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
plus end minus end
filament actin of an of polarity demonstration
00 nm
1 experimental experimental for tool a as domain head S1 Myosin 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