The cytoskeleton is a network of protein polymers that maintains cell shape and enables intracellular transport and movement. It consists of microtubules, microfilaments, and intermediate filaments. Microtubules are hollow tubes involved in structural support, transport, and cell organization. Microfilaments are thin filaments involved in motility and contractility. Intermediate filaments provide structural support. Motor proteins such as kinesins and dyneins interact with these cytoskeletal elements and convert chemical energy from ATP into mechanical force and movement.

1. Cytoskeleton

2. • Form an elaborate interactive network of
polymers of protein subunits held together by
weak, noncovalent bonds
• Although they appear stationary in
micrographs, they are highly dynamic
structures capable of dramatic reorganization
• Recent study suggested that prokaryotes
prokaryotes have proteins that carry
cytoskeletal-like activities

3. Microtubules
• Long, hollow, and stiff unbranched tubes
found in all eukaryotes that are distributed in
the cytoplasm
• Functions in support, intracellular transport,
and cell organization
• Composed of subunits of tubulin

4. Intermediate filaments
• Tough, ropelike fibers found in the nucleus
and cytoplasm of animals
• Functions in structural support
• Composed of a variety of proteins

5. Microfilaments
• Solid, thin structures found in the cytoplasm
of eukaryotes that are often organized into a
branching network
• Functions in motility and contractility
• Composed of actin

6. Study of the cytoskeleton
Live-cell fluorescence imaging
• allows researchers to directly observe
molecular processes in living cells—an
approach known as live-cell imaging

7. FIGURE 9.4 Dynamic changes in length of microtubules within
an epithelial cell. The cell was injected with a small volume of
tubulin that had been covalently linked to the fluorescent dye rhodamine.
After allowing time for the cell to incorporate the labeled
tubulin into microtubules, a small portion of the edge of the living cell
was examined under the fluorescence microscope.

8. In Vitro and In Vivo Single-molecule Assays
• high-resolution video microscopy has led to
the development of in vitro motility assays
• Single-molecule assays have allowed
researchers to make measurements that were
not possible with standard biochemical
techniques that average the results obtained
from large numbers of molecules

9. In some of the earlier assays, microtubules
were attached to a glass coverslip. Then,
microscopic beads containing attached motor
proteins were placed directly onto the
microtubules using aimed laser beams. The
laser beams are shone through the objective
lens of a microscope, producing a weak
attractive force near the point of focus.
Because it can grasp microscopic objects, this
apparatus is referred to as optical tweezers.

10. When ATP is present as an energy source, the
movements of a bead along a microtubule can
be followed by a video camera, revealing the
size of individual steps taken by the motor
protein. Focused laser beams can also be used
to “trap” a single bead and determine the
minute forces (measured as a few
piconewtons, pN) generated by a single motor
protein as it “tries” to move the bead against
the force exerted by the optical trap

11. In this experiment a purified GFP-labeled kinesin
molecule is seen to move processively along a
microtubule whose plus end is labeled with a
red fluorescent dye named Cy5.
The clarity of these images is made possible by
the use of a specialized type of laser-based
fluorescence microscopy called TIRF (total
internal reflection microscopy)

12. Atomic force microscopy
-measures the mechanical properties of the
cytoskeletal elements themselves. AFM is an
instrument that uses a nanosized tip to probe
the surface of a macromolecular specimen

13. - embed the tip of an AFM into a single intermediate
filament and pull on the end or the middle of the
filament to test its extensibility and tensile strength.
- a segment of filament can be mechanically stretched
up to 3.5 times its normal length before it breaks into
two pieces

14. Microtubules
• components of a diverse array of structures,
including the mitotic spindle of dividing cells and
the core of cilia and flagella
• outer diameter of 25 nm and a wall thickness of
approximately 4 nm, and may extend across the
length or breadth of a cell
• wall of a microtubule is composed of globular
proteins arranged in longitudinal rows,
protofilaments, that are aligned parallel to the
long axis of the tubule
• 13 protofilaments aligned side by side in a
circular pattern within the wall

15. • Each protofilament is assembled from dimeric building
blocks consisting of one alpha-tubulin and one beta-
tubulin subunit
• The tubulin dimers are organized in a linear array along
the length of each protofilament
• All of the protofilaments of a microtubule have the
same polarity. Consequently, the entire polymer has
polarity. One end of a microtubule is known as the plus
end and is terminated by a row of beta-tubulin
subunits. The opposite end is the minus end and is
terminated by a row of alpha- tubulin subunits(p325,
d)

16. • contain additional proteins, called
microtubule-associated proteins (MAPs)
• The binding of one of these MAPs to the
surface of a microtubule connects
microtubules to each other, thus maintaining
their parallel alignment. MAPs generally
increase the stability of microtubules and
promote their assembly

17. Motor Proteins that Traverse the Microtubular
skeleton
• convert chemical energy (stored in ATP) into
mechanical energy, which is used to generate
force or to move cellular cargo attached to the
motor
• Collectively, motor proteins can be grouped
into three broad superfamilies: kinesins,
dyneins, and myosins. Kinesins and dyneins
move along microtubules

18. Kinesins
- discovered in 1985 by Ronald Vale and
colleagues when they isolated a motor protein
from the cytoplasm of squid giant axons
- tetramer constructed from two identical
heavy chains and two identical light chains
• The routes followed by cytoplasmic vesicles and
organelles are largely defined by microtubules,
and members of the kinesin superfamily are
strongly implicated as force-generating agents
that drive the movement of this membrane
bounded cargo

19. Cytoplasmic Dynein
- discovered in 1963 as the protein responsible
for the movement of cilia and flagella
-a huge protein composed of two identical
heavy chains and a variety of intermediate
and light chains. Each dynein heavy chain
consists of a large globular head(force
generating agent) with an elongated
projection (stalk)

20. • As a force-generating agent in positioning the
spindle and moving chromosomes during
mitosis
• As a minus end–directed microtubular motor
with a role in positioning the centrosome and
Golgi complex and moving organelles, vesicles,
and particles through the cytoplasm

21. Intermediate filaments
• strong, flexible ropelike fibers that provide
mechanical strength to cells that are subjected
to physical stress, including neurons, muscle
cells, and the epithelial cells that line the
body’s cavities with a diameter of 10–12 nm
• chemically heterogeneous group of structures
that, in humans, are encoded by
approximately 70 different genes

22. • FIGURE 9.40 Cytoskeletal elements are connected to one another by protein
cross-bridges. Electron micrograph of a replica of a small portion of the
cytoskeleton of a fibroblast after selective removal of actin filaments. Individual
components have been digitally colorized to assist visualization. Intermediate
filaments (blue) are seen to be connected to microtubules (red) by long wispy
cross-bridges consisting of the fibrous protein plectin (green).

23. Types of IF
Types I and II: Acidic Keratin and Basic Keratin, respectively. Produced
by different types of epithelial cells (bladder, skin, etc)
Type III. Intermediate filaments are distributed in a number of cell
types, including: Vimentin in fibroblasts, endothelial cells and
leukocytes; desminin muscle; glial fibrillary acidic factor in
astrocytes and other types of glia, and peripherin in peripheral
nerve fibers
Type IV Neurofilament H (heavy), M (medium) and L (low). Modifiers
refer to the molecular weight of the NF proteins. Another type IV
is "internexin" and some nonstandard IV's are found in lens fibers of
the eye (filensin and phakinin).
Type V are the lamins which have a nuclear signal sequence so they
can form a filamentous support inside the inner nuclear
membrane. Lamins are vital to the re-formation of the nuclear
envelope after cell division