Prednáška 1 Mikrofilamenty I The A fibroblast stained with Coomassie blue ( 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

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

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 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 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 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