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Exam Wednesday covers molecular biology through endocytosis Review sessions here today, Monday, 6-8PM I will upload exam 3 from Gard last fall

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“-” Actin filament 2.

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“-” Myosin head Thick filament

ATP

“+” Myosin is an “actin-dependent” ATPase that acts as a “molecular motor” 1. No nucleotide. Myosin head is tightly bound to actin ( “rigor” ) 2. ATP binding releases myosin from actin 3. ATP hydrolysis “cocks” myosin Pi

ADP

“+” 4. Pi is released, strengthening binding of myosin to actin 5. Myosin binds actin tightly and undergoes “power stroke” releasing ADP Myosin heads “walk” towards “barbed” (“plus”) -end of actin filament 17.7-myosin.mov

Muscle contraction involves actin-myosin II sliding

ECB 17-41

Thick filaments are bipolar; myosin heads on two sides ratchet in opposite directions Both sides ratchet toward + end of actin ( myosin is a + end directed motor ) Causes actin filaments to slide in opposite directions Actin filaments don’t slide back because other myosins are bound

Myofibril contraction results from sliding of thin and thick filaments ECB 17-44 Relaxed myofibril Z + M + Z A-band I-band Z M Sarcomere shortens… I-bands shorten… A-bands unchanged… Z Z Z A-band I-band Thick filaments +ATP +Ca 2+ Thin filaments Contracted myofibril Filaments slide as myosin heads walk toward plus-ends of thin filaments (towards Z-lines)…

Filament sliding leads to contraction

Contraction is regulated by toponin and tropomyosin

ECB 17-48

Tropomyosin filament binds along actin filament Troponin complex binds to tropomyosin In the absence of Ca 2+ , tropomyosin blocks myosin binding In presence of Ca 2+ , Troponin C binds Ca 2+ Conformational change of troponins and tropomyosin uncovers myosin binding site Myosin walks on actin and myofibril contracts Removal of Ca 2+ restores inhibition Where does Ca 2+ come from?

Myofibril contraction is stimulated by release of Ca 2+ from the “sarcoplasmic reticulum Myofibril 17.13-muscle_contraction.mov

Plasma membrane T-system T-tubules formed from invaginations of plasma membrane. The T-system carries “nerve impulse” into muscle fiber… “Sarcoplasmic reticulum (SR)” Derivative of ER SR serves as a Ca 2+ reservoir Signal from neuron causes Ca thru voltage-gated Ca 2+ 2+ release channels.

Ca 2+ stimulates myofibril contraction.

Contraction is terminated by pumping Ca 2+ back into the SR…

ECB 17- 46

Calcium release occurs through a voltage gated channel

ECB 17-47

L19 Non-Muscle Actin

“Non-muscle” actin is abundant in non-muscle cells Microvilli Stress fibers and focal contacts Filopodia and lamellapodia Contractile ring Adapted from ECB figure 17-29 Microvilli: “brush border” epithelia of intestine (increased surface area), “hair cells” of inner ear (sound detection) Stress fibers: adhesion and cell shape (fibroblasts growing in vitro) Filopodia and lamellipodia: at leading edge of moving cells in vitro and in vivo Contractile ring: division of the cytoplasm in animal cells Cortical actin: just beneath plasma membrane of most eukaryotic cells Intracellular transport: myosin-coated organelles move along actin filaments in plants

Actin filaments in cells are often dynamic

Grow and shorten rapidly (minutes), but not as fast as microtubules Regulated by 1 - ATP binding to actin - end ECB 17-31 + end ATP bound actin adds at + end ATP is hydrolyzed and ADP actin destabilizes filament Filaments with more ADP-actin are less stable and tend to depolymerize 2 - Dynamics also regulated by actin-binding proteins

Modulating the assembly/function of actin:actin-binding proteins G-actin ARP 2/3 complex Nucleating proteins Monomer binding Thymosin b 4 proteins Side-binding proteins Tropomyosin Motors Myosins Cross-linking proteins Filamin F-actin End-capping proteins CapZ Severing proteins Gelsolin/villin Bundling proteins a -Actinin

ECB 17- 32

The surface of a moving cell is very dynamic Filopodia Lamellipodia 01.1-keratocyte_dance.mov

Lamellipodia (“ruffles”) : sheet-like extensions of the cell’s leading edge Filopodia (“microspikes”): “finger-like” Motility is dependent upon actin assembly:

inhibited by cytochalasins and latrunculin (fungal toxins that block actin assembly)

How can we visualize actin in cells?

