3. The cytoskeleton:
• protein filament complex
identified by electron microscopy
Isolation: cells treated with a nonionic
detergent (f.e. Triton X-100)
the cytoskeleton
microtubules: 25 nm thick
microfilaments: 7 nm thick
intermediate filaments: 10 nm thick
Zdroj: Audesirk, Teresa and Gerald, Biology, Prentice Hall, 1999
4. PALADE CLAUDE PORTER deDUVE
LEDBETTER a PORTER (1963): MICROTUBULES in E.M.
ALLEN a KAMIYA (1964): MICROFILAMENTS in E.M.
5. Globular protein actin Globular proteins tubulins Various fibrous proteins
= G actin
Weber et al. (1975) Lazarides and Weber(1974) Hynes a Destree (1978)
immunofluorescence Plant Cell Cytoskleton
Gretchen Robinson
6. Bacteria, cyanobacteria:
Cytoskeleton
occurence: - no cytoskeleton;
- only proteins related to
Animal cells: tubulin (FtsZ) and actin
- microtubules
- actin filaments (microfilaments)
- intermediate filaments Plant cells
and fungi:
- microtubules
- actin
filaments
(microfilaments)
Alberts et al. ( 2004)
7. MICROTUBULES
• Microtubule structure:
hollow cylindres
thickness - 25 nm
wall contains 13
protofilaments
Monomers: α-tubulin
β-tubulin
α and β tubulins form
tubulin dimers = building
units of microtubules
Polymerization
proceeds from αβ-tubulin
dimers cont.GTP
Plus ends (+), minus ends(-)
Alberts et al. 2004
8. In vitro polymerization of microtubules proceeds
more rapidly at + ends, depolymerization at – ends
In vitro experiments showed that microtubules polymerize more rapidly
at their plus ends from tubulin dimer containing GTP,
while polymerization is slower or depolymerization proceeds at minus
ends (tubulin dimers containing GDP).
Dimers move from one end to the other (tubulin treadmilling). When some
structure is joined to microtubule, it is translocated along microtubule
(like man standing on escalator).
Tubulin dimer
with GTP (red)
Tubulin dimer
with GDP(green)
Polymerization + end Depolymerization - end Alberts et al.
2004
9. In vivo animal microtubules
polymerize from
centrosomes
containing gama–tubulin
rings
(red circles)=teplates for
αβ-tubulin dimers Polar MTs
→polymeration of
microtubules proceeds
Centrosome contains:
- two centrioles,
- pericentriolar matrix,
- gama-tubulin rings.
Alberts et al. 2004
10. Functions of microtubules in the cell (examples)
- animal shape determination,
- position of cell organelles,
- intracellular transport,
- polar growth,
- chromosome segregation,
- cytokinesis (fragmoplast-
plant cells),
- cell movement by cilia
and flagella,
- compression-resisting
„girders”,
- information medium…
Alberts et al. 2004
11. The plant cell: centrosomes and centrioles absent.
Microtubules are nucleated from single gama-tubulin ring
complexes (i) at the plasma membrane, (ii) at the nuclear
membrane, and (iii) at fragmoplast.
Preprophase band
Mitotic spindle
Fragmoplast MTs
Cortical MTs
12. Microtubule-associated proteins (MAPs):
- non-motor proteins: MAPS 1 – 4, Tau protein…
- molecular motors (motor proteins): kinesins, dyneins
• Non-motor:
MAP1 – 4 neurites and dendrites
polymeration of MTs
elongation of MTs
Tau neurites
Motor proteins: kinesins → +
dyneins → -
•
13. Cilia and flagella contain 9 pairs of microtubule doublets circularly
arranged, and two central single microtubules (9+2).
Basal bodies of cilia and flagella contain 9 microtubule triples (9+0).
Dynein ATPase is involved in the movement cilia and flagella.
8 categories of „ciliary“ diseases exists in man caused by mutations in
the various genes coding for the axonemal proteins (Afzelius BA, J.Pathol. 2004).
Campbel et al. 2002
14. ACTIN CYTOSKELETON
Monomer:
G-actin cont. ATP -
globular protein
Structure:
two helical
chains form one
microfilament of
F-actin
7 nm thick.
Plus (+) end,
minus(-) end.
Alberts et al. 2004
15. Actin is phylogenetically very old and 89% homology of
yeast and mammalian actin was detected.
Gabriel M., Microbiology (UK)
• Budding yeast has one actin
gene that has 89% homology to
actin of mammals (non-muscle).
• Homo sapiens has 6 actin genes
coding α, β and γ actin isoforms.
16. Myosin I and myosin II in the eukaryotic
cells (examples)
Transport of vesicles along
microfilaments.
Telescopic sliding of
microfilaments (contraction).
Transport ofsubmembrane
microfilaments
17. Polymeration of actin filament:
nucleation complex at the plasma membrane
filopodium growing filopodium
+end
Nucleation complex
18. Structures and functions of actin cytoskeleton
(examples)
A. Actin filaments in microvilli of the intestinal epithel.
B. Actin stress fibres.
C. Filopodia (pointed protrusions), lamellipodia (flatted
protrusions), pseudopodia (false foot); fagocytosis; ameboid
movement.
D Actin cytokinetic contractile ring in cytokinesis of animal
and fungal cells.
Alberts et al. 1998
19. INTERMEDIATE FILAMENTS
Monomers: various proteins (see later). Structure: monomer
- dimer - tetramer = protofilament. 8 protofilaments helically
arranged into one intermediate filament. Thickness: 10 nm.
Campbell, Reece (2002)
20. INTERMEDIATE FILAMENT PROTEINS
(examples)
• Cytokeratins – epithelial cells (mechanical
strenth)
• Vimentin - mesenchymal cells (cell shape
determination)
• Desmin – muscle cells
(structural support of muscle fibres)
• Proteins of neurofilaments - in neurons
• Nestin - neurons
• Lamins A, B, C – under the nuclear envelope
(shape of the nucleus, chromosome
positioning, gene expression…)
22. Cytoskeleton and medicine: Human diseases
caused by mutations in the cytoskeletal genes
Microtubular cytoskeleton: „ciliary and flagellar diseases“:
Immobile flagella of sperm cells, immobile cilia of ciliary
epithel in respiratory tract (Kartagener syndrome); in
Falopian tube, in embryonal development,
neurodegenerative diseases (Alzheimer disease)…
Actin cytoskeleton: myopathia, kardiomyopathia, malignant
tumors...
Intermediary filaments: skin diseases (epidermolysis
bullosa),other diseases: amyotrofic lateral sklerosis, inborn
cardiomyopathia, liver cirhosis, pulmonal fibrosis…
Membrane cytoskeleton: abnormalities of erythrocytes in
anemia (spherocytosis, eliptocytosis)…
Nuclear cytoskeleton: laminopathia (Progeria syndrome)….
23. Present clinical correlates
Diagnosis:
antibodies against intermediate filament
proteins – diagnosis of the origin of malignant
tumors
Therapy of malignant tumors:
Inhibitors of microtubules used as drugs to
inhibit proliferation of cells of malignant
tumors (Taxol, Vinca- alkaloids….)
24. Glossary
Actin filament. Protein filament 7- nm wide, formed from globular actin molecules. A major
constituent of the cytoskeleton of all eucaryotic cells, especially abundant in muscle cells.
Centriole. Short cylindric array of microtubules, usually found in pairs at the center of a
centrosome in animal cells. Also found at the base of cillia and flagella (called basal bodies).
Centrosome. Centrally located organelle of animal cell that is the primary microtubule organizing
center (MTOC) and acts as the spindle pole during mitosis. In most animal cells it contains a
pair of centrioles.
Ciliate. Type of single-celled eucaryotic organism (protozoan) characterized by numerous cillia
on its surface. The cillia are used for swimming, feeding, or capture of prey.
Cilium. Hairlike extension on the surface of a cell with a core bundle of microtubules and
capable of performing repeated beating movements. Cillia, in large numbers, drive the
movement of fluid over epithelial sheets, as in the lungs.
Cytoskeleton. System of protein filaments in the cytoplasm of a eucaryotic cell that gives the cell
shape and the capacity for directed movement. Its most abundant components are actin
filaments, microtubules and intermediate filaments.
Dimer. A structure composed of two equivalent halves. The term „heterodimer“ is sometimes
used when the two halves are not perfectly identical.
Dynein. Member of a family of large motor proteins that undergo ATP-dependent movement
along microtubules. Dynein is responsible for the movement of cilia and flagella.
Fibrous protein. A protein with an elongated shape. Typically one such as collagen or
intermediate filament protein that is able to associate into long filamentous structures.
Filopodium. Long thin actin-containing extension on the surface of an animal cell. Sometimes
has an exploratory function, as in a growth cone of neuron.
Flagellum. A long whipelike protrusion that drives a cell through a fluid medium by its beating.
Eucaryotic flagella are longer versions of cilia; bacterial flagella are completely different,
being smaller and simpler in construction.
Globular protein. Any protein with an approximately rounded shape. Most enzymes are globular.
Intermediate filament. Fibrous protein filament (about 10 nm in diameter) that forms ropelike
networks in animal cells. Often used as a structural element that resists tension applied to the
cell from outside.
Kinesin. One member of a large family of motor proteins that uses the energy of ATP hydrolysis
to move along a microtubule.
25. Lamellipodium. Dynamic sheetlike extension on the surface of an animal cell, especially one
migrating over a surface.
Microtubule. Long, stiff, cylindrical structure composed of the protein tubulin. Used by
eucaryotic cells to regulate their shape and control their movement.
Myofibril. Long, highly organized bundle of actin, myosin and other proteins in the cytoplasm of
muscle cells that contracts by a sliding filament mechanism.
Motor protein. Protein such as myosin or kinesin that uses energy derived from ATP hydrolysis
to propel itself along a protein filament or polymeric molecule. Myosin - type of motor
protein that uses ATP to drive movements along actin filaments. Myosin II is a large protein
that forms the thick filaments of skeletal muscle. Smaller myosins, such as myosin I, are
widely distributed, and responsible for many actin-based movements.
Nuclear lamina. Fibrous layer on the inner surface of the nuclear membrane made up of a
network of intermediate filaments made from nuclear lamins.
Phagocytic cell. A cell such as a macrophage or neutrophil that is specialized to take up particles
and microorganisms by phagocytosis.
Phagocytosis. The process by which particulate material is engulfed („eaten“) by a cell (f.e.
Amoeba proteus, macrophages, neutrophils).
Polarity. Refers to a structure such as an actin filament or a fertilized egg that has an inherent
direction – so that one can distinguish one end from the other.
Polymer. Large and usually linear molecule made by the repetitive assembly, using covalent
bonds, of multiple identical or similar units (monomers).
Pseudopodium. (Latin for „false foot“). Large cell-surface protrusion formed by ameboid cell as
they crawl. More generally, any dynamic actin-rich extension of the surface of an animal cell.
Sarcomere. Repeating unit of a myofibril in a muscle cell, about 2.5 μm long, composed of an
array of overlapping thick (myosin) and thin (actin) filaments).
Tubulin. Protein from which microtubules are made.
Gama-tubulin ring. Protein complex in centrosomes that nucleates microtubule assembly.
Gama-tubulin ring complex (γ-TU-RC). Protein complex, nucleating microtubules in the plant
cell ( that does not have centrosomes) at the plasma membrane, nuclear membrane and
fragmoplast.
