epithelial cells -do-

III Vimentin; Fibroblasts, WBC Can polymerise

either with one or
desmin; Muscle cells,
two type III proteins.
Glial fibrillary acidic protein Glial cells They do not
(GFAP) copolymerise with
keratins.

IV Neurofilaments -NF-L; NF-M; Neurons The three NFs can

NF-H copolymerise.

V Nuclear lamins- A, B and C Nuclear lamina Assemble into 2D

sheets.

VI Nestin Stem cells of -

central nervous
system (CNS)

Although IFs differ in size, sequence and distribution yet they share a common
structural design (Fig.7.12). They all possess a central α-helical rod of more
than 300 amino acids flanked by non helical variable sized amino (head) - and
carboxyl (tail)-terminal ends. The common central domain is largely
responsible for filament assembly and the variable ends determine the
specialised role of different IF proteins.

The central rod has tandem repeats of a seven amino acid sequence (heptad
repeat) that promotes the formation of coiled-coil dimers between parallel α-
152 helices. The dimer may be a homo-or hetero-dimer. Then two dimers
Unit 7 Cytoskeleton Proteins and Cell Movement

associate to form tetramers; the dimers have a staggered alignment and anti
parallel. The resultant tetramer lacks polarity. They do not possess (+) or (-)
ends. Eight of these tetramers further associate laterally to form a unit length
filament. The unit length filaments are supercoiled end to end to form a mature
full-length filament about 11 nm in diameter (Fig. 7.13).They form a rope like
structure.

Fig. 7.12: a) Major classes of IFs b) Domain structure of IFs (helical central
domain).

Fig. 7.13: (a) Assembly of Intermediate filaments; b) Electron micrograph of IFs.

(Source: Gerald Karp: Cell and Molecular Biology: Concepts and Experiments) 153
Block 2 Structure and Function of the Cell

IFs in cells are dynamic and reorganise in response to cell cycle specific or
differentiation specific cues. The assembly and disassembly of some
intermediate filaments can be modified by phosphorylation. The nuclear lamins
are disassembled by phosphorylation during mitosis that results in breakdown
of the nuclear envelope. The process reverses at the end of cell division.
Similarly cytoplasmic. Vimentin is also phosphorylated in a cell cycle
dependent fashion. All phosphorylation do not regulate filament assembly and
disassembly. The phosphorylation of NF may affect their functional
interactions.

Functions of intermediate filaments

1. Intermediate filaments provide a scaffold to organize the contents of the cell.

They resist mechanical and shear stresses.

2. Intermediate filaments known as lamins A, B, and C provide strength to

nuclear envelope and help in distribution of chromatin. These lamins form a
meshwork that reinforces the inside of the nuclear membrane.

3. Hard keratins are found in hair, scales and nails.

4. Neurofilaments along with MTs and microfilaments provide high tensile strength
to compression, twisting, stretching and bending forces to stabilize the extended
axons of nerve cells.

5. Desmosomes and hemidesmosomes anchor IFs to regions of cell-cell and cell-

matrix contacts (Refer to unit 6).

SAQ 4
Match the cells/ sub cellular structure in column I with Ifs in Column II.
Column I Column II

I. Nerve cells a. Keratins

II. Glial cells b. Neurofilaments

III. Skin cells c. Desmins

IV. Muscle cells d. lamins

V. Nuclear membrane e. GFAP

7.6 STRUCTURE AND FUNCTION OF CILIA

AND FLAGELLA
Recall the structure of microtubules described in section 7.2. All eukaryotic
cilia and flagella are bundle of microtubules. Cilia are generally small and hair-
like structures that extend from the body of a variety of cells while flagella are
long hair-like structure and generally found in prokaryotic cells. They vary in
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terms of length and numbers in different types of cells. The central core of
doublet microtubules is called an axoneme in which nine pairs of outer
doublet microtubules are organized in a circle around two central singlet
microtubules. This arrangement of MTs is termed “9 + 2” pattern (Fig. 7.14).

At its point of attachment to the cell the minus ends of axoneme connects to
basal body which like centrioles consists of nine triplets of MTs. Each triplet
(A, B and C) has one complete (A tubules) and two incomplete protofilaments
(B and C tubules).

The basal body initiates the growth of axonemal microtubules. The A and B
tubules of each triplet extend into the axoneme and so we have a doublet of
nine MTs. The outer MT doublets are connected to the central pair by radial
spokes. The axoneme is also associated with other proteins like nexin that
joins adjacent doublets and dynein to power movement. The two arms of
dynein are attached to A- tubule. The central pair is surrounded by a fibrous
sheath (inner sheath).

Fig. 7.14: a) Structure of cilia and flagella b) Electron micrograph of cilia.

