Content of the present lecture 1. Chemical and physical structure of cytoskeleton 2. Intermediate filaments, microtubules, centrosom, actin filaments 3. Self-assembly and dynamic structure of cytoskeletal filaments 4. How cells regulate their cytoskeletal filaments 5. Molecular motors prof. MUDr. Oldřich Nečas, DrSc. Nečas, O et al.: Cytoskelet. Academia, 1991 Scientists in Brno Cytoskeleton the dynamic system of proteins filaments and tubules which main functions are transport of substances and cell components, cell support and participation on cell division System of filaments situated in cytoplasm and nucleus The cytoskeleton – fibrillar structures Fixed and labelled cell in culture The cytoskeleton – fibrillar structures Microtubules (green) Actin filaments (red) DNA in nucleus (blue) Microtubules (determine the positions of membrane-enclosed organelles and direct nuclear transport) Microfilaments, actin filaments (determine the shape of the cell's surfaces + cell locomotion) Intermediate filaments (provide mechanical strength) Major types of cytoskeleton filaments ? Cytoskeleton is dynamic structure Cytoskeleton the dynamic system of proteins filaments and tubules which main functions are transport of substances and cell components, cell support and participation on cell division System of filaments situated in cytoplasm and nucleus The cytoskeleton – fibrillar structures Cytoskeleton is reorganised by rapid changes, for example during cell division Crawling fibroblast with polarized dynamic actin cytoskeleton (red). The polarization is assisted by microtubule cytoskeleton (green). Chromosomes are drawn brown. Rapid changes in cytoskeleton Rapid changes in cytoskeletal organisation observed during the development of Drosophila early embryo Rapid changes in cytoskeleton Actin filaments = red Microtubules = green Clump of bacteria (white arrow) is about to be captured by a neutrophil A neutrophil in pursuit of bacteria As the bacteria move, the neutrophil quickly reassembles the dense actin network at the leading edge (red) to push toward the location of the bacteria Although the actin bundles must maintain their stable organisation for the entire lifetime of the animal, the individual actin filaments remain strikingly dynamic and are continuously remodelled and replaced on average every 48 hours Cytoskeleton can form also stable structures ➢ stable structures are typical for such cells that have achieved a stable, differentiated morphology ➢ typically neurons or epithelial cells Polarity enabling cells to tell the difference between ➢ top and bottom ➢ front and back Cytoskeleton is responsible for large-scale cellular polarity Besides forming stable specialised cell surface protrusions Polarized epithelial cells maintain the critical functions between ➢ apical surface = absorbs nutrients ➢ basolateral surface = transfer nutrients through plasma membrane to the bloodstream All the components of the cytoskeleton cooperate to produce the characteristic shapes of specialized cells Cytoskeleton in polarized epithelia Apical surface form microvilli that increase the cell surface (formed by actin filaments, red) Below the microvilli band of actin filaments form cell-cell junctions Intermediate filaments (blue) are anchored to other structures Cytoskeleton in polarized epithelia Apical surface form microvilli that increase the cell surface (formed by actin filaments, red) Below the microvilli band of actin filaments form cell-cell junctions Intermediate filaments (blue) are anchored to other structures Microtubules (green) provide a global coordinate system Structural support of the eukaryotic cell ➢ mechanical stability of the cell ➢ cell shape ➢ internal arrangement (spatial organization) of the organelle Enables the cell movement Regulate the cell movement The cytoskeleton functions 1) Cytoskeleton components are freely available in the cell 2) They create organelles and their parts 3) They participate on building of eukaryotic flagellum, centrioles, spindle body, etc. 4) Microtubules freely penetrate the whole cell 5) Actin filaments form dense network directly under the cell surface Where one can find the cytoskeleton? Microtubules (determine the positions of membrane-enclosed organelles and direct nuclear transport) Microfilaments, actin filaments (determine the shape of the cell's surfaces + cell locomotion) Intermediate filaments (provide mechanical strength) Major types of cytoskeleton filaments Topography of filaments microtubulesIntermediate filaments microfilaments Cytoskeleton filaments are constructed from smaller protein subunits The formation of protein filaments from much smaller subunits allows regulated filament assembly and disassembly to reshape the cytoskeleton Cytoskeleton during changes Filament formation from a small protein subunits Cytoskeleton reorganization Rapid reorganization of the cytoskeleton in a cell in response to an external signal ➢ are long, hollow cylinders made of the protein tubulin ➢ have an outer diameter of 25 nm ➢ consist of 13 parallel protofilament ➢ protofilaments are polymers of tubulin α and β, which form heterodimers Microtubules Structure of microtubules ➢ microtubules are long and straight and typically have one end attached to a single microtubuleorganising centre called a centrosome Functions of microtubules ➢ they run out the centrosome to outer part of cell ➢ organise organelle movement and determine the positions of membrane-enclosed organelles ➢ direct nuclear transport ➢ help forming cell shape and cause as a support skeleton of cell Microscopy of microtubules Dynamic instability of microtubule end Growing and shrinking of microtubule end ➢ Colchicine is so named „mitotic toxin“ 1) It inhibits the polymerisation of tubuline protomers → inhibits mitotic spindle formation → stops mitosis in metaphase 2) It stops the cell movements → slow down leukocyte movement → curing of acute phase of holarthitis ➢ The