Visualizing actin organization in cells… Filopodia Filopodia Lamellipodia Stress fibers lamellipodium Fixed (dead cells) • Electron microscopy… high resolution but limited area 1. Fluorescence microscopy a. Antibodies and immunofluorescence microscopy… b. Fluorescent Phalloidin • Toxin from the “death angel” mushroom, specifically binds F-actin .

Live cells Fluorescent actin

F-actin is concentrated in filopodia and lamellipodia… Filopodia Lamellipodia + Branched meshwork of short actin filaments in lamellipodia Bundles of actin filaments in filopodia Barbed (+) end of actin filaments oriented towards plasma membrane

Orientation of actin filaments in migrating cell

Arrowhead points to + end

ECB 17-34

Misleading, actually a branched network What do you guess drives extension of lamellipodia and filopodia?

Actin filament assembly - Arp 2/3 complex;

(all eukaryotes?) ARP2 + Rapid elongation ARP2/3 complex F-actin More proteins ARP3 Assembly of actin filaments from “pure” subunits is very slow Actin assembly is facilitated by the ARP2/3 complex Two actin-related proteins (ARPs 2 and 3) …and several other polypeptides in a macromolecular complex.

G-actin VERY SLOW!

ARP2/3 “nucleates” actin filament assembly by providing a template or “seed” that can be elongated by subunit addition.

The ARP2/3 complex caps the minus-end of actin filaments…

ARP2/3 promotes actin assembly at the plasma membrane Plus-ends (barbed) ARP2/3 promotes nucleation of actin filaments Filaments continue to elongated by addition of subunits at their plus ends Continued elongation drives membrane extension Bundling and cross-linking proteins bind to and organize actin filaments Subunits add to plus-ends + + + Filopodia Activated ARP2/3 complex G-actin F-actin ARP2/3 Bundling/cross linking protein End binding protein May also drive extension of tip-growing cells of non-animals (fungal hyphae, pollen tubes)

Arp 2/3 promotes actin nucleation & branching;

ECB 17-36

Activated ARP2/3 binds to side of exisiting actin filaments …and nucleates new filaments from the side (branches) New, elongating filaments are not yet capped Network depolymerizes in rear Branching networks are common in all eukaryotes thus far examined

Actin filament assembly drives extension of lamellipodia Actin filament assembly drives forward membrane extension… Actin filaments disassemble behind the leading edge… Leading edge of cell membrane G-actin F-actin ARP2/3 Capping protein

Actin filament assembly drives extension of lamellipodia Actin filament assembly drives forward membrane extension Actin filaments disassemble behind the leading edge G-actin F-actin ARP2/3 Capping protein

Actin filament assembly drives extension of lamellipodia Actin filament assembly drives forward membrane extension… Actin filaments disassemble behind the leading edge… Actin assembly also drives movement of intracellular parasites 17.9-listeria_parasites.mov

G-actin F-actin ARP2/3 Capping protein

Actin assembly is regulated by Rho family GTPases

“molecular switches” Rac activation causes formation of massive lamellipodium Cdc42 (GTPase family) causes formation of filapodia

ECB 17-39

Modulating the assembly and function of actin: actin-binding proteins G-actin ARP 2/3 complex Nucleating proteins Monomer binding proteins Thymosin b 4 Side-binding proteins Tropomyosin Motors Myosin Cross-linking proteins Filamin F-actin End-capping proteins CapZ Severing proteins Gelsolin/villin Bundling proteins a -Actinin