(Image Courtesy: Gerald Karp: Cell and Molecular Biology: Concepts and Experiments)

Recall that dynein is a minus end directed motor protein. It is also responsible
to drive the movement of cilia and flagella. This process requires ATP.

Look at Fig. 7.15 in which dynein arms bind to A-microtubules while dynein
head interacts with B microtubules of adjacent doublets. The basis of
axonemal movement is sliding of protein filaments relative to one another. As
dynein heads move, it causes the A tubule of the doublet to slide towards the
basal body of adjacent B tubule. Since multiple nexin links hold adjacent MT
doublets together, a sliding movement is converted into a bending motion. The
activity of dynein is regulated to coordinate these movements.

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Fig. 7.15: Dynein dependent movement of cilia and flagella.

SAQ 5
Differentiate between cilia and flagella.

7.7 SUMMARY
In this unit so far, you have studied that:

 In eukaryotes the cytoskeleton consists of three types of protein fibres

(MTs, microfilaments and IFs) and accessory proteins. They form a
network that establishes interconnected paths of communication. The
cytoskeleton is a dynamic structure and reorganises in response to
changing demands.

 The cell skeleton help cells to maintain shape; in intracellular transport

and positioning of subcellular organelles; cell movement; cell division and
above all to withstand stress.

 Bacteria also encode proteins that bear resemblance to cytoskeletal

proteins in eukaryotes. In 1992, a bacterial homolog of tubulin; FtsZ
protein was shown to bind and hydrolyse GTP. It has a seven amino acid
tubulin signature sequence and can also assemble into protofilaments.
Later bacterial proteins (FtsA, MreB), related to actin superfamily and IFs
(crescentin) were discovered. They can assemble into actin like or IFs,
respectively.

 Microtubules (MTs) are 25nm rigid hollow cylindrical tubules of variable

length. They are built by polymerisation of tubulin heterodimers (α and β)
that are closely related and tightly linked globular proteins. In mammalian
cells a cylindrical MT is formed from 13 protofilaments aligned in parallel
with the same polarity. It is a polar structure with a plus (fast growing) and
minus (slow growing) end. MTs exhibit dynamic instability. The
centrosome nucleates the growth of microtubules and determines their
number and distribution.
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Unit 7 Cytoskeleton Proteins and Cell Movement

 MTs and associated motor proteins (dynein and kinesin) help in transport
and positioning of organelles and vesicles; cell division; cell motility and
cell polarity.

 Actin filaments (F actin) are thin, thread like structures, about 6-7 nm in
diameter. The actin monomer (G-actin) is a globular protein (43Kd) that
binds ATP and polymerises to form actin filaments. The filament has a
polar structure with a slow growing minus or pointed end and a fast
growing plus or barbed end. ATP hydrolysis triggers depolymerisation.
Actin filaments engage in an ATP dependent dynamic behaviour called
treadmilling.

 Actin filaments are cross linked with the help of actin binding proteins to
form bundles and complex network. They also interact with plasma
membrane and anchor the cell at regions of cell-cell contact. The actin
bundles may be closely packed or loosely cross linked bundles and even
contractile. The motor protein for actin filaments is a myosin family
member.

 The nonmuscle myosin participates in cytokinesis, cell crawling,

intracellular transport, etc.

 Intermediate filaments (IFs) are about 10nm non polar filaments. They
constitute a heterogeneous group of fibrous proteins with variable
expression profile. Although IFs differ in size, sequence and distribution
yet they share a common structural design. These proteins do not bind
nucleotides to regulate their assembly and disassembly. They provide
mechanical and tensile strength to cells.

 All eukaryotic cilia and flagella are bundles of microtubules. The

arrangement of microtubules is termed “9 + 2” pattern. The motor protein
dynein powers the movement of cilia and flagella. They are used for
multiple purposes such as locomotion, to collect food, move mucus along
with trapped particles up towards the mouth to be swallowed and to propel
egg.

7.8 TERMINAL QUESTIONS

1. Compare (a) intermediate filaments, (b) actin filaments and (c)
microtubules in terms of size, polarity, building blocks and distribution in
an animal cell.

2. What is the mode of action of the following inhibitors?

i) Colchicine ii) Cytochalasins iii) Phalloidin

3. Compare the structure and role of motor proteins associated with MTs.

4. Why MTs are polar structures? What is the difference between the plus
and minus ends?

5. Indicate similarities between MTs and microfilaments. 157

Block 2 Structure and Function of the Cell

6. Explain how ATP hydrolysis after actin addition to microfilament affects

filament elongation dynamics.

7. Why are intermediate filaments more stable than other

cytoskeletalproteins?

