The Cytoskeleton

Marie Kopecká mkopecka@med.muni.cz 2007/2008

Heuser J.E., Kirschner M.W. J. Cell Biol. 86, 212-234 (1980)

Zdroj: Audesirk, Teresa and Gerald, Biology, Prentice Hall, 1999

microtubules: 25 nm thickmicrofilaments: 7 nm thickintermediate filaments: 10 nm thick

The The cytoskeletoncytoskeleton::• protein • protein filamentfilament complexcomplex

identifiedidentified by by electronelectron microscopymicroscopyIsolationIsolation: : cellscells treatedtreated withwith a a nonionicnonionic

detergent (f.e. Triton Xdetergent (f.e. Triton X--100)100)

the cytoskeleton

PALADE CLAUDE PORTER deDUVE

LEDBETTER a PORTER (1963): MICROTUBULES in E.M.ALLEN a KAMIYA (1964): MICROFILAMENTS in E.M.

Plant Cell CytoskletonGretchen Robinson

Lazarides and Weber(1974)Weber et al. (1975) Hynes a Destree (1978)

immunofluorescence

Globular protein actin= G actin

Globular proteins tubulins Various fibrous proteins

Cytoskeletonoccurence:

Animal cells:- microtubules- actin filaments (microfilaments)- intermediate filaments

Bacteria, cyanobacteria:

- no cytoskeleton;

- only proteins related to tubulin (FtsZ) and actin

Alberts et al. ( 2004)

Plant cells

and fungi:

- microtubules

- actin filaments(microfilaments)

• Microtubule structure:hollow cylindresthickness - 25 nm wall contains 13 protofilaments

Monomers: α-tubulinβ-tubulin

α and β tubulins formtubulin dimers = buildingunits of microtubules

Polymerizationproceeds from αβ-tubulin dimers cont.GTPPlus ends (+), minus ends(-)

Alberts et al. 2004

MICROTUBULES

In vitro polymerization of microtubules proceedsmore 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 somestructure is joined to microtubule, it is translocated along microtubule(like man standing on escalator).

+ end - end

Tubulin dimer with GTP (red)

Polymerization

Tubulin dimerwith GDP(green)

Depolymerization Alberts et al. 2004

In vivo animal microtubules

polymerize from centrosomes

containing gama–tubulin rings

(red circles)=teplates for

αβ-tubulin dimers →polymeration of microtubules proceeds

Centrosome contains: - two centrioles,- pericentriolar matrix,- gama-tubulin rings.

Alberts et al. 2004

Polar MTs

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 ring complexes (i) at the plasma membrane, (ii) at the nuclearmembrane, and (iii) at fragmoplast.

Cortical MTs

Preprophase band

Mitotic spindle

Fragmoplast 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 MTselongation 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 in the various genes coding for the axonemal proteins (Afzelius BA, J.Pathol. 2004).

Campbel et al. 2002

ACTIN CYTOSKELETON

Monomer:G-actin cont. ATP -globular proteinStructure:two helicalchains form onemicrofilament ofF-actin 7 nm thick.Plus (+) end, minus(-) end.

Alberts et al. 2004

Actin is phylogenetically very old and 89% homology of yeast and mammalian actin was detected.

• 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.

Gabriel M., Microbiology (UK)

Myosin I and myosin II in the eukaryoticcells (examples)

Transport of vesicles alongmicrofilaments.

Telescopic sliding of microfilaments (contraction).

Transport ofsubmembranemicrofilaments

Polymeration of actin filament:nucleation complex at the plasma membrane

+end

filopodium growing filopodium

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 animaland 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, chromosomepositioning, gene expression…)

Isolatednuclearskeleton

Nuclear lamina

Hozák P., Exp.Cell Res. (1996)

Cytoskeleton and medicine: Human diseasescaused by mutations in the cytoskeletal genes

Microtubular cytoskeleton: „ciliary and flagellar diseases“:Immobile flagella of sperm cells, immobile cilia of ciliaryepithel in respiratory tract (Kartagener syndrome); in Falopian 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 in anemia (spherocytosis, eliptocytosis)…Nuclear cytoskeleton: laminopathia (Progeria syndrome)….

Diagnosis: antibodies against intermediate filamentproteins – diagnosis of the origin of malignanttumors

Therapy of malignant tumors:Inhibitors of microtubules used as drugs to

inhibit proliferation of cells of malignanttumors (Taxol, Vinca- alkaloids….)

Present clinical correlates

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 cilliaon 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 sometimesused when the two halves are not perfectly identical.

Dynein. Member of a family of large motor proteins that undergo ATP-dependent movementalong 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. Sometimeshas 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 hydrolysisto move along a microtubule.

Lamellipodium. Dynamic sheetlike extension on the surface of an animal cell, especially onemigrating 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 hydrolysisto 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 particlesand 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 inherentdirection – 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 anarray 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.