The cytoskeleton is composed of three main components - actin filaments, microtubules, and intermediate filaments. Actin filaments are made up of actin monomers that polymerize to form double helical structures. They play important roles in cell shape, movement, division, and intracellular transport. Microtubules are composed of tubulin dimers that assemble into hollow rods. They are involved in determining cell shape, movement, and transport of organelles. Both actin and microtubules undergo dynamic instability, rapidly assembling and disassembling in response to cellular signals.

2. Cytoskeleton
 Cytoskeleton in the structure of the protoplasm was
postulated in 1928 by koltzoff
 Dynamic three- dimensional scaffolding
 Proteins
 Provides – structures, strength and motility
 Internal transport of organelles and other (Mitotsis)
 Composed of three types- actin filaments,
microtubules and intermediate filament
 Linked subcellular organelles and the plasma
membrane

3. Actin filaments
• They are made up of many monomers of a protein called
actin
• Combined in a structure that resembles a double helix
• They are made of actin monomers, microfilaments – actin
filaments
• Globular proteins
• Within the cell, actin filament – called microfilaments
• Abundantly beneath the plasma membrane
• Mechanical support, cell shape, movement –enable the cells
to migrate, engulf particles and divides

4. Functions
• They serve as tracks for the movement of a motor protein called
myosin
• During cell division , a ring made of actin and myosin pinches the cell
apart to generate two new daughter cells
• Actin and myosin are also plentiful in muscle cells and which the
organized structures of overlapping filaments called sacromeres
• It serves as a inside cell transport of cargoes, including protein
containing vesicles and even organelles
• Major role in cell motility (WBC –during immune system)

5. Organization of actin filaments- two major
structural types
Actin bundles Actin networks filaments
Cooper and Hausman

6.  In actin bundles , actin filaments are cross-linked in
packed parallel arrays
 In networks, the actin filaments are cross-linked in
orthogonal arrays that form three-dimensional
meshwork with the properties of semisolid gel
 Governed - by a variety of actin binding proteins that
cross-link actin filament in distinct pattern
 All these actin binding protein in cross-linking contain
at least two domains, bind to actin and allowing them
to bind other actin filament

7.  Actin binding proteins- small-rigid proteins, helps in
closely alignment (in case of bundle)
 Actin binding proteins- large flexible proteins that
can cross-link perpendicular filaments

8. Actin filament bundles – two types
 There are atleast two structurally and functionally
distinct types of actin bundles involving different actin
–binding proteins
 Alpha- actinin cross –links actin filaments into loose
bundles
 Fimbrin cross-links filaments into tight bundles
Cooper and Hausman
Contractile bundle Parallel bundle

9. Parallel Bundle
In the parallel bundle, actin filaments aligned in
parallel and closely spaced
Seen adjacent to the Plasma membrane
Ex- Fimbrin, 68kD protein containing two adjacent
Actin –binding domains
It binds to actin filaments as a monomer, holding two
parallel filaments close together
( two neighboring domains (ABD) which bind actin)
Tight bundles –distance between the filaments 14nm
Frist isolated in the intestinal microvilli
Cooper and Hausman

10. Contractile Bundle
 The second one contractile
bundle – these actin filaments
cross-linked are widely spread
 Alpha –Actinin
 These Alpha –Actinin, binds
actin as a dimer each subunit
consist 102kD protein containing
single actin binding site
 Filament distance 40nm, which
allows contraction of these
bundles
 Interaction of myosin with actin
Cooper and Hausman

11. Assembly and disassembly –Actin filaments
 Each individual are globular actin (G) ….375 aa
 Two binding sites for other two monomers…..head to tail
 Polymerization ---Filamentous (F) actin
 Each monomer rotate by 166º …that makes the appearance of double –
stranded helix
 All the actin monomers are oriented in the same direction with distinct
polarity
 Polarity –which plays an important role in their assembly and also
establishing specific direction of myosin movement relative to actin
Barbed end Pointed end

13. Polymerization
Nucleation, is the formation small aggregate consisting /formation of three actin
Able to reversible by addition of monomer of both end, but barbed end grows
/elongates five to ten times more than pointed end
ATP bind actin monomers are added rapidly to the barbed end ATP is hydrolyzed to
ADP after polymerization
The ADP actin monomer is less tightly bound and it is dissociate from the pointed end
This phenomenon – Threadmilling

14.  Cytochalasins- bind to the barbed ends of actin
filaments and block their elongation
 Changes the cell shapes, as well as inhibition of cell
movement
 Phalloidin – Binds tightly to actin filaments and
prevents their dissociation into individual actin
molecules
 Within the cell, the assembly and disassembly of actin
filaments is regulated by a diverse group of actin
binding proteins
 Regulation and stability of the actin cytoskeleton at
different level by actin binding proteins