8. How do IFs differ from other cytoskeletal proteins?

7.9 ANSWERS
1. i) FtsZ protein resembles tubulin; MreB and FtsA proteins are actin like
proteins; crescentin of Caulobacter is like IF proteins.

ii) The classification of cytoskeleton is based on their diameter and subunit

structure.

2. a)

i) α β tubulin heterodimers. ii) the two ends are asymmetric

iii) Mitchison and Kirschner iv) tracks; motor proteins

v) kinetochore on duplicated chromosomes

vi) kinesin vii) depolymerisation

viii) nucleate the growth of MTs

ix) binds to free tubulin; depolymerisation of MTs

x) bundles of MTs.

b).

3. a)

i) True. ii) False iii) True iv) False v) False vi) False

b)

Fig. (ii) shows the actin filaments.

4.

I.- b; II. – e; III.- a; IV.-c; V.- d

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Unit 7 Cytoskeleton Proteins and Cell Movement

5. Cilia are short, hair-like structures and usually more numerous that may
cover the entire cell surface. In contrast, flagella are longhair-like structures,
generally one or two.

TERMINAL QUESTIONS
1.
Type of Diameter Building Filament Location in
cytoskeletal blocks polarity the cell
Proteins
25 nm Tubulin Polar with a (+) Present cell
Microtubule
dimers & (-) end interior

7-9 nm keratins, Non-polar present in

Intermediate
Vimentin; the entire cell
Filaments
lamins; NF

10-12 nm Polar with a (+) Found at the

& (-) end inner edges
Actin Filaments G-actin
of the cell

2. i) Colchicine: It binds to free tubulin and prevents polymerization. It causes

the disintegration of the mitotic spindle in a dividing cell

ii) Cytochalasins: It binds to the plus end of actin filaments and block further
polymerisation.

iii) Phalloidin: It binds actin filaments tightly and stabilise them.

3. i) Kinesins are relatively less complex motor proteins (MW:110,000 –

135,000) while dyneins are large and structurally complex proteins (MW:
1,000,000).

ii) They move in opposite direction along MT tracks; kinesins are plus end
directed while dynein are minus end motors.

4.

The MT is a polar structure built from a related heterodimer. In a microtubule,

α-tubulin is present at the minus (-) end and β-tubulin is at the plus (+) end of a
protofilament. The two ends grow at different rates. The minus ends are
stabilised by embedding them in centrosomes while the plus end are directed
away from the MTOC.

5.

i) Both are built by polymerisation of a single globular protein encoded by

multigene families; MT has tubulin dimers and actin has G-actin. (ii) Both
exhibit dynamic behaviour. (iii) Both are polar structures. (iv)Both require
specific motor proteins for movement related functions, for instance
intracellular transport. 159
Block 2 Structure and Function of the Cell

6.

The actin subunits in a filament can hydrolyse ATP and depolymerise by

releasing ADP-actin due to a decrease in affinity with adjacent actin
monomers. In the cell this process is regulated. The rate of addition and
removal dictates the extent of growth or shrinkage respectively.

7.

IFs are more stable because they do not bind and hydrolyse nucleotides,
responsible for the dynamic behaviour of MTs and microfilaments. IFs in cells
are dynamic and reorganise in response to cell cycle specific or differentiation
specific cues such as disassembly by phosphorylation of lamins.

8.

i) IFs are intermediate in diameter to MTs and actin filaments. ii) They are self
assembled from a variety of fibrous proteins, either alone or in combination. iii)
They do not bind NTPs. iv) They are non polar 10nm filaments. v) They are
not associated with motor proteins. vi) Their dynamic behaviour is dictated by
modifications or association with other proteins.

7.10 SUGGESTED READINGS

1. Gerald Karp, Janet Iwasa, Wallace Marshall (2010). Cell and Molecular
Biology: Concepts and Experiments. VIII Edition. John Wiley and Sons.
Inc. ISBN: 978-1-118-88614-4.

2. De Robertis, E.D.P. and De Robertis, E.M.F. (2006). Cell and

Molecular Biology. VIII Edition. Lippincott Williams and Wilkins,
Philadelphia.

3. Cooper, G.M. and Hausman, R.E. (2013). The Cell: A Molecular

Approach. VI Edition. ASM Press and Sunderland, Washington, D.C.;
Sinauer Associates.

4. Harvey Lodish, Arnold Berk, S Lawrence Zipursky, Paul Matsudaira,

David Baltimore and James Darnell (2016). Molecular Cell Biology, VIII
Edition, W. H. Freeman & Company; New York:ISBN-10:1-4641-8339-
2.

5. Arshad Desai and Timothy J. Mitchison. Microtubule Polymerization

Dynamics.Annu. Rev. Cell Dev. Biol. 1997. 13:83–117.

6. Molecular Biology of the Cell (© Garland Science 2008).

160