ECB 17- 32

Bundling vs. cross-linking: a -actinin vs filamin a -Actinin (dimer) is rod-shaped with two actin binding sites: – Forms loose parallel bundles of actin filaments… – Z-line of striated muscle – Stress fibers and focal contacts Filamin (dimer) is has two actin binding sites on long flexible arms: – Forms cross-linked actin “gels” – Smooth muscle … – Stress fibers and focal contacts

Actin filament bundles called “stress fibers” are common in cultured fibroblasts See ECB figure 17-37 Actin bundling proteins ( a -actinin) Myosin II Actin cross-linking proteins (filamin) Polarity of actin filaments in bundle is not uniform Organized by actin binding proteins… Bundling ( a -actinin) Crosslinking (filamin) Type II myosin ECB 17-37

Stress fibers are lnked to the extracellular matrix at “focal contacts” MCB figure 22 10 © Freeman Publishing See ECB figure 16-30 Actin bundling proteins ( a -actinin) Myosin II Actin cross-linking proteins (filamin) Polarity of actin filaments is not uniform “Focal contacts” aka “focal adhesions”

Integrins link actin filaments to the extracellular matrix in animals cells Actin filaments Linker proteins Plasma membrane Integrins Extracellular matrix There are many other linkages from actin to ECM in animal cells Actin filaments in plant cells are also linked to the ECM (cell wall), but the linking molecules are different

Modulating the assembly and function of actin: actin-binding proteins G-actin ARP 2/3 complex Nucleating proteins Monomer binding proteins Thymosin b 4 Side-binding proteins Tropomyosin Motors Myosins use energy of ATP hydrolysis to walk along actin filaments… Cross-linking proteins Filamin F-actin End-capping proteins CapZ Severing proteins Gelsolin/villin Bundling proteins a -Actinin Adapted from ECB figure 16-27

Non-muscle cells contain multiple myosins

Conventional (Type II) Single-headed “Type I” myosin Myosin I Unconventional myosin Small bipolar filaments of ~30-40 molecules. What roles do different myosins play in cells?

Myosin-I and intracellular transport

Main form of transport in many non-animal cells (animal cells primarily use microtubule based motility)

ECB 17-38

Myosin-I on vesicles moves vesicle toward + end of actin filament Always ratchets towards + end Anchored myosin-I can move filament

Myosin-I powers cytoplasmic streaming in green algae and plants Cytoplasmic streaming in Elodia… Vesicles, ER, and other organelles move along actin cables in subcortical cytoplasm Powered by myosin-I; speeds up to 100 µm per second!

Myosins and cell motility

Myosin I In Dictyostelium (cellular slime mold) •Type II myosin (red) is found in the “tail” •Type I myosin (green) is found in the leading edge Myosin II

A model for motility using actin assembly and myosin motors Actin network in cortex Lamellipodium Actin Myosin I Cortex under tension Focal contact Actin polymerization extends lamellipodium (myosin-I?) Movement of G-actin Actin assembly New focal contact Focal contact and actin disassembly Contraction Myosin II New focal contact Myosin II MBoC (4) figure 16-93 Movie 1… Movie 2…

ECB 17-33

Non-muscle cells contain multiple “unconventional” myosins 17 “subfamilies” of myosins: Specialized for specific functions Adapted from MBoC (4) figure 16 54 © Garland Publishing See ECB figure 16-32

Actin Binding Proteins in Plants G-actin ARP 2/3 complex Nucleating proteins Tropomyosin Side-binding proteins F-actin Monomer binding Profilin proteins Cap32 End-capping proteins Motors Myosin Severing proteins Gelsolin/villin Cross-linking proteins Filamin Bundling proteins a -Actinin

ECB 17- 32

By genome sequencing of Arabidopsis, all major classes are present Function of most have not yet been studied Why tropomyosin in plants?