15.  ABP – stabilizes the actin filament by capping the ends
and preventing the dissociation of actin monomers
 Some of the proteins binding along the length of actin
filaments stabilizing them /cross-linking them to one
another
 Some of them will disassemble actin filaments either
by serving them/stimulating their depolymerization
 Binding with monomer actin, control the assembly of
filaments by regulating the exchange of ATP for ADP

16. Stimulating the nucleation…
 Formin and Arp2/3 complex… actin related protein ,
which determines where the filaments are formed
within cell
 Formin –large family 140-200kD, barbed end tracking
protein (EU)—adding new monomer to end
 Formins nucleate long unbranched actin filaments
that make up stress fibers , the contractile ring,
filopodia and the thin filaments of muscle cells
 It is stabilized by the Filament Stabilizing Protein-
such as tropomyosin family
 It s 30-36 kd fibrous proteins that bind lengthwise
along the groove of actin filaments

17.  During the cell processes or moving cells, actin
filaments both actively turn over and also branch
extensively
 Branched actin filaments is nucleated by the Arp2/3
complex, which binds ATP actin near the barbed ends
of filaments
 This complex contains seven proteins----that activate
the Arp2/3 complex binds to side of an existing actin
filament near barbed end and form new branch

18.  Another type of Actin binding protein remodels
 Within in the cell one of the protein –ADF/Cofilin(Actin
polymerizating factor) family
 ADF/Cofilin-Two different activity
1. Actin depolymerization factor -This protein binds to actin
filament and enhance the rate of dissociation of ADP-actin
monomers from the pointed end
2. Preventing the reassembly into filaments
• Profilin
1.Actin binding protein, profilin-reverse the effect of cofilin
and stimulates the incorporation of actin monomers into
filaments
2.Profilin can stimulate the exchange of bound ADP to ATP,
resulting in the formation repolymerized into filaments

19.  Cell signaling mechanisms –The activities of these
proteins control the actin polymerization
 Regulated in response to environmental signals
 ADF/cofilin,profilin,formin, and Arp2/3 complex-
rapid turnover of actin filaments and remodeling of
the actin cytoskeleton
 Which is required for a variety of cell movements and
changes in cell shape

20. Microtubules
 Second principal component of the
cytoskeleton
 Rigid hollow rods approximately 25nmin
diameter
 Tubulin dimers polymerize to form
microtubules, 13 linear protofilaments
assembled around a hollow core
 Undergo assembly and disassembly within
the cell
 Function-determine cell shape and cell
movements, including some forms of cell
locomotion, intracellular transport of
organelles, separation of chromosomes
during meiosis

21.  Microtubules are nucleated
From MTOC Microtubule Organizing centre at their
minus end and the plus end is growing outward from
each MTOC

22. Microtubule Assembly

23. Structure and organization of microtubules….
 Microtubules are composed of single type of globular
protein called tubulin
 Building blocks of microtubules- tubulin dimers
consisting of two closely related 55kd polypeptides-
alpha tubulin and beta tubulin (6 and 7 genes)
 Third type gamma – tubulin – concentrated in the
centrosome , plays major role in initiating microtubule
assembly

24. Structure and Assembly

25.  The protofilaments, which are composed of head to tail
arrays of tubulin dimers are arranged in parallel
 Two distinct ends- growing plus end and non-growing
minus end
 This polarity determine the direction of molecular motor
movement along the microtubules
 GTP bounded beta tubulin ( not to alpha tubulin), which
regulates the polymerization
 Then it is hydrolysed to GDP (shortly), which weakness
the bind affinity of tubulin dimer for each other
 Causing rapid depolymerization
 The microtubules , must be protected in order to prevent
rapid depolymerization- by anchoring the minus end in the
microtubule organizing the centre or centrosome

26. http://publication.letstalkacademy.com/formation-of-microtubules

27. www.frontiersin.org/articles/10.3389/fpls.2014.00511/full

29. • Dynamic instability – stabilized at the minus end, rapid
GTP hydrolysis to beta-tubulin during the shortly after
polymerization end (which reduces the binding
affinity)behavior known as dynamic instability
• Growth- continues as long as new GTP bound tubulin
molecules are added more rapidly than GTP is hydrolysed.
GTP cap is retained at the plus end and continues the
growth of microtubule
 Shrinkage – GTP hydrolysed more rapidly than new
subunits are added, the presence of GTP plus end of the
microtubule leads to disassembly and shrinkage
To remember…..
 Rapid GTP hydrolysis at the plus end results – dynamic
instability
 Catastrophe – rapid depolymerization shrinkage of the
microtubule
Growth – dynamic instability - shrinkage

