1. Department of Natural Sciences
University of St. La Salle
Bacolod City

2. CYTOSKELETON
 The cytoskeleton is a network of connected
filaments and tubules extending from the
nucleus to the plasma membrane.
 Dynamic: dismantles in one spot and
reassembles in another to change cell shape
 It maintains the shape of the cell- fibers act
like a geodesic dome to stabilize and balance
opposing forces.
 Anchor organelles
 Move the cell and control internal movement
of structures.

3. Motility of cells is determined by special
organelles for locomotion.
Internal movements
(cytoplasmic
streaming or cyclosis)
by cytoskeleton
components.

5. The ACTIN cytoskeletal
machinery (red) is
responsible for maintaining
cell shape and generating
force for movements.
Polymerization and
depolymerization of actin
filaments (1) drives the
membrane forward,
whereas actin cross-linking
proteins organize bundles
and networks of filaments
(2) that support overall cell
shape. Movements within
the cell and contractions at
the cell membrane (3) are
produced by myosin motor
proteins. The actin (red) and
intermediate filament
(purple) cytoskeletons
integrate a cell and its
contents with other cells in
tissues (4) through
attachments to cell
adhesions. Another type of
intermediate filament, the
nuclear lamins (5) are
responsible for maintaining
the structure of the nucleus.

6. Mechanical properties of Networks composed of
actin, tubulin, and microtubules, actin filaments, or an
intermediate filament called
intermediate vimentin, all at equal concentration,
filament polymers. were exposed to a shear force in a
viscometer, & the resulting degree
of stretch was measured. The
results show that microtubule
networks are strong, rigid hollow
tubes that are easily deformed but
rupture (red starburst) and begin to
flow without limit when stretched
beyond 150% of their original
length. Actin filament networks are
much more rigid, but they also
rupture easily. Intermediate filament
networks, by contrast, are not only
easily deformed, but they withstand
large stresses and strains without
rupture; they are thereby well suited
to maintain cell integrity.

7.  Actin is encoded by a large, highly conserved
gene family. Humans have 6 actin genes, which
encode isoforms of the protein.
 Although differences among isoforms seem
minor, the isoforms have different functions: α-
actin is associated with contractile structures;
γ-actin accounts for filaments in stress fibers;
and ß-actin is at the front, or leading edge, of
moving cells where actin filaments polymerize.
 Sequencing of actins from different sources
has revealed that they are among the most
conserved proteins in a cell, comparable with
histones, the structural proteins of chromatin.

8.  They form a dense complex web under the cell membrane. In
intestinal cell microvilli, they act to shorten the cell
 In plant cells, actin filaments form tracts along which
chloroplasts circulate.
 Involved in cell rigidity, tensile strength and resilience,
cellular movement (pseudopodia and mesenchyme cell
migration, platelet activation)
 Pseudopodia are associated with actin near the moving edge
of the cell. Actin filaments move by interacting with myosin
changing the configuration to pull the actin filament forward.
 Similar action accounts for pinching off cells during cell
division and for amoeboid movement.
 Other arrangements of microfilaments in association with
accessory proteins are possible. Ex: contractile rings of cell
division; parallel bundles are found in stress fibers of
fibroblasts, filopodia and other cell projections; gels of short
randomly oriented filaments are found in egg cortical
regions.

9. ACTIN AT
THE CROSS
ROADS

10.  All cytoskeleton types
form as helical
assemblies of subunits
that self-associate using
a combination of end-to-
end and side-to-side
protein contacts.
 They grow fastest from
the plus end than the
minus end of the
assembly.
 F-actin is polymerized
through addition of
globular actin or G-actin
monomers at the
growing (+) end, bearing
a stabilizing ATP cap.

11. Nucleation of new actin
filaments (red) is mediated
by ARP complexes (orange)
at the front of the web.
Newly formed filaments are
thereby attached to the
sides of preexisting
filaments. As these
elongate, they push the
plasma membrane forward.
The actin filament plus ends
will become protected by
capping proteins (blue),
preventing further assembly
or disassembly from the old
plus ends at the front of the
array. Hydrolysis of ATP
bound to the polymerized
actin subunits promotes
depolymerization at the rear
end of the actin complex by
a depolymerizing protein
(green). The spatial
separation of assembly and
disassembly allows the
network as a whole to move
forward at a steady rate.

12. Treadmilling. Actin subunits can flow through the filaments by
attaching preferentially to the (+) end and dissociating
preferentially from the (-) end of the filament. Removal of
monomers at the (–) end and addition of monomers at the (+)
end leaves the filaments at the same overall length. The oldest
subunits in a treadmilling filament lie at the (-) end. Treadmilling
occurs at intermediate concentrations of free subunits.

13. Microfilaments in a cell. A crawling cell with 3 areas showing the
arrangement of actin filaments. The actin filaments are shown in red,
with arrowheads pointing toward the plus end. Stress fibers are
contractile and exert tension. Filopodia are spike-like projections of the
plasma membrane that allow a cell to explore its environments. The
cortex underlies the plasma membrane.

14. A model of how forces generated in
the actin-rich cortex move a cell
forward.
The actin-polymerization-
dependent protrusion and firm
attachment of a lamellipodium at
the leading edge of the cell moves
the edge forward (green arrows at
front) and stretches the actin
cortex. Contraction at the rear of
the cell propels the body of the cell
forward (green arrow at back) to
relax some of the tension (traction).
New focal contacts are made at the
front, and old ones are
disassembled at the back as the cell
crawls forward. The same cycle can
be repeated, moving the cell
forward in a stepwise fashion.
Alternatively, all steps can be tightly
coordinated, moving the cell
forward smoothly. The newly
polymerized cortical actin is shown
in red.

15. Platelet activation. (A) Platelet activation is a controlled sequence of actin
filament severing, uncapping, elongation, recapping, and cross-linking that creates
a dramatic shape change in the platelet. (B) SEM of platelets prior to activation. (C)
An activated platelet with its large spread lamellipodium. (D) An activated platelet
at a later stage than the one shown in C, after myosin II-mediated contraction.

16. Several actin-binding proteins influence deployment of
filaments in the cytoplasm:
 Profilin binds to G-actin
monomers to regulate
polymerization
 Capping protein limits
length increase by binding
to the end of actin filament
 Fimbrin binds adjacent actin
filaments to form bundles
 Filamin stabilizes filament 3-D network by
intersecting with microfilaments
 Gelsolin breaks filament into shorter segments by
inserting between subunits
 Vinculin & actinin mediate binding of actin to cell
membrane at intercellular junctions and cell base.

18. The modular structures of four actin-cross-linking proteins
Each of the proteins shown has two actin-binding sites (red) that are
related in sequence. Fimbrin has two directly adjacent actin-binding sites,
so that it holds its two actin filaments very close together (14 nm apart),
aligned with the same polarity. The two actin-binding sites in α-actinin are
separated by a spacer around 30 nm long, so that it forms more loosely
packed actin bundles. Filamin has two actin-binding sites with a Vshaped
linkage between them, so that it cross-links actin filaments into a network
with the filaments oriented almost at right angles to one another. Spectrin
is a tetramer of two α and two ß subunits, and the tetramer has two actin-
binding sites spaced about 200 nm apart

19. Twisting of an actin filament induced by cofilin.
(A) Three dimensional reconstruction from cryo-EM of filaments made of
pure actin. The bracket shows the span of two turns of the actin helix.
(B) Reconstruction of an actin filament coated with cofilin, which binds in
a 1:1stoichiometry to actin subunits all along the filament. Cofilin is a
small protein (14 kilodaltons) compared to actin (43 kilodaltons), and so
the filament appears only slightly thicker. The energy of cofilin binding
serves to deform the actin filament lattice, twisting it more tightly so that
the distance spanned by two turns of the helix is reduced.

20. Filamin cross-links actin filaments into a three-dimensional
network with the physical properties of a gel
(A) Each filamin homodimer is about 160 nm long when fully
extended and forms a flexible, high-angle link between two
adjacent actin filaments. (B) A set of actin filaments cross-linked
by filamin forms a mechanically strong web or gel.

21. INTERMEDIATE FILAMENTS
 Are structurally similar but biochemically distinct,
with diameters intermediate between microtubules
and microfilaments (about 10 nm).
 They associate with polypeptides fillagrin (binds to
keratin), plectin (links vimentin), and synamin (also
links vimentin, but found in muscle).
 5 types are:
1. Glial filaments – found in non-neural cells of the
CNS: astrocytes, oligodendrocytes, microglia.
2. Keratin filaments – characteristic of epithelial
cells; called tonofilaments are often associated
with desmosomes at the cell surface. They
participate in the formation of keratin in
keratinizing epithelia.

22. 3.Desmin – characteristic of smooth, striated & cardiac
muscle; keep sarcomeres of neighboring myofibrils in
register across the width of the fiber; link Z-bands of
peripheral myofibrils to the sarcolemma; ensures uniform
distribution of tensile strength throughout the muscle cell.
4.Vimentin – abundant in fibroblasts and mesenchymal
derivatives, in bundles or randomly oriented in a network
throughout the cytoplasm.
5.Neurofilaments – present in nerve cell processes with a
cytoskeletal function; helps to maintain the gel state of the
axoplasm; involved in intracellular metabolite transport.

23. A model of intermediate
filament construction
The monomer shown in (A)
pairs with an identical
monomer to form a dimer (B)
in which the conserved
central rod domains are
aligned in parallel and
wound together into a coiled
coil. (C) Two dimers then line
up side by side to form the
tetramer soluble subunit of
intermediate filaments. (D)
Within each tetramer, the 2
dimers are offset with
respect to one another, thereby allowing it to associate with
another tetramer. (E) In the final 10-nm rope-like filament,
tetramers are packed together in a helical array, which has 16
dimers in cross-section. Half of these dimers are pointing in each
direction.

24. Keratin filaments in
epithelial cells
• Immunofluorescence
micrograph of the network
of keratin filaments (green)
in a sheet of epithelial
cells in culture.
• The filaments in each cell
are indirectly connected to
those of its neighbors by
desmosomes.
• A 2nd protein (blue) has
been stained to reveal the
location of the cell
boundaries.

25. Blistering of the skin caused by a mutant keratin gene.
A mutant gene encoding a keratin protein was expressed in a transgenic
mouse. The defective protein assembles with the normal keratins and
thereby disrupts the keratin filament network in the basal cells of the
skin. LM of cross sections of normal (A) and mutant (B) skin show that
the blistering results from the rupturing of cells in the basal layer of the
mutant epidermis (small red arrows). (C) Cells in the basal layer of the
mutant epidermis, as observed by EM. As indicated by the red arrow, the
cells rupture between the nucleus & the hemidesmosomes, which
connect the keratin filaments to the underlying basal lamina.

26. Two types of intermediate filaments in cells of the nervous system.
(A) Freeze-etch EM image of neurofilaments in a nerve cell axon, showing
the extensive cross-linking through protein cross-bridges an arrangement
believed to give this long cell process great tensile strength. The cross-
bridges are formed by the long, nonhelical extensions at the C-terminus of
the largest neurofilament protein (NF-H). (B) Freeze-etch image of glial
filaments in glial cells, showing that these intermediate filaments are
smooth and have few cross-bridges. (C) Conventional EM of a cross
section of an axon showing the regular side-to-side spacing of the
neurofilaments, which greatly outnumber the microtubules.