Prednáška 1 Mikrofilamenty I The cytoskeleton A fibroblast stained with Coomassie blue Microfilaments (actin filaments): 5-9 nm Microtubules: 25 nm Intermediate filaments: 10 nm Actinomyosin complex is not limited to muscle cells „To see what everyone has seen, but think what no one else has thought.“ 1927: C6H8O6 Ignose Godnose Kyselina askorbová Albert Szent-Györgyi Alžbetínska univerzita (1893-1986) (1914-1919) Description of two forms of myosin in muscle Banga & Szent-Györgyi (1942) Muscle myosin 20 min salt extraction overnight salt extraction myosin A myosin B (low viscosity) (high viscosity) +Boiled muscle juice myosin A myosin B (low viscosity) (low viscosity) fibres unchanged +shortened fibres Difference between myosin A and B preparations? +/- ACTIN Substance in boiled muscle juice? ATP Wilhelm Kühne (1864): first description of myosin in muscle Needham, J. et al. (1942). Nature 150: 46-49. Microfilaments (actin filaments; ø5-9 nm) Cellular structures containing microfilaments are involved in cell motility Michael Abercrombie Abercrombie & Heaysman (1953): Observation on the social behaviour of cells in tissue culture. Exp. Cell Res. 5: 111-131. JohnAbercrombie Abercrombie Cell Motility Video 1 Oriented movement of a cell toward an attractant Various forms of actin microfilaments in animal cells lamellipodium Leading edge Various forms of actin microfilaments in metazoan cells Filopodium Lamellipodium Ruffle Lamellum Microvillus Cortical actin Podosome Endosome Phagocytic cup Stress fibre Endocytic pit Golgi ass.-actin Cadherin-based adherent junction Nuclear actin Chhabra & Higgs (2007). Nature Cell Biol. 9: 1110-1121. Scanning electron micrograph showing lamellipodia at the leading edge of a human fibroblast migrating in culture Lamellipodium being extended by a fibroblast in anticipation of locomotion Red: actin Green: Ena Fish keratocyte: Network of actin filaments extending the lamellipodium in the direction of cell movement Lamellipodium formation is regulated by growth factors and their receptors Invasion of filopodia to explore the environment Filopodia as sensors for the advancing cell Actin bundles in a filopodium Roles of microfilaments in eukaryotic cell: Cell division Cell adhesion Cell movement Cell shape Two forms of actin: G- a F-actin minus end plus end Bottom line: F-actin has structural and functional polarity Each actin filament is a two-stranded helix with a twist repeating every 37 nm Myosin S1 head domain as a tool for experimental demonstration of polarity of an actin filament Pointed end Myosin-I S1 head domain Myosin-II Barbed end minus end plus end filament experimental an actin of a tool for as polarity of head domain plus end minus end demonstration 00 nm 1 Myosin S1 Prokaryotic origin of the actin cytoskeleton? MreB of Thermotoga maritima as a model Nature 413: 39-44 (2001) Mbl, a paralogue of MreB in B. subtilis forms helical filaments under cell membrane Cell 104: 913–922 (2001) Prokaryotic origin of the actin cytoskeleton? Comparison between animal G-actin (left) and MreB (right) Nature 413: 39-44 (2001) Effect of addition of pre-formed actin filaments on actin polymerization Nucleation Elongation Steady state s t n +nuclei e m a l i f f -nuclei o s s a M Time The critical concentration (CC) is the concentration of G-actin monomers in equilibrium with actin filaments ) F + Filament G ( n i t c a l Monomer a t o t f o s s a M - C C Actin concentration Polymerization of actin in vivo is modulated by actin-capping proteins Protein binds and keeps actin in ADP-bound form site that binds to Protein bound over actin filament actin-binding site CAN POLYMERIZE CANNOT POLYMERIZE Examples: (+) Capping protein: CapZ (capping inhibited by PIP2) (-) Capping protein: Tropomodulin Blocking the (-) or (+) ends of a filament with acting-capping proteins permits growth only at the opposite ends (-) end (+) end + CC=C C (0.1 mM) (-) Capping protein - CC=C C (0.8 mM) (+) Capping protein Treadmilling filament - + C C > G-actin concentration > C C (-) end (+) end Results from in vitro analysis of actin dynamics are difficult to interpret in a context of a living cell + C C = 0.1 mM Total actin concentration in vivo = ~0.5 mM Yet, 40% of actin is unpolymerized Thymosin is a ATP-G-actin-sequestering protein thus preventing it from polymerization Thymosin: ~5 kDa protein Intracellular concentration ~0.55 mM Binds ATP-G-actin in 1:1 ratio F-actin « G-actin + thymosin « G-actin/thymosin.