30.  Dynamic instability- Tim Mitchison and Marc
Kirschner in 1984- continual and rapid turnover of
many microtubules but---- some of which have half
life only several minutes
 Colchicine and colcemid –bind to tubulin and inhibit
microtubule polymerization ….blocks mitosis
 Vincristine and Vinblastine – cancer chemotherapy ,
selectively inhibit the rapidly dividing cells
 Taxol- it stabilizes microtubules, cell division ----
prevents or blocks
 Because it stabilizes …the microtubules rather than
inhibiting the assembly
 MAPs – notes

31. MAPs …….
Cooper ,2000

32. Stabilization of Microtubules
 Dynamic instability
 Disassembled within the cell
 Interact with proteins ----disassemble microtubules
 Rate of tubulin depolymerization
 Other proteins binds to microtubule
 Increase stability
 Determining cell shape and polarity

33. MAP s
 MAPs identified ---large number
 MAP-1, MAP-2 and tau ---neuronal cell
 MAP-4 all non neuronal
 tau ---lesions ..in brain of Alzheimer patients

34. Formation of the Mitotic Spindle
The centrioles and centrosomes duplicate
during interphase
Prophase of mitosis the duplicated centrosome
separate and move to opposite sides of the nucleus
The nuclear envelope then disassembles, and
microtubules reorganize to form the mitotic spindle
Kinetochore microtubules are attached
to the condensed chromosomes, polar microtubules
overlap with each other in the center of the
cell, and astral microtubules extend outward to
the cell periphery
At metaphase, the condensed chromosomes
are aligned at the center of the spindle

35. Organization of microtubules in nerve cells
Two distinct types of processes extend from the cell body of nerve cells (neurons).
Dendrites are short processes that receive stimuli from other nerve cells.
The single long axon then carries impulses from the cell body to other cells, which
may be either other neurons or an effector cell, such as a muscle.

36. Nerve cells – two distinct types
 Axons and dendrites
 Organized differently and associated with distinct
types MAPs
 Axons – plus end ---oriented away from the cell body
 Minus end are not anchored in the centrosome
 Both are capped and terminate in the cytoplasm of the
axon
 Contain tau proteins but no MAP2

37. Dendrites..
 Both direction …. Microtubule s
 Plus ends pointed towards cell body.. Some are pointed
towards periphery
 Capped
 Contains MAP2, no tau proteins

38. Alzherimer’s disease
 Tau protein is abnormal and microtubule structure
collapse
 Hyperphosphorylated tau – excessive/ abnormal
phosphorylation of tau results in the PHF- tau and
NFTs
 Destory neurons
 Causes --- loss of memory, …cerebral cortex---
language…social behaviour
 Misfolding –tau protein
 Clump forming NFTs

39. Organization of microtubules
within the cells
 MAPs –regulates the behaviour ofmicrotubules
 Binds to plus end--- control dynamic instability
 Along the length of microtubules
 Microtubule stability – post translation modification
of tubulin
• Phosphorylation, acetylation, palmitoylation
• Removal/ addition of carboxy terminal tyrosines
• Addition of multiple glutamines/glycines
• Post translation sites are important ----binding MAPs

42. Intermediate filament proteins contain a central α-helical rod domain of
approximately 310 amino acids (350 amino acids in the nuclear lamins)
The N-terminal head and C-terminal tail domains vary in size and shape
Intermediate Filament
Geoffrey M Cooper 2000

43.  Diameter – 10-12nm
 Intermediate filaments are composed of several
families of proteins
 Expressed in different types of cells
 70 different intermediate filament proteins have been
identified and classified into five groups
 Based on similarities between their amino acid
sequences

44.  Types I and types II
 Type I acidic and one type II neutral/basic- two groups
of keratins
 Hard keratins- hair, nails and horns
 Soft keratins – cytoplasm of epithelial cells
 Type III includes vimentin… fibroblasts, smooth
muscle cells ..

45.  Another type III – desmin.. Muscle cells where
connects the Z discs
 Type IV neurofilament proteins – NF
 Type IV, nestin … expressed in embryonic
development
 Types V … nuclear lamins.. Found in all eukaryotic
cells

46. Cooper ,2000

47. Assembly of IF …
 The central rod domains of two polypeptides wind
around each other in a coiled-coil structure to form
dimers
 Dimers then associate in a staggered antiparallel
fashion to form tetramers
 Tetramers associate end to end to form protofilaments
and laterally to form filaments
 Each filament contains approximately eight
protofilaments wound around each other in a rope like
structure

48.  Modified by phosphorylation- regulates their assembly
and disassembly
 Ex- nuclear lamins
Disassembly
• Ex- Vimentin
Disassembly

49. Organization IF…
 Network in the cytoplasm – extending fro the nucleus
to PM
 Associated with other elements of cytoskeleton
 Scaffolding – integrates the components of the
cytoskeleton and organizes- internal structure of the
cell
 Keratin – anchored to PM at two areas of specialized
cell contacts – Desmosomes and hemidesmosomes

50. (B)Schematic of a desmosome. Intermediate filaments are anchored to sites of
cell-cell adhesion by desmoplaskin. (C) Schematic of a hemidesmosome.
Intermediate filaments are anchored to an integrin by plectin. (A, Don
Fawcett/Photo Researchers, Inc.)

51. Desmosomes……..cell to cell interaction

52. Experimental demonstration of keratin function
A plasmid encoding a mutant keratin that interferes with the normal assembly of keratin
filaments was microinjected into one pronucleus of a fertilized egg.
Microinjected embryos were then transferred to a foster mother, and some of the offspring were
found to have incorporated the mutant keratin gene into their genome.
Expression of the mutant gene in these transgenic mice disrupted the keratin cytoskeleton of cells
of the epidermis, resulting in severe skin blistering due to cell lysis following mild mechanical
stress.

53.  Experimental evidence for such an in vivo role of intermediate
filaments was first provided in 1991 by studies in the laboratory
of Elaine Fuchs
 These investigators used transgenic mice to investigate the in
vivo effects of expressing a keratin deletion mutant encoding a
truncated polypeptide that disrupted the formation of normal
keratin filaments
 This mutant keratin gene was introduced into transgenic mice,
where it was expressed in basal cells of the epidermis and
disrupted formation of a normal keratin cytoskeleton
 This resulted in the development of severe skin abnormalities,
including blisters due to epidermal cell lysis following mild
mechanical trauma, such as rubbing of the skin
 The skin abnormalities of these transgenic mice thus provided
direct support for the presumed role of keratins in providing
mechanical strength to epithelial cells in tissues

54.  Human genetic disease, epidermolysis bullosa simplex
(EBS)
 Like transgenic mice expressing mutant keratin genes,
patients with this disease develop skin blisters resulting
from cell lysis after minor trauma
 Role of keratins in allowing skin cells to withstand
mechanical stress
 ALS, known as Lou Gehrig’s disease – results from
progressive loss of motor neurons, which in turn leads to
muscle atrophy, paralysis and eventual death
 ALS and other types of motor neuron disease are
characterized by the accumulation and abnormal assembly
of neurofilaments
 Overexpression of NF-L or NF-H – in transgenic mice –
similar to ALS (suggest the involvement of neurofilaments
in the pathogenesis of motor neuron disease

55. Abnormalities of neurofilaments in diseases of motor
neurons
 Other studies in transgenic mice have implicated abnormalities
of neurofilaments in diseases of motor neurons, particularly
amyotrophic lateral sclerosis (ALS).
 ALS, known as Lou Gehrig's disease and the disease afflicting the
renowned physicist Stephen Hawking, results from progressive
loss of motor neurons, which in turn leads to muscle atrophy,
paralysis, and eventual death.
 ALS and other types of motor neuron disease are characterized
by the accumulation and abnormal assembly of
neurofilaments, suggesting that neurofilament abnormalities
might contribute to these pathologies.
 Consistent with this possibility, overexpression of NF-L or
NF-H in transgenic mice has been found to result in the
development of a condition similar to ALS.
 Suggest the involvement of neurofilaments in the pathogenesis
of motor neuron disease.

56. Stability..
 Keratin monomers appear very stable in most biological settings
 Keratin assembly is more stable, undergo rapid turn over/
remodeled depending on he circumstances of the cell
 PTM- keratin
 Phosphorylation, o-linked glycosylation, acetylation
 Phosphorylation has been extensively than other , serine
/threonine residues located head and tail of the domains of the
keratins
 Keratin phosphorylation ---enhance keratin solubility, which in
turn triggers reorganization of the keratin filament network …
increased in migration
 Cytoprotection by keratin phosphorlyation---server as a sponge
/sink and thereby shunt undesirable phosphorylation of
proapoptotic proteins by stress activated kinases

57. https://www.ncbi.nlm.nih.gov/books/NBK21594/
https://www.ncbi.nlm.nih.gov/books/NBK9908/