similar effects have vinblastine and vincristine from Vinca rosea ➢ Taxans from Taxus brefifolia accelerate tubules formation, stabilised them and inhibit depolymerisation Microtubules and colchicine http://www.youtube.com/watch?v=5rqbmLiSkpk&featur e=related Microtubules Cytoskeleton microtubules.wmv Microtubule dynamics 16.5-microtubule_dynamics.mov ➢ two-stranded helical polymers of the protein actin ➢ they appear as flexible structures with a diameter of 5-9 nm ➢ are organised into a variety of linear bundles, twodimensional networks, and three-dimensional gels ➢ actin filaments are dispersed through the cell ➢ but most highly are concentrated in the cortex, just beneath the plasma membrane Actin filaments, microfilaments ➢ mechanically supporting function ➢ altogether with myosin form the contractile apparatus ➢ responsible for a lot of type of intracellular movements ➢ the cytoplasm flux ➢ the cell protrusions and invaginations forming ➢ on higher levels the actin and myosin are the components of muscle cells Actin filaments functions Microfilaments structure Microfilaments structure Filaments resist thermal breakage Single protofilament: thermally unstable Filaments resist thermal breakage Multiple protofilament: thermally stable ➢ bind to polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move steadily along it ➢ transport shipment along microtubules ➢ organise movement of organelles, vesicles, etc. ➢ function is strongly connected with ATP hydrolysis ➢ kinesins and cytoplasmic dyneins Molecular motors Kinesin and dynein Kinesin Movement to plus end of microtubule, from centrosome to cell periphery Dynein Movement to minus end of microtubule, to centrosome microtubule kinesin (dimer) ATP organelle Kinesin microtubule kinesin (dimer) ATP organelle Kinesin http://www.biomach.org/Molecular_Devices/Biological_Motors/Ion_Channels_and_Nanopores/SEC_YEG.jpg Phases during translocation http://www.youtube.com/watch?v=lLxlBB9ZBj4 Kinesin explanation Kinesin.avi + 16.7 Kinesin.mov Organelle movement 16.6-organelle_movement.mov ➢ rope-like fibbers with a diameter of around 10-15 nm ➢ made by intermediate filament proteins ➢ different sizes and constitutions in different cell types; also in the same cells of different animal species ➢ extend across the cytoplasm giving cells mechanical strength ➢ in an epithelia strength the entire epithelium ➢ nuclear lamina just beneath the inner nuclear membrane Intermediate filaments 1) Vimentin-like = cells of mesenchymal origin 2) Desmin – muscle cells 3) Neurofilament proteins = neurons 4) Glial fibrillary acidic proteins = glial cells 5) Cytokeratins = epithelial cells and their derivatives (hair, feathers, nails, skin, …) According to basic protein subunit Types of intermediate filaments http://www.youtube.com/watch?v=FoDniO676Dw Intermediate filament Intermediate filaments.wmv Centrosome – mitotic spindle body ➢ organelle of animal cells and the cell of lower plant ➢ associated with the nuclear membrane ➢ consists of microtubules and associated proteins ➢ serves as the main microtubule organizing centre ➢ participate in separation of chromosomes during nucleus division ➢ undermine orientation of chromosomes and their movement to poles of mitotic spindle ➢ during egg cell maturation disappears ➢ it is transport to zygote by sperm Centrosome function • centriole - central body • centrosphere - dense unstructured net surrounding the centriole • astrosphere - slurry fibers of cytoplasm protruding from the centrosphere centriole pair centrosphere astrosphere Centrosome structure Details of centriole structure A total of 9 microtubule triplets is in each centriole Centriole structure http://www.nature.com/nrm/journal/v2/n9/slideshow/nrm0901_688a_F1.html More information on … Centriole during mitosis Mitotic spindle 17.7-mitotic_spindle.mov 9 pairs of microtubules from protein tubulin + protein dynein Flagellum of eukaryotic cells Cytoskeleton in prokaryotic cells? ➢ the cytoskeleton was considered to be specific feature of eukaryotic cell ➢ it was not possible to find anything inside of prokaryotic cells either by electron microscope JANUARY 2009 Salje et al. found cytoskeletal filaments responsible for DNA segregation during division The method used was cryo-electron microscopy !! Cytoskeleton in prokaryotic cells? J. Salje et al., Science 323, 509 -512 (2009) ParM filaments in frozen E. coli cells Direct observation of bundles cytosol Transmission image of a vitreous cryosection of a cell containing overexpressed ParM filaments J. Salje et al., Science 323, 509 -512 (2009) view over the longitudinal axis ParM filaments ParM filaments Direct observation of bundles J. Salje et al., Science 323, 509 -512 (2009) detail of the filament electron cryotomogram Direct observation of bundles J. Salje et al., Science 323, 509 -512 (2009) in vitro ParM bundles cryotomogram of intact cell ParM filaments cytosol Direct observation of bundles J. Salje et al., Science 323, 509 -512 (2009) Direct observation of bundles Small bundles of ParM filaments are involved in plasmid-DNA segregation Bundles of ParM filaments involved in R1 plasmid DNA segregation lie at the periphery of the nucleoid and may indicate plasmid capture therein nucleoid plasmid nucleoid plasmid nucleoid plasmid Direct observation of bundles J. Salje et al., Science 323, 509 -512 (2009) ➢ filament ParM bundles which enables plasmid segregation lie at the nukleoid periphery ➢ plasmid molecules are entrapped here and are subsequently distributed into daughter cells Model of plasmid DNA segregation pilus of E. coli flagellum of r. Salmonella → pili, flagella Microtubules in prokaryotic cell Flagellum 14.6 bacterial flagellum.mov Who is moving faster, macro or micro organisms? Organism Km/h Body length/s Cheetah Human Bacterium Organisms' movement Who is moving faster, macro or micro organisms? Organism Km/h Body length/s Cheetah 111 Human 37,5 Bacterium 0,00015 Organisms' movement