1. Cytoskeleton: components

2. Cytoskeleton proteins revealed by Commassie staining
Cytoskeleton: filament system
Three types of filaments
and accessory proteins
(assembly of cytoskeleton, motor
proteins that move organelles
or filaments)
Internal order
Shape and remodel surface
Move organelles
Movement
Cell division

3. Dynamic and adaptable
Actin filaments:
shape of the cell’s surface
and whole cell locomotion
5-9 nm diameter
Microtubules:
positions of membrane-
enclosed organelles,
intracellular transport
25 nm diameter
Intermediate filaments:
mechanical strength and
resistance to shear stress
10 nm diameter

6. Cytoskeletal filaments are all constructed from
smaller protein subunits
Intermediate filaments: smaller
elongated and fibrous subunits
Actin and microtubule filaments:
compact and globular subunits
All form as helical assemblies
of subunits
Noncovalent interactions:
rapid assembly and disassembly

7. Multiple protofilaments give strength and adaptability
Ends are dynamic

8. Intermediate filaments are resistent to bending or stretching forces

9. The time course of actin polymerization in a test tube

10. The structure of a microtubule and its subunits
hollow and cylindrica and polar
GTP!
GTP
heterodimer
13 parallel protofilaments

11. The structure of an actin monomer and actin filament
two parallel protofilaments
that twist around each other
in a right-handed helix
polar
ATP
monomer
Flexible but cross-linked and
bundled together by accessory
proteins in a living cell

13. The preferential growth of microtubules at the plus end
Plus end: polymerize and
depolymerize faster than
minus end
Microtubules:
Plus end- b subunit
Minus end- a subunit
Actin filaments
Plus end- barbed end
Minus end- pointed end

15. The treadmilling of an actin filament
Structural difference between the two ends
D form polymer leans towards disassembly

23. Treadmilling behavior of a microtubule
as in a living cells
The extent of treadmilling inside the cell
Is uncertain.
Actin treadmilling is observed in vitro.
A treadmilling-like phenomenon is seen in
living cells for microtubules
Tubulin conjugated with fluorescent dye
1/20 subunit is fluorescent
“Microtubule lattice”

24. Dynamic instability:predominant in microtubules
GTP hydrolysis “catch up”
Treadmilling: predominant in actin filaments

25. GTP hydrolysis causes
filament to curve

26. Lateral bonds force GDP-containing
protofilaments into a linear conformation

27. Direct observation of the dynamic instability of
microtubules in a living cell
Dynamic instability of individual actin filaments cannot be
observed readily-difference between two ends are not so extreme
However the actin filament turn over is rapid: individual filament
persists for a only few minutes

28. The dynamic behavior of filaments allows cells to
change structures rapidly and
Giant multinuclear
cell of a fly early
embryo
Actin filaments:red
Mircotubules:green
One division
per 10 minutes

29. Actin and tubulin are highly conserved: they have to bind to
many proteins directly and indirectly
Accessory proteins and intermediate filament proteins
are not as conserved

30. A model of intermediate filament construction
Intermediate filaments
are only found in some
metazoans:vertebrates,
nematodes,molluscs
Parallel
Antiparrel
Not required in
every cell type
Ancesters: nuclear lamins
“subunit”
No polarity!
Easily bent
Hard to break
8 parallel protofilaments

33. Mechanical properties of actin, tubulin and
Intermediate filament polymers
viscometer
Microtubules: easily
deformed and then rupture
Actin filaments are more
rigid and also rupture
easily
Intermediate filaments:
easily deformed and don’t
rupture--maintain cell integrety

34. Intermediate filaments impart mechanical stability
to animal cells
Keratin filaments in epithelial cells
The most diverse family
20 in human epithelial cells
10 more in hair and nails
Diagnosis of epithelial
cancers (carcinomas)
“desmosomes”

35. Blistering of the skin caused by mutant keratin genes
Epidermolysis bullosa simplex:the skin blisters in response to
very slight mechanical stress
Truncated keratin (missing both the N- C- domains) Tg mice
Other blistering diseases:
mouth, esophageal lining and cornea of the eye--
mutations of different keratins

36. Two types of intermdiate filaments in cells of the nervous system
Neurofilaments:axons
Regular spacing
NF-L, NF-M, NF-H proteins coassemble
axon
glia
NF-M and NF-H have long C-terminal tails
That bind to neighboring filaments:uniform spacing
When axons grow, subunits are added at the filament ends
and along the filament length; axon diameter increase 5 fold
In ALS (Lou Gehrig’s Disease), there is an accumulation and abnormal assembly of
Neurofilaments in motor neuron cell bodies and axon--interfere with normal axon transport

37. Actin filaments and microtubules are targets of
many plant toxins
Amanita Phalloids (death cap)
(Eat raw meat)

38. Effect of the drug taxol on microtubule organization
treatment of cancers

44. Summary
Three types of cytoskeletal filaments, protofilaments;
Subunits, polymerization, treadmilling, dynamic
instability;
Intermediate filaments, cell integrity, diseases caused
by mutations in the intermediate filament genes
4. Natural toxins and cytoskeleton