The cytoskeleton:• protein filament complex identified by electron microscopyIsolation: cells treated with a nonionic detergent (f.e. Triton X-100) the cytoskeletonmicrotubules: 25 nm thickmicrofilaments: 7 nm thickintermediate filaments: 10 nm thick Zdroj: Audesirk, Teresa and Gerald, Biology, Prentice Hall, 1999
PALADE CLAUDE PORTER deDUVELEDBETTER a PORTER (1963): MICROTUBULES in E.M. ALLEN a KAMIYA (1964): MICROFILAMENTS in E.M.
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
Bacteria, cyanobacteria: Cytoskeleton occurence: - no cytoskeleton; - only proteins related toAnimal cells: tubulin (FtsZ) and actin- microtubules- actin filaments (microfilaments)- intermediate filaments Plant cells and fungi: - microtubules - actin filaments (microfilaments) Alberts et al. ( 2004)
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
In vitro polymerization of microtubules proceeds more rapidly at + ends, depolymerization at – endsIn 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
In vivo animal microtubulespolymerize from centrosomescontaining gama–tubulin rings(red circles)=teplates forαβ-tubulin dimers Polar MTs →polymeration of microtubules proceeds Centrosome contains: - two centrioles, - pericentriolar matrix, - gama-tubulin rings. Alberts et al. 2004
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 ciliaand flagella,- compression-resisting „girders”,- information medium… Alberts et al. 2004
The plant cell: centrosomes and centrioles absent.Microtubules are nucleated from single gama-tubulin ringcomplexes (i) at the plasma membrane, (ii) at the nuclearmembrane, and (iii) at fragmoplast.Preprophase band Mitotic spindleFragmoplast MTsCortical MTs
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 → - •
Cilia and flagella contain 9 pairs of microtubule doublets circularlyarranged, 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 inthe various genes coding for the axonemal proteins (Afzelius BA, J.Pathol. 2004). Campbel et al. 2002
ACTIN CYTOSKELETONMonomer:G-actin cont. ATP -globular proteinStructure:two helicalchains form onemicrofilament ofF-actin7 nm thick. Plus (+) end, minus(-) end. Alberts et al. 2004
Actin is phylogenetically very old and 89% homology ofyeast 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.
Myosin I and myosin II in the eukaryoticcells (examples) Transport of vesicles along microfilaments. Telescopic sliding of microfilaments (contraction). Transport ofsubmembrane microfilaments
Polymeration of actin filament:nucleation complex at the plasma membrane filopodium growing filopodium +end Nucleation complex
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
INTERMEDIATE FILAMENTSMonomers: various proteins (see later). Structure: monomer- dimer - tetramer = protofilament. 8 protofilaments helicallyarranged into one intermediate filament. Thickness: 10 nm. Campbell, Reece (2002)
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…)
Cytoskeleton and medicine: Human diseasescaused by mutations in the cytoskeletal genesMicrotubular cytoskeleton: „ciliary and flagellar diseases“:Immobile flagella of sperm cells, immobile cilia of ciliaryepithel in respiratory tract (Kartagener syndrome); inFalopian tube, in embryonal development,neurodegenerative diseases (Alzheimer disease)…Actin cytoskeleton: myopathia, kardiomyopathia, malignanttumors...Intermediary filaments: skin diseases (epidermolysisbullosa),other diseases: amyotrofic lateral sklerosis, inborncardiomyopathia, liver cirhosis, pulmonal fibrosis…Membrane cytoskeleton: abnormalities of erythrocytes inanemia (spherocytosis, eliptocytosis)…Nuclear cytoskeleton: laminopathia (Progeria syndrome)….
Present clinical correlatesDiagnosis:antibodies against intermediate filamentproteins – diagnosis of the origin of malignanttumorsTherapy of malignant tumors:Inhibitors of microtubules used as drugs to inhibit proliferation of cells of malignant tumors (Taxol, Vinca- alkaloids….)
GlossaryActin 